Supramolecular Hydrogelators and Hydrogels: From Soft Matter to Molecular Biomaterials

Abstract

In this review we intend to provide a relatively comprehensive summary of the work of supramolecular hydrogelators after 2004 and to put emphasis particularly on the applications of supramolecular hydrogels/hydrogelators as molecular biomaterials. After a brief introduction of methods for generating supramolecular hydrogels, we discuss supramolecular hydrogelators on the basis of their categories, such as small organic molecules, coordination complexes, peptides, nucleobases, and saccharides. Following molecular design, we focus on various potential applications of supramolecular hydrogels as molecular biomaterials, classified by their applications in cell cultures, tissue engineering, cell behavior, imaging, and unique applications of hydrogelators. Particularly, we discuss the applications of supramolecular hydrogelators after they form supramolecular assemblies but prior to reaching the critical gelation concentration because this subject is less explored but may hold equally great promise for helping address fundamental questions about the mechanisms or the consequences of the self-assembly of molecules, including low molecular weight ones. Finally, we provide a perspective on supramolecular hydrogelators. We hope that this review will serve as an updated introduction and reference for researchers who are interested in exploring supramolecular hydrogelators as molecular biomaterials for addressing the societal needs at various frontiers.

1. Introduction

1.1. Hydrogelators and Hydrogels

Molecular self-assembly is a ubiquitous process in nature, and is also believed to play an essential role in the emergence, maintenance, and advancement of life.¹⁻³ While the primary focus of the research on molecular self-assembly centers on the biomacromolecules (proteins, nucleic acids, and polysaccharides) or their mimics, the self-assembly of small molecules in water (or an organic solvent) also has profound implications from fundamental science to practical applications. Because one usual consequence of the self-assembly of the small molecules is the formation of a gel (or gelation), a subset of these small molecules is called gelators. Depending on the solvents in which they form gels, these small molecules are further classified as hydrogelators⁴ (using water as the liquid phase) and organogelators⁵ (using an organic “solvent” as the liquid phase). More precisely, hydrogelators (i.e., the molecules) self-assemble in water to form three-dimensional supramolecular networks that encapsulate a large amount of water to afford an aqueous mixture. The aqueous mixture is a supramolecular hydrogel because it exhibits viscoelastic behavior of a gel (e.g., unable to flow without shear force). Unlike the conventional polymeric hydrogels that are mainly based on covalently cross-linked networks of polymers (i.e., gellant), the networks in supramolecular hydrogels are formed due to noncovalent interactions between the hydrogelators (Figure 1A).⁶ Considering that water is the unique solvent to maintain life forms on earth, it is important and necessary to distinguish water from organic solvents. Because supramolecular hydrogels are a type of relatively simple heterogeneous system that consists of a large amount of water, it is not surprising that the applications of hydrogels and hydrogelators in life science have advanced most significantly. Thus, in this review we mainly focus on the works that study the properties and explore the applications of supramolecular hydrogels and hydrogelators in biomedical science. Because of the rapid advancement of the field, it is unavoidable that some works are inadvertently absent from this review. Here we offer our sincere apology in advance and hope readers will let us know those deserving works so we can include them in future reviews.

Figure 1.

(A) Illustration of the process for creating polymeric hydrogels via cross-linking (left), or formation of supramolecular hydrogels via a chemical or physical perturbation initiated self-assembly (right). Adapted with permission from ref (6). Copyright 2006 Wiley-VCH Verlag GmbH & Co. KGaA. (B) Molecular structures of 1 and 2. (C) Molecular structure of Nap-FF (3). (D) Optical image and negatively stained TEM image of the hydrogel of 3. Adapted from ref (14). Copyright 2011 American Chemical Society.

1.2. History and Serendipity

According to the report by Hoffman in 1921, the first small molecule hydrogelator was dibenzoyl-l-cystine (1) (Figure 1), which was able to form “a gel of 0.1% concentration [that] was rigid enough to hold its shape for a minute or more when the beaker containing the gel was inverted”.⁷ Interestingly, the same hydrogel was reported by Brenzinger almost 20 years earlier.⁸ However, not until a century later did Menger et al. use modern physical methods in chemistry (e.g., X-ray crystallography, light and electron microscopy, rheology, and calorimetry) to examine the hydrogel of 1 again and provide invaluable molecular details that reveal many fundamental design principles for creating effective hydrogelators made of small molecules. Impressively, among the 14 aroyl-l-cystine derivatives studied by Menger in the seminal work in 2000,⁹ the best hydrogelator (2) is able to self-assemble and to rigidify aqueous solutions at 0.25 mM, ca. 0.01 wt %, in less than 30 s, which probably still holds the record in terms of the lowest concentration of hydrogelators and the fastest rate for gelation.¹⁰ One of the most revealing design principles in the study of 1 is that aromatic moieties are highly effective for enhancing intermolecular interactions in water. This principle is largely responsible for the successful use of aromatic–aromatic interactions to design hydrogelators of small peptides.^11,12 Not surprisingly, nature has already used aromatic–aromatic interactions to evolve proteins.¹³ These facts imply that the use of aromatic–aromatic interactions is an effective and biomimetic way to enhance hydrogen bonds and other interactions in water for molecular self-assembly in water that usually lead to supramolecular hydrogels.¹² A notable example of this principle is that a small dipeptide derivative (3) is capable of enabling many other molecular motifs to self-assemble in water to form supramolecular hydrogels (Figure 1).¹⁴

Despite the above seemingly obvious general principle of supramolecular hydrogelation, one common theme mentioned by the researchers who study gelators, intriguingly, is that their work on gelators started from an accidental discovery of a particular molecule that forms a gel in a solvent. For example, the first small molecule hydrogelator reported by Hoffman unlikely was the intended goal. The research of organogelators also began with a surprise. In 1987, Weiss et al., while investigating the photochemistry of small organic molecules, serendipitously observed that small amounts (usually ca. 1% by weight) of cholesteryl 4-(2-anthryloxy)butyrate (CAB) are able to cause reversible gelation in a wide variety of organic liquids, the different types of functional groups of which in different positions can interact with CAB to affect gelation.¹⁵ Similarly, Shinkai et al., in the process of developing a new synthetic route, have made a surprising finding that the recrystallization of a calix[8]arene derivative from certain solvents (e.g., n-hexane, 1-butanol, and carbon disulfide) results in gels, which is the first example of a gelator derived from macrocycles.¹⁶ In fact, in the research of oligomeric peptides, it is rather common to obtain serendipitous hydrogels of the peptides even if the intended goals are something else.¹⁷⁻²¹ It is quite intriguing that serendipity is a common occurrence throughout the encounter of small molecule gelators.²² These fortuitous happenstances, paradoxically, imply that the formation of supramolecular hydrogels via the self-assembly of small molecules in water is, undeniably, a general phenomenon and a rather common process.²³ Therefore, the exploration of supramolecular hydrogels unlikely will be fruitless, and the applications of supramolecular hydrogelators will have a more profound impact than a mere serendipitous observation, as we intend to illustrate in this review.

1.3. Scope and Arrangement

The development of supramolecular hydrogels in the past two decades not only has underscored the above implication, but also has provided a fundamentally new approach for chemists to control the properties of soft materials via the molecular engineering of a diverse set of substrates for a wide range of applications.^4,24−35 To further realize the far-reaching impact of supramolecular hydrogels in many fields of science and technology, from chemistry to physics and to biology, and from materials to pharmacy to health, it would be helpful to review the progress made so far and to consider possible new directions.³⁶⁻⁴⁴ To contribute to this objective, in this review we intend to provide a relatively comprehensive summary of the work of supramolecular hydrogelators after 2004 and to put emphasis particularly on the applications of supramolecular hydrogels/hydrogelators as molecular biomaterials. We make this rather arbitrary choice for several reasons: (i) The excellent review by Hamilton in 2004⁴ has covered the works on hydrogelators prior to 2004. (ii) While the early research of gelators has made significant progress in the elucidation of the physiochemical properties of hydrogelators and the corresponding small molecule hydrogels,^4,45,46 the past decade has witnessed significant and exciting advances in the exploration of the applications of hydrogelators and hydrogels in biomedicine. (iii) The recent excellent reviews^{25,36,47−55} on gelators (including hydrogelators) have already provided considerable insights into the structure–property relationships of supramolecular hydrogelators; thus, the emphasis on the use of supramolecular interactions and hydrogelators for various applications will complement other review papers and provide a broader perspective at the interface of chemistry and other fields of molecular science. We hope that, by summarizing the development of hydrogelators within 10 years or so and with the emphasis on the design of molecular biomaterials and the relevant applications, in this review we will provide a potential starting point not only for expanding the knowledge base of supramolecular hydrogels as soft molecular biomaterials, but also for attempting to address fundamental questions, perhaps providing a venue for chemists to address the holy grail question in chemistry, that is, the origin of life.⁵⁶

We have arranged the review in the following order. After the brief introduction of the methods for generating supramolecular hydrogels, we discuss the supramolecular hydrogelators on the basis of their categories, such as small organic molecules, coordination complexes, peptides, nucleobases, and saccharides. After the introduction of the molecular building blocks for supramolecular hydrogels, we focus on the various potential applications of the supramolecular hydrogels as molecular biomaterials, classified by their applications in cell cultures, tissue engineering, cell behavior, imaging, immunology, and unique applications of hydrogelators. Particularly, we also discuss the applications of supramolecular hydrogelators after they form supramolecular assemblies but prior to reaching the critical gelation concentration (CGC) because this subject is less explored but may hold equally great promise for helping to address fundamental questions about the mechanisms or the consequences of the self-assembly of small molecules. Finally, we provide our (probably biased) perspectives on supramolecular hydrogelators. We hope that this review will serve as an updated introduction and reference for researchers who are interested in exploring the potentials of supramolecular hydrogelators for discovering, inventing, and creating innovative molecular assemblies, including soft matter and molecular biomaterials. We believe such molecular biomaterials will contribute to addressing the societal needs at various frontiers.

2. Stimuli for Hydrogelation

Despite the fact that they share a prominent appearance and properties (e.g., soft and wet) with polymeric hydrogels, supramolecular hydrogels differ from polymeric hydrogels in many subtle ways. One essential difference is that supramolecular hydrogels, unlike the polymeric hydrogels that originate from a randomly cross-linked network made of strong covalent bonds, are the consequence of molecular self-assembly driven by weak, noncovalent interactions among hydrogelators in water. This subtle yet fundamental difference not only renders more ordered molecular arrangement in the supramolecular hydrogels, but also manifests itself in the process of hydrogelation. While simple swelling usually confers a polymeric hydrogel, a stimulus or a triggering force is necessary to bias thermodynamic equilibrium for initiating the self-assembly process or phase transition to obtain a supramolecular hydrogel. Therefore, there are many forms of stimuli or triggers for manipulating the weak interactions. For the transition from a nongel state to a hydrogel to occur, the free energy must be negative. Thus, the overall impact of the stimuli or triggers usually is negative ΔH or positive ΔS or both, which can be achieved by either physical methods (e.g., changing the temperature, applying ultrasound, or modulating the ionic strength) or chemical methods (e.g., pH change, chemical or photochemical reactions, redox, and catalysis). The following sections briefly introduce the commonly used methods for generating supramolecular hydrogels.

2.1. Temperature or Ultrasound

One of the key features of a supramolecular hydrogel, especially when compared to most cases of gels formed by polymers, is the apt thermal reversibility of the self-assembly process, during which the strengths of hydrophobic interactions and/or hydrogen bonding are temperature dependent. In this kind of hydrogel, the temperature of gelation (T_gel) is one most often reported parameter. Multiple methods are able to determine T_gel, including the “dropping ball” experiment, differential scanning calorimetry (DSC), and/or various rheological measurements.⁵⁷ The data collected via these methods are useful for comparing structurally diverse hydrogelators and evaluating the potential practical applications according to the thermodynamic features of given hydrogels. One notable point is that organogels and hydrogels may differ thermodynamically. For example, Miravet et al. found that the aggregation process is enthalpy driven in an organic solvent but entropy driven in water when studying the molecular hydrogels from bolaform amino acid derivatives 4 (Scheme 1) on the basis of the thermodynamics of gel solubilization.^58,59 This observation, also reported by others,⁶⁰⁻⁶² underscores the fundamental thermodynamic differences between supramolecular organogels and hydrogels, which deserve the attention of researchers.

Scheme 1. Representative Molecular Structures of Hydrogelators To Form Hydrogels after Receiving Different Stimuli.

Though cooling from an elevated temperature is a common approach for making supramolecular hydrogels, an increase of the temperature of a supramolecular hydrogel can give quite opposite consequences: the usual one is formation of a well-dissolved solution, but it is also possible that precipitation will occur at higher temperature. For example, Nandi et al. have applied a range of techniques to demonstrate the effects of temperature and elucidated the activation barriers for the assembly of riboflavin–melamine hydrogels,⁶³⁻⁶⁵ the formation of which is triggered by cooling a homogeneous solution of the mixtures from 80 or 120 to 30 °C. Bhattacharya et al. reported another two-component hydrogel comprising fatty acids and amines, the spacer length of which in the di/oligomeric amine dictates the gel melting temperature.⁵⁵ However, Xu et al. observed that the increase of temperature induces a hydrogel, formed by a dipeptide derivative (Fmoc-d-Ala-d-Ala; Fmoc = (fluoren-9-ylmethoxy)carbonyl), starting syneresis and finally collapsing into a precipitate.¹¹ This behavior is similar to that of lower critical solution temperature (LCST) polymers,⁶⁶ indicating that the increase of the entropy drives the self-assembly of the hydrogelator to a kinetically stable state. A change of temperature also results in many other hydrogels explored for various kinds of applications.⁶⁷⁻⁷² During the past decade, many thermally reversible supramolecular hydrogels have emerged for potential applications in various fields,⁷³⁻⁷⁷ such as drug delivery.^78,79 Regardless of a particular molecular system, gaining a more comprehensive understanding of the thermodynamic properties of supramolecular hydrogels by a change of temperature is always beneficial for the optimization and applications of supramolecular hydrogels.

In chemical laboratories or in industry, ultrasound commonly serves as a convenient physical stimulus to speed dissolution or dispersion or clean up the surface by disrupting weak intermolecular interactions. In fact, it is quite common to use ultrasound to assist the formation of supramolecular hydrogels, but the systematic study of ultrasound to control the properties of soft materials is a rather recent event. In essence, the force of ultrasound readily rearranges the aggregation of molecules by cleaving self-locked intramolecular hydrogen bonds or π stacking to form interlocked structures through intermolecular interactions, usually involving the participation of water molecules. The interest in using ultrasound for gelation has apparently received more attentions in generating organogels. Naota et al. reported an association-inert binuclear Pd complex which, being stabilized by intramolecular π–π stacking interactions, can instantly form gels in a variety of organic solvents upon a brief irradiation with ultrasound.⁸⁰ Later, Naota et al. assumed that ultrasound could destroy intramolecular H-bonding of metal-containing peptides and consequently initiated polymerization under the intermolecular H-bonding in the semistable system.⁸¹ Recently, Ratcliffe et al. found that ultrasound may reshape sheetlike dipeptide particles into elongated molecular assemblies, due to the sonocrystallization effect, as the origin of gelation.⁸² Usually, with the treatment of ultrasound, it is easier for the gelators to induce fibril formation.⁸³ For example, Feng et al. reported that ultrasound can promote cyclo(l-Tyr-l-Lys) (5) to form a hydrogel when its aqueous solution is cooled (Scheme 1), although it normally precipitates in water and gels a number of polar organic solvents, including N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).⁸⁴ In addition, Gu et al. concluded that ultrasound not only accelerates the gelation process and recovers the properties of an l-lysine-based hydrogelator, but also induces the self-assembly of fibrils to entangle and to form 3D networks.⁸⁵ However, ultrasound may also trigger precipitation instead of fibril formation. Nandi et al. reported a metastable bicomponent hydrogel of thymine and 6-methyl-1,3,5-triazine-2,4-diamine that slowly converts into a crystalline precipitate depending on the method of its preparation (e.g., sonication induced).⁸⁶ The similar rich phase behavior seems to be common when ultrasound, combined with a change of temperature, is applied to supramolecular hydrogels.⁸⁷

2.2. pH

A change of pH probably is the most effective and the simplest chemical method to trigger supramolecular hydrogelation because a small amount of acid or base easily and rapidly can lead to a large pH shift via a diffusion-limited process. As an attractive chemical method, a change of pH is particularly useful for generating hydrogels since the pH of an aqueous solution not only is well-defined, but also can be determined easily by a pH paper and precisely by a pH meter. Although pH-triggered hydrogelation largely relies on reversible protonation/deprotonation of basic or acidic group(s) in a particular hydrogelator, a change of pH also may affect the intensity and strength of hydrogen bonding between the hydrogelator and water molecules. Moreover, pH may affect the conformation of molecules to favor hydrogelators to grow from a homogeneous solution to a fibrillar structure in water via noncovalent forces, including aromatic–aromatic interactions, hydrogen bonding, and hydrophobic interactions. Normally, the self-assembly of small molecules, especially molecules possessing acidic or basic groups, requires altering the pH to dissolve in the aqueous phase before adjusting the pH of the solution for screening the charge repulsion to result in hydrogels.

Among a variety of supramolecular hydrogelators, peptide-based hydrogelators are the most common ones that form supramolecular hydrogels on the basis of a change of pH. Specifically, according to the molecular structures of the peptides or peptide derivatives, a change of pH, by affecting the state of charges on the peptidic hydrogelators, usually results in three kinds of hydrogels. (i) Hydrogels that form at low pH: most of the N-terminal-blocked peptides or peptide derivatives result in this type of hydrogel.⁸⁸ (ii) Hydrogels that are stable at physiological pH: many small amphiphilic molecules self-assemble to form hydrogels of this category, and they usually are suitable for certain biological applications or share certain features with natural biomaterials.⁸⁹⁻⁹² (iii) Hydrogels that exist at a high pH: the hydrogelators serving as building blocks of this type of hydrogel likely have a very hydrophobic group or primary amine groups.⁹³⁻⁹⁵ One intriguing and often overlooked fact of supramolecular hydrogelators is that the pK_a of the monomeric hydrogelator may differ from the pK_a of the assemblies of the hydrogelators. For example, Ulijn et al. recently reported that a decrease of the solution pH^96,97 of Fmoc-diphenylalanine (Fmoc-FF, 6; Scheme 1) induces the self-assembly of 6 to form an entangled network of flexible fibrils or flat rigid ribbons, only the former of which results in a weak hydrogel. According to the authors, the self-assembly of 6 to form fibrils consisting of antiparallel β-sheets results in two apparent pK_a shifts, which are ∼6.4 and ∼2.2 pH units above the theoretical pK_a (3.5) of the monomeric 6.⁹⁷

Although protons or hydroxide anions diffuse fast, the self-assembly of the hydrogelators during hydrogelation introduces inhomogeneity. Thus, it is rather necessary and common to combine acid or base titration with mechanical mixing (including ultrasound) to achieve a homogeneous pH change. Recently, Adams and Donald et al. utilized the hydrolysis of glucono-δ-lactone (GdL) to gluconic acid as a means of adjusting the pH gradually in a solution of small molecule hydrogelators, which allows the specific targeting of a certain final pH. This method achieves a uniform pH change of the solution by slowing the release of protons, which appears to be particularly useful for the hydrogelation of the hydrogelators that are soluble at high pH and gel at a lower pH. One notable advantage of this approach is reproducibility of self-assembly and hydrogelation,⁹⁸⁻¹⁰¹ which may be particularly important in the study of the biological functions of the assemblies of small molecules. The same principle should be applicable to the slow release of hydroxide anion for the hydrogelation of amine-containing hydrogelators, which remains to be demonstrated. It is noteworthy that a change of pH usually influences other physiochemical properties of the hydrogelators (e.g., fluorescence^102,103 of the hydrogels or the morphology¹⁰⁴ of the matrixes of the hydrogels).

2.3. Chemical Reactions

Chemical reactions, which often yield products with properties different from the reactants, have become important tools in the production of soft materials, such as hydrogels. Particularly, the incorporation of chemical functional groups into biological molecules can create unique sites of addressable reactivity in even large and complex targets. Although many kinds of chemical reactions have found applications to generate polymeric hydrogels,^105,106 such as click chemistry,¹⁰⁷ redox reactions,¹⁰⁸ Michael addition,¹⁰⁹ ligation reactions,¹¹⁰ acid–base reactions,¹¹¹ and ring-opening metathesis polymerization (ROMP),^112,113 the use of similar approaches to produce supramolecular hydrogels has received much less attention. Recently, Xu et al. reported that a simple chemical modification of a small molecule (8) could generate another molecule (7) with excellent solubility at physiological pH. The solution of 7 turns into a hydrogel upon the addition of a strong base (NaOH) for the hydrolysis of the carboxylic ester bond of 7 to produce 8 (Scheme 2). The unusual property of the hydrogel of 8 is that it is kinetically stable over a wide pH range.¹¹¹ This result illustrates a simple method to produce supramolecular soft materials and may be particularly useful in designing a robust system of prodrugs that can maintain a constant release rate against abrupt changes in the environment. Moreover, Hamachi et al. demonstrated the use of a retro-Diels–Alder reaction to convert a bolaamphiphile to a hydrogelator. Simple heating triggers the reaction and results in a morphological transformation (from 2D nanosheets to a network of 1D nanofibers, as proved by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS)) to give a new heat-set supramolecular hydrogel.⁴³

Scheme 2. Representative Molecular Structures of Precursors and Hydrogelators To Form Hydrogels after Chemical Reactions.

Besides hydrolysis, redox reaction^114,115 provides another useful method for controlling the self-assembly of small molecules. For example, Nilsson et al. demonstrated that reduction of a disulfide bond in a cyclic peptide is a viable strategy for controlling peptide self-assembly to form a hydrogel.¹¹⁶ As shown in Scheme 3, Xu et al. reported that a tripeptide derivative (Nap-FFK, 9), a versatile self-assembly motif, could be integrated with a ruthenium(II) tris(bipyridine) complex to afford the first supramolecular metallohydrogelator (10). As a hydrogelator, not only do the molecules of 10 self-assemble in water to form a hydrogel, but also the hydrogel exhibits a gel–sol transition upon oxidation of the metal center.¹¹⁷ McNeil et al. developed a convenient and portable triacetone triperoxide (TATP) sensor by utilizing a thiol-to-disulfide oxidation to trigger a solution-to-gel phase transition.¹¹⁸ Lu et al. introduced a cysteine-containing small peptide, Ac-I₃CGK-NH₂ (11). Under an oxidative environment, not only do the molecules of 11 form hydrogels at low concentrations, but also the hydrogels exhibit a tunable strength according to the degree of oxidation.¹¹⁵ Recently, Das et al. used native ligation to generate a peptide that forms a dimer upon the oxidation of O₂ in air, and the dimer acts as a gelator in a mixed solvent of methanol/water.¹¹⁹ These arbitrarily selected examples illustrate that there is hardly a limitation of using chemical reactions for generating supramolecular hydrogels. Although any aqueous chemical reaction potentially can generate hydrogels, it is likely that atom-economy reactions¹²⁰ will be particularly more suitable for creating supramolecular hydrogels.

Scheme 3. Representative Molecular Structures of Hydrogelators.

2.4. Photochemical Reactions

For chemical reactions, light is a simple stimulus for activating the reactants and starting a chemical transformation. Particularly, the invention of coherent light sources (e.g., laser¹²¹) has greatly advanced photochemistry,^122,123 which has laid a perfect foundation for the application of photochemical reactions in the creation of materials. Besides being extensively used in the fabrication of a diverse array of materials that include industrial membranes and coatings, dental adhesives, and optical and electronic materials,^124−126 photochemical reactions have already found application in producing polymeric hydrogels.^45,127,128 Because photochemical reactions allow the hydrogels to be defined with both temporal and spatial resolution, it is not surprising that light-derived hydrogels have received increasing attention for broad biomedical applications that include drug delivery, wound healing, tissue engineering, and construction of high-density cell arrays.^129−132 Recently, researchers have explored the use of light to initiate self-assembly for generating supramolecular hydrogels. For example, Yamamoto et al. described the formation and biodegradation of cross-linked natural and related polymer hydrogels, fibers, and capsules with photoinduced methods. The irradiation of the aqueous solutions of copoly[LysLys(Cou)] containing 5–10 mol % ε-[(7-coumaryloxy)acetyl]-l-lysine [Lys(Cou)] residues causes a photo-cross-linking reaction between coumarin moieties in the side chains and turns the solutions to transparent hydrogels.¹³³ As shown in Scheme 4, Schneider et al. developed a photocaged peptide, MAX7CNB (12), which remains unfolded and unable to self-assemble when being dissolved in an aqueous medium. The irradiation (260 nm < λ < 360 nm) of the solution releases the photocage, α-carboxy-2-nitrobenzyl protection, and triggers peptide folding to produce amphiphilic β-hairpins that self-assemble to generate viscoelastic hydrogels.¹³⁴ Besides initiating folding and self-assembly and regenerating rigid, nontoxic soft materials, this photocage chemical method is also used for other applications (e.g., photorelease of functional compounds) since light can be easily controlled in intensity, direction, and duration in both space and time, as illustrated by the widely used calcium photocages.^135,136 Wang et al. presents a photo-cross-linking strategy, based on the ruthenium-complex-catalyzed conversion of tyrosine to dityrosine upon light irradiation, to enhance the mechanical stability of a peptide-based hydrogel by 10⁴-fold with a storage modulus of around 100 kPa, which, according to the authors, is one of the highest reported so far for hydrogels made of small peptide molecules at a concentration of 0.5 wt %.¹³⁷ Considering the well-established photochemistry of [Ru(bpy)₃]²⁺, this method is convenient and versatile for enhancing the mechanical stability of tyrosine-containing peptide-based photo-cross-linked supramolecular hydrogels. Khan et al. used ultraviolet (UV) light to cross-link alginate hydrogels modified with methacrylate groups.¹³⁸ By using a rheometer to monitor the hydrogelation during UV exposure, they illustrated a potentially powerful tool to elucidate the dynamics of gelation and predict the mechanical properties of the hydrogels. Obviously, it is impossible to numerate all the cases of photochemically generated hydrogels in this section. While these examples illustrate the use of photochemical reactions for generating hydrogels, designing new hydrogelators, and predicting the properties of new molecules, we shall mention specific cases when we discuss the building blocks and applications of supramolecular hydrogels in the subsequent sections.

Scheme 4. Molecular Structures of a Photochemical Precursor of a Hydrogelator.

2.5. Catalysis and Enzymes

Catalysis, especially enzymatic reactions, undoubtedly is a prominent dynamic feature of life. Considering that self-assembly is the molecular foundation of life, and soft and wet are another two obvious characteristics of most types of cells, it is not surprising that catalysis and enzymes are attracting increased attention and are achieving many unexpected successes in the generation and applications of supramolecular hydrogelators and hydrogels.

To this day, the reports of catalytic control over self-assembly processes mostly deal with biocatalytic formation hydrogels (e.g., enzyme-instructed hydrogelation),^{6,34,39,139−141} and much room remains for achieving directed self-assembly by catalytic action in fully synthetic systems. Recently, van Esch et al. reported the procedure for preparation of low-molecular-weight hydrogels in the presence of an acid or aniline, which acts as the catalyst for the in situ formation of a hydrogelator. The concentration of the catalyst controls the conjugation of two water-soluble precursors, an oligoethylene-functionalized benzaldehyde and a cyclohexane-derived trishydrazide, thus tuning the gelation time and mechanical properties of the final gels (also see Figure 3).^142,143 Recognizing catalyst-assisted self-assembly as a common process in nature to achieve spatial control over structure formation, they developed an ingenious way to generate a spatially controlled supramolecular hydrogel using a micropatterned catalyst on a surface.¹⁴⁴ According to their design, the precursors (cyclohexane-1,3,5-tricarbohydrazide and 3,4-bis[2-(2-methoxyethoxy)ethoxy]benzaldehyde, 3:1) of a gelator (trishydrazone derivative¹⁴⁴) react on micropatterned catalytic sites on a surface to form building blocks of self-assembled nanofibers that act as the matrixes of the hydrogels. Unlike homogeneous catalysis, this method apparently can achieve multilevel organization among the nanofibers, which is uniquely promising for further development. Liu et al. introduced Cu²⁺ into a glutamic acid-based bolaamphiphilic lipid (N,N′-hexadecanedioyldi-l-glutamic acid, l-HDGA, 13; Scheme 5) to form nanotubes with multilayer walls.¹⁴⁵ Providing a high density of catalytic sites (Cu²⁺), such nanotubes showed enhanced-asymmetry catalytic behavior and accelerated the asymmetric Diels–Alder cycloaddition between cyclopentadiene and azachalcone. While any aqueous catalytic reaction may find applications for generating supramolecular hydrogels, the development of catalytic supramolecular hydrogelation for targeted applications likely will be most useful.

Figure 3.

Catalytic formation of trishydrazone hydrogelator 70 from soluble building blocks 68 and 69 leads to supersaturation followed by formation of fibers that eventually cross-link to form a network that traps the surrounding solvent, leading to gelation: blue, hydrophilic functional groups; red, hydrophobic functional groups. Adapted with permission from ref (142). Copyright 2014 Nature America.

Scheme 5. Representative Molecular Structure of Hydrogelators.

The pivotal importance of enzymes in a variety of cellular processes, including self-assembly and self-organization, justifies the exploration of enzymatic supramolecular hydrogelation. Although the application of enzymes to cross-link covalent polymers is an effective process for generating hydrogels,¹⁴⁶ the use of enzymes to prepare supramolecular hydrogels has several distinct advantages, such as the opportunity to achieve sophisticated secondary structure, adaptability to structural modification, and, most importantly, excellent accessibility to enzymes both in vitro and in vivo due to the fast diffusion of small molecules. Despite the huge diversity of enzymes, so far only a handful of enzymes have been explored for catalyzing hydrogelation. These enzymes are phosphatase,^147−158 β-lactamase,¹⁵⁹ esterase,¹²⁸⁹ matrix metalloproteinase-9 (MMP-9),^164,165 α-chymotrypsin,¹⁶⁶ thrombin,¹⁶⁷ chymotrypsin,¹⁶⁷ and β-galactosidase¹⁶⁸ (for catalyzing bond cleavage reaction), lipase,¹⁶⁹ microbial transglutaminase (MTGase),¹⁷⁰ and thermolysin^{160−163,171} (for catalyzing bond-forming reactions), and some other enzymes such as glucose oxidase,¹⁷² peroxidase,^173−175 and tyrosinase.¹⁷⁶ Regardless of the types of reactions or enzymes, the essential feature for enzymatic hydrogelation of small molecules involves the enzymatic conversion of a precursor into a hydrogelator (normally via bond cleavage or bond formation, but not limited to these two). The self-assembly of the hydrogelators to form supramolecular nanostructures (usually nanofibers), and the entanglement or alignment of the nanofibers, affords the matrixes of the hydrogel. The first case of enzymatic formation of supramolecular hydrogels is the use of an alkaline phosphatase¹⁷⁷ to dephosphorylate a precursor, Fmoc-tyrosine phosphate (14), under slightly basic conditions to form a hydrogelator (15), which self-assembles in water to form a supramolecular hydrogel (Scheme 6).¹⁵³ Besides promising a new methodology for the creation of hydrogels in situ, this process also builds up a platform for screening enzyme inhibitors^158,159 and detecting the presence of an enzyme.¹⁵⁹ Instead of catalytically breaking bonds for supramolecular hydrogelation, Ulijn et al. took a different approach by triggering the self-assembly of peptide hydrogels via reverse hydrolysis using thermolysin.¹⁶² Utilizing the fact that certain proteases can thermodynamically favor the formation of peptide bonds, they used thermolysin to catalyze the coupling of two amino acid precursors to form a hydrogelator, which then self-assembled to form a hydrogel. A major advantage of employing reverse hydrolysis is that no byproducts except water are formed, although the use of hydrophobic precursors may be problematic in water. The demonstration of enzymatic supramolecular hydrogelation has sparked relatively active research of bioresponsive materials. For example, Yang et al. reported the use of an enzymatic dephosphorylation process to assist the formation of supramolecular hydrogels.¹⁵⁷ McNeil et al. recently developed a modular system for detecting protease activity.¹⁶⁷ They designed and developed a precursor (16; Scheme 6) which is unable to form a hydrogel under most conditions, but turned into a translucent gel upon the treatment of a protease (i.e., thrombin). The past decade has witnessed a considerable success in preparation of supramolecular hydrogels using enzymatic transformation. Certainly, the promises of enzymatic hydrogelation are far from fully realized. We hope that the more detailed discussion of the applications of the specific cases of enzymatic hydrogelation in the later sections will provide stimulation for further development.

Scheme 6. Representative Molecular Structures of Precursors and Hydrogelators To Form Hydrogels on the Basis of Catalysis.

3. Characterization of Supramolecular Hydrogels

The increased number of hydrogelators and the requirement of more information on supramolecular hydrogels at both the nanoscale and molecular levels require more accurate analysis and characterization of the hydrogels. In this review, we first give a brief introduction of various techniques generally used to characterize supramolecular hydrogels before discussing the classifications and potential applications of hydrogelators. Especially, we focus on the high-resolution techniques that elucidate the molecular self-assembly processes leading to gelation. Unavoidably, some sample preparation methods may affect the native nanostructures of the hydrogels. Thus, among all the characterization techniques, those methods that preserve the native properties of hydrogels should be preferred over the ones that need to dry or/and to stain the samples. Generally speaking, the analysis and characterization of supramolecular hydrogels aim to help scientists better understand how the small molecules are arranged and organized in the matrixes of hydrogels, which may lead to new approaches not only for rational design of supramolecular hydrogelators but also for the development of various functional molecular biomaterials.

3.1. Visual Inspection

The classic “inverting-vial” method is still the simplest way to initially assess a supramolecular hydrogel.^178,179 Visual inspection of the material just by flipping the vial upside down, which acts as a “zeroth-order” characterization technique, provides an intuitive impression for researchers on the shape and strength of the hydrogels. Thus, according to the visual inspection, one can easily classify the material as a solution, viscous liquid, half-gel, or solidlike gel, which may contribute to selection of more suitable techniques for further characterizing the hydrogels. Despite its simplicity, this assay by the “naked eye”, in fact, provides important information—whether a small molecule self-assembles in water.

3.2. Microscopy

With the rapid development and easier operation of microscopic instruments, researchers rely more and more on the microscopy techniques to study the morphology of micro- and nanostructures that act as the matrixes of supramolecular hydrogels. Among all the microscopy techniques, atomic force microscopy (AFM) or scanning force microscopy (SFM), as high-resolution scanning probe microscopies, can achieve a resolution on the order of fractions of a nanometer, which is more than 1000 times better than the optical diffraction limit. Using AFM, one may analyze hydrated samples in situ under high humidity conditions or even without dehydration.^180,181 In addition, it is possible to measure the roughness of a sample surface at a high resolution, which helps classify a sample on the basis of its mechanical properties (e.g., hardness and roughness) and offers new capabilities for the microfabrication of a sample (e.g., an atomic manipulation). However, scanning force microscopy can be misleading due to multiple factors,¹⁸² and it is imperative to use multiple techniques to verify uncommon observations obtained by AFM or SFM.

Electron microscopy techniques, including transmission (TEM)¹⁸³ and scanning (SEM)¹⁸⁴ microscopies, utilize a beam of accelerated electrons as a source of illumination. Since the wavelength of an electron is rather short, electron microscopy has the capacity to reveal the structures of small objects with resolution up to a nanometer. For example, TEM can achieve better than 50 pm resolution.¹⁸⁵ On the basis of these properties, electron microscopy can provide valuable information about the morphology of the molecular aggregates/nanofibrils leading to hydrogelation. However, the requirement of completely dried samples under the operating conditions (e.g., high vacuum) makes TEM and SEM less reliable for inferring the native molecular arrangement in the hydrogel state since the dehydration process may result in artifacts that are difficult to explain. Furthermore, staining agents (e.g., urnayl acetate, phosphotungstate, or osmium tetroxide), which are used to increase the electron density of TEM samples to improve the quality/contrast of the images, may interact with hydrogelators to change the self-assembly morphology and to induce artifacts. Cryogenic techniques have already been used in TEM for studying the self-assembled structures. For cryo-electron microscopy (cryo-EM) or electron cryomicroscopy, the samples are studied at cryogenic temperatures (generally liquid nitrogen temperatures) through creation of thin vitrified ice films.¹⁸⁶ Cryo-EM has the advantage of reducing or eliminating the artifacts by making nanometer resolution images of the native gel state feasible. Indeed, several research groups have already successfully determined the structures of supramolecular hydrogels and their related fibrous assemblies with cryo-EM.^187,188 Particularly, a seminal work of the use of cryo-EM to solve the structures of peptide nanofibers¹⁸⁹ has demonstrated the capability of EM for studying the self-assembly of small molecules in water, which is emerging as a new frontier of supramolecular chemistry at the intersection of chemistry and cell biology.¹⁹⁰ In addition, the development of another technique, environmental scanning electron microscopy (ESEM),¹⁹¹ also provides a useful approach to characterize supramolecular hydrogels under a certain humidity.¹⁹²

3.3. Oscillatory Rheometry and Differential Scanning Calorimetry

Oscillatory rheometry, as a comprehensive technique to characterize viscoelastic materials, is becoming a routine measurement of supramolecular hydrogels. Rheology, which studies the flow of supramolecular hydrogels, can provide tertiary structural information about the type, number, and strength of networks responsible for the observed hydrogelation.¹⁹³ Several types of setups, such as parallel plates, concentric cylinders, and cone-and-plate systems, are suitable for the measurement of the rheological properties of hydrogels. All the setups contribute to making a thin layer of supramolecular hydrogels between a stationary and a movable component. The basic principle of oscillatory rheometry is to measure the response of supramolecular hydrogels to an applied oscillatory stress, which is quantified by the elastic properties, such as G* (complex modulus), G′ (storage or elastic modulus), and G″ (loss modulus or viscosity). Meanwhile, the relationship between these variables and the oscillatory frequency, imposed stress, temperature, time, or hydrogelator concentration usually contributes to the studies of certain key characteristics (e.g., critical strains, thermodynamic nature of gelation) of hydrogels. The temperature of gelation (T_gel) is one of the most often studied characteristics of a gel, which is determined by the point that noncovalent cross-links or global molecular rearrangements are broken by thermal energy. Differential scanning calorimetry (DSC)¹⁹⁴ is a well-established characterization method for the test of T_gel, especially when there is a sharp phase transition associated with hydrogelation. Although the rheological, elastic, and thermodynamic properties of supramolecular hydrogels provide limited insight into the atomistic molecular arrangement and understanding of how small molecules self-assemble to form hydrogels, the combination of physical characterization with a systematic structural modification of the hydrogelators would contribute to establishing the structure–property relationships of supramolecular hydrogels. Clearly, this approach requires the synthesis of molecules, which is a core activity of chemistry. Moreover, the synthesis of new molecular entities provides opportunities for discovering new materials, especially supramolecular hydrogels. This kind of approach, together with additional techniques and greater correlation of various techniques, ultimately should help infer the nanostructures of supramolecular hydrogels in great molecular detail.

3.4. X-ray Diffraction

Small-angle X-ray scattering (SAXS) is another technique for characterizing supramolecular hydrogels with a resolution close to that of TEM.^195,196 Different from TEM, which focuses on the local morphology of gel matrixes, SAXS mainly provides averaged information on the matrixes of supramolecular hydrogels by measuring the spatiality of the matrixes. In addition, wide-angle X-ray powder diffraction (XRD) contributes to the elucidation of the molecular organization and nanostructures of supramolecular hydrogels, especially when microcrystals are formed in the hydrogels.¹⁹⁷ The long d spacing obtained from XRD represents the longest repeat distance in the ordered structures by molecular self-assembly, which may provide insight into the packing of small molecules in either an extended or a bent conformation. A related technique for the characterization of gels is small-angle neutron scattering (SANS).^198,199 Although it is a rather specialized technique to which researchers have limited access, SANS is able to provide useful information about the average sizes and shapes of the nanostructures in a supramolecular hydrogel.^200−202

3.5. Other Physical Methods

Besides the techniques referred to above, some other physical methods, such as circular dichroism (CD), UV/vis, infrared (IR) spectroscopy, fluorescence, or NMR, may also provide certain information about the molecular arrangement in supramolecular hydrogels through detection of the molecule–molecule or molecule–water interactions in primary or secondary structures. For instance, CD has a wide range of applications in many different fields, such as the study of the secondary structures of proteins, or the investigation of charge-transfer transitions. In the cases of soft materials such as hydrogels, CD is able to study the self-assembled superstructures in the gel phase or at the gel-to-sol transition.^203,204 However, it always remains a challenge to make any precise conclusion from the CD spectra alone, which means that it is better to combine CD with other techniques for studying secondary structures of supramolecular hydrogels. UV/vis is the technique used for investigating π–π stacking (or aromatic–aromatic interactions) or metal coordination in the process of hydrogelation.^18,205−207 UV/vis, in combination with CD, may provide information for certain molecular arrangements in hydrogels. IR spectroscopy, dealing with the infrared region of the electromagnetic spectrum, is suitable for confirming the presence of hydrogen bonding and determining the protonation state of carboxylic acids.^208−211 Fluorescence is a useful tool for the investigation of the aggregation between aromatic groups and the formation of hydrophobic pockets inside hydrogels. In addition, the incorporation of fluorescent probes into supramolecular hydrogelators usually results in large, flat aromatic surfaces for self-assembly, thus providing a reliable approach for the design of an effective strategy to understand the gelation process and to discover more biological applications of supramolecular hydrogels.²¹² Furthermore, solution-state NMR can identify the chemical shift changes in the aggregation process from the solution spectra to the gelled ones.²¹³ Solid-state magic angle spinning NMR (MAS NMR), being extensively used for characterizing structures of protein or peptide aggregates, may be useful for elucidating the structures of supramolecular hydrogels. Recent reports on the use of solid-state NMR to elucidate the packing of Aβ in Aβ amyloids²¹⁴ indicate solid-state NMR as a powerful method for elucidating the molecular arrangement of aggregates. However, the requirement of isotope labeling has limited the routine use of solid-state NMR for characterization of supramolecular hydrogels. In summary, it is always beneficial to analyze all the data collected via multiple methods for elucidating the nanostructures and potential applications of a given supramolecular hydrogel since data from various techniques are usually complementary to each other.

3.6. Modeling

On the basis of the molecular structural data collected from microscopy, XRD, SANS, or rheology, it is possible to use modeling for proposing a plausible arrangement of the molecular organization in supramolecular hydrogels.²¹⁵ Actually, researchers have already developed some relevant model systems from computer simulation about the gelation process of gelators in organic solvents.^216−220 However, few modeling approaches, currently, are reliable in describing the self-assembly of small molecules in water because of the inherent kinetic nature of hydrogels²²¹ and the lack of an accurate description of hydrophobic interactions, which, interestingly, are the major driving forces for small molecules self-assembling in water to form supramolecular hydrogels.

4. Molecular Design

While implying that the formation of supramolecular hydrogels via the self-assembly of small molecules in water is a common process, the serendipitous discoveries of many supramolecular hydrogelators also paradoxically indicate that, presently, it is still impossible to predict a hydrogelator a priori on the basis of its molecular structure. In fact, many designs of supramolecular hydrogelators only become possible after the serendipitous discovery of a particular hydrogelator. The inability of the prediction, in our opinion, mainly originates from the inaccurate evaluation of the interactions between water molecules and the hydrogelators (and their assemblies). Despite this currently unsolved problem, supramolecular hydrogelators, indeed, share common features despite their different molecular structures. Like certain proteins that self-assemble, supramolecular hydrogelators possess amphiphilicity and require noncovalent interactions (π–π interactions, hydrogen bonding, and charge interactions among the molecules, among others) that allow effective building up of three-dimensional networks as the matrixes of hydrogels.

Scheme 7 shows a few classical examples of hydrogelators that certainly are the products of multiple weak interactions. Being derived from an existing family of low-molecular-weight organic gelators (urea derivatives),^222,22317, an effective hydrogelator, maintains the intermolecular hydrogen bonds provided by its bisurea motif²²⁴ and allows its free carboxylic acid groups for both solubility in water and pH control. Consisting of a saccharide (instead of using urea) for hydrogen bonding, 18 also relies on the long alkyl chain to enhance intermolecular hydrophobic interaction and to promote intermolecular hydrogen bonding among hydroxyl groups and amide bonds,^225,226 which results in a gelator that gels a diverse range of solvents (including water).²²⁷ Incorporating l-lysine, which is easily made into an amphiphile, 19(228) and 20(37) not only act as hydrogelators, but also have inspired a wide range of other hydrogelators^{76,211,229−232} based on l-lysine. Along the notion of synthetic amphiphiles,²³³21 consists of two alkyl tails and self-assembles to form micelles, which result in hydrogelation.^234,235 Instead of relying on alkyl chain(s), 22(11) utilizes aromatic–aromatic interactions of (fluoren-9-ylmethoxy)carbonyl (Fmoc) to promote intermolecular hydrogen bonding for supramolecular hydrogelation. Because the Fmoc group is commonly used as a protection group for peptide synthesis, this convenience has led to many other Fmoc-based peptide hydrogelators.²³⁶ These explorations and other related studies,^12,237,238 undoubtedly, establish aromatic–aromatic interaction as an effective hydrophobic force to enhance intermolecular hydrogen bonding for self-assembly of small molecules in water.^12,239 An intriguing and unexpected candidate as a hydrogelator is a vancomycin derivative (23).¹⁸ Although a vancomycin analogue (ramoplanin) is able to form nanofibers upon binding to its receptor (a lipid I analogue),²⁴⁰ it is still unusual for 23(18) to act as a hydrogelator. This case, indeed, reflects the essential role and the diverse origins of multiple weak interactions for supramolecular hydrogelation. Despite the immense diversity of the hydrogelators, an essential requirement of a supramolecular hydrogelator is amphiphilicity. Although adequate intermolecular interactions among the hydrogelator are necessary for the self-assembly of the hydrogelators in water, one should avoid excessive intermolecular interactions that may result in the precipitation of the molecules in water. Since several excellent reviews^4,5,36 have already discussed molecular design in depth, interested readers are recommended to consult those reviews. Instead of prescribing a set of detailed rules of the design of a hydrogelator, we simply introduce the hydrogelators and hope the readers will formulate their own intuition on the aspect of the molecular design of supramolecular hydrogelators.

Scheme 7. Representative Molecular Structures of Hydrogelators.

4.1. Hydrogels Based on Small Organic Molecules

Because the discovery of supramolecular hydrogels was made with small organic molecules,⁴ we first discuss the small organic molecules that act as the molecular building blocks of supramolecular hydrogels (Table S1). The large and diverse pool of building blocks makes categorizing these small molecules rather subjective; thus, we arrange the hydrogelators according to their resemblance in molecular structure and start with the hydrogelator having the lowest molecular weight within each type. However, it is not necessarily that the hydrogelator having the lowest molecular weight would be the most effective hydrogelator. The most effective one should be the one that occupies the least volume fraction to form a hydrogel. Thus, highly effective hydrogelators should be able to gel water at a very low weight percentage. Another reason for this arrangement is that the structural similarity of the hydrogelator may offer a feasible starting point for the theoreticians who are interested in supramolecular hydrogels and strive to formulate principles for predicting supramolecular hydrogelation on the basis of molecular structures, which is still a challenge. In the following, we first discuss supramolecular hydrogels made of homotypic hydrogelators, and then introduce hydrogels consisting of a mixture of small molecules.

4.1.1. Urea-Containing Hydrogelators

By attaching a pyridyl group to the urea motif, Dastidar et al. synthesized a small hydrogelator (24; Scheme 8) that forms a hydrogel with the CGC of 0.8 wt %.^241,242 It was found that the urea group has to be at the para position of the pyridine to form the hydrogel. Because ethylene glycol molecules interact with both 24 and water, the authors were able to grow the crystals of 24 in a mixed solvent of water/ethylene glycol. The crystal structure contains both water and ethylene glycol and reveals valuable details about the intermolecular interactions that involve 24, water, and ethylene glycol. Scanning electron microscopy (SEM) shows the fibrils formed by 24 in water are much thinner than the fibrils of this hydrogelator formed in water/ethylene glycol, suggesting the addition of ethylene glycol promotes the interfibrillar interactions. There are many other hydrogelators based on the urea motif developed during this decade.^243−256 For example, John et al. reported a urea-containing hydrogelator, 1-[3-(decyloxy)phenyl]urea (25 (n = 10)) that not only forms a hydrogel in water at 0.1 wt %, but also serves as a matrix for preparing and stabilizing gold nanoparticles by in situ reduction.²⁵⁷ Steed et al. reported the gelation ability of a series of chiral bisurea gelators (26).²⁵⁸ When n is an even number in 26, the molecules act as a gelator (1 wt %) in a mixed solvent (e.g., CHCl₃–MeCN–DMSO:H₂O = 7:1), but 26 fails to form a gel when n is an odd number. According to the crystal packing diagrams, the antiparallel urea tape motif appears to be necessary for the formation of hydrogels, which consist of matrixes made of microcrystals. Shimizu et al. designed second-generation self-assembling bisurea macrocycles (e.g., 27), which consist of more flexible building blocks that form columnar structures in the solid state.²⁵⁹ van Esch et al. reported a class of efficient hydrogelators based on a simple attachment of hydrophilic hydroxyl or amino functionalities to cyclohexane bisurea organogelators. They found that 28 in 1 N NaOH forms a hydrogel with a CGC of 0.5 wt %. Interestingly, after the formation of the hydrogel, the pH decreases to around 11.2. Further lowering the pH to 10.1 results in a gel–sol transition. While the pure enantiomer of 28 results in a more stable hydrogel than that made of racemic 28, the hydrogel of racemic 28 melts, almost being independent of the concentration. The authors observed that the racemic hydrogel became turbid upon heating, a commonly observed phenomenon for an entropy-driven hydrogel.²⁶⁰

Scheme 8. Representative Molecular Structures of Urea-Containing Hydrogelators.

4.1.2. Pyridine-Containing Hydrogelators

As shown in Scheme 9, Tang et al. synthesized a small gelator, 29, from 3-hydroxy-2-aminopyridine and glutaric anhydride. 29 forms a supramolecular hydrogel at a concentration of 1.5 wt %. The authors found that an increase of the power of the ultrasound from 200 to 500 W decreases the width of the self-assembled fibers from 8 to 2 μm, accompanied by an increasing network density in the hydrogels.²⁶¹ Dastidar et al. synthesized a series of bisamides derived from l-(+)-tartaric acid as potential hydrogelators. Among 14 bisamides synthesized, dipyrid-3-yltartaramide (30) displays an intriguing nanotubular morphology of its gel network in the gel made in DMF/water. Bearing a pyridyl group, 30 is able to coordinate with Cu(II)/Zn(II) salts under suitable conditions to afford metallogels. One unique aspect of this study is that the authors managed to obtain a considerable amount of single-crystal structures of those gelators. While polymorphism likely exists in the gel phase, these structural details have provided useful insights to understand the plausible intermolecular interactions among the gelators.²⁶²

Scheme 9. Representative Molecular Structures of Pyridine-Containing Hydrogelators.

While 30 fails to form a gel at pH below 7.0, another pyridine-containing amino acid-based gelator (31) forms gels in aqueous media in the presence of hydrochloric acid. Besides the fact that it forms a transparent gel in a water/ethanol mixture at a CGC of 0.2 wt %, the solution of the gelator successfully detects and traps hydrogen chloride gas, likely due to the sol–gel transition when the pH is lowered.²⁶³ Tang et al. synthesized a hydrogelator, 2,6-bis[N-[(carboxypropyl)carbonyl]amino]pyridine (32), from 2,6-diaminopyridine and glutaric anhydride by a one-step procedure. 32 forms a self-supporting hydrogel at a concentration of 4 wt %, which contains microcrystalline networks to immobilize water.²⁶⁴ Sambri et al. reported a class of terpyridine derivatives (e.g., 33), in their bisprotonated forms, to act as versatile hydrogelators upon ultrasound irradiation. Although the terpyridine ligand chelates with metal cations, resulting in stable gels with tunable emissive properties, the SEM images of the hydrogels exhibit only a slight change before and after the chelation.^265,266

McNeil et al. designed an innovative class of pyridine-based gelators that formed gels in a mixed solvent of water and DMSO. For example, 34 forms a gel at 3 wt % in 1:1 DMSO/water. Besides investigating the relationship between molecular structure and gelation ability of these pyridine-based compounds,²⁶⁷ the authors discovered that some of the gelators are able to sense nitric oxide.²⁶⁸ If this class of compounds can act as hydrogelators with a reduced use of DMSO, they likely will find broader applications. Bhattacharya et al. reported an effective hydrogelator (35) based on (phenylenedivinylene)bispyridinium. With two n-octyl chains, 35 forms a hydrogel in water with a CGC of 0.12 wt %. This hydrogelator self-assembles to give a morphological transition from fiber to coil to tube, depending on the concentration of the gelator. Because the emission of the chromophore is sensitive to the environment, self-assembly of the gelator and a change of the ionic strength lead to the aggregates fluorescing in different colors.²⁷⁴ This type of fluorescent colloid, recently rediscovered and termed “aggregation-induced emission” by Tang et al.,^269,270 was reported three decades ago or earlier.^271,272 In fact, the restriction of bond rotation to generate fluorescent colloids had already found applications in molecular imaging about two decades ago.²⁷³ The generation of a white-light emission from a single chromophore in a single solvent (water),²⁷⁴ indeed, agrees with the polymorphism of the assemblies of the hydrogelators, which illustrates the versatility of supramolecular hydrogels. The authors also observed a similar emission switch when the solvent was a mixture of ethanol and water and reported that the color of the emission depends on the temperature.²⁷⁵

4.1.3. Alkyl-Chain-Containing Hydrogelators

As shown in Scheme 10, Schmidt et al. designed and synthesized a series of derivatives of N-amidated 3- and 4-aminobenzoic acids with linear alkyl chains ranging between 3 and 13 methylene units long, among which the 4-(octanoylamino)benzoic acid sodium salt is able to form supramolecular hydrogels thermoreversibly in aqueous solutions of alkaline sodium salts at a concentration of 1 wt % 36 and 1 N NaOH. Moreover, a mold-casting/drying process can transfer the supramolecular assemblies to produce self-supporting, macroscopic, supramolecular, nanofiber mats, which are thermally and mechanically stable, and resistant to a large variety of organic solvents. On the basis of SEM, XRD, and cryo-TEM, the authors proposed the mechanism of the formation of the nanofibers of 36, involving the transformation of spherical micelles into ribbons and platelets of multiple stacks of bilayers of the sodium salt of 36.²⁷⁶ Araki et al. reported an asymmetrically substituted sulfamide (37) that forms a hydrogel in water at a CGC of 1.0 wt %. SEM and XRD suggest the formation of lamellar superstructures via a hydrogen-bond-directed amphiphilic 2D sheet. 37 can also gel an organic solvent, such as benzene. One intriguing property is that the casted film of 37 and the xerogel of 37, from benzene or water, result in almost identical XRD patterns. More interestingly, this gelator is able to form homogeneous and heterogeneous biphasic gels when the solvents are benzene and water. It would be useful to develop applications of the biphasic gels.²⁷⁷ On the basis of a similar concept, Araki reported that 38 forms a hydrogel at a CGC of 0.5 wt % upon protonation of the tertiary amine groups. The authors also observed lamellar superstructures and suggested the formation of 2D sheetlike assemblies by the 2D hydrogen bond networks between sulfamide moieties. The authors also reported that, with an increase of the concentration of 38 to 2.0 wt %, the hydrogel exhibits relatively high mechanical stability.²⁷⁸

Scheme 10. Alkyl-Chain-Containing Hydrogelators.

Patnaik et al. reported that cetylpyridinium chloride (CPC) (39) forms a gel with a CGC of 6 wt % in a mixed solvent of chloroform and water. On the basis of SAXS, the authors suggested that the packing of the molecules is polymorphic, which also leads to a lamellar organization.²⁷⁹ The authors also reported a two-component gel resulting from 39 in the presence of a structure-forming bolaamphiphilic additive, 6-aminocaproic acid (6-ACA), and the CGC remains at 6 wt % for the mixture of 39 and 6-ACA. The authors used SAXS to infer that the gelators assemble as a lamellar organization of a loosely interdigitated bilayer structure of 39 and 6-ACA molecules predominantly due to charge transfer, hydrogen bonding, and hydrophobic interactions.²⁸⁰ Alanne et al. recently reported a simple hydrogelator²⁸¹ based on bisphosphonates (BPs), a well-known class of compounds used for treating osteoporosis. Similar to the incorporation of bisphosphonates in both polymeric hydrogels²⁸² and supramolecular hydrogels,^283,28440 is a new supramolecular hydrogelator consisting of bisphosphonates which forms a transparent hydrogel (at 4 wt %) that contains lamellar structures.²⁸¹

Baskar et al. reported that N-octadecylmaleamic acid (41) formed hydrogels with a CGC of 0.75 wt % in basic conditions. Small-angle X-ray diffraction indicates lamellar structures in the hydrogels. The hydrogels likely are lyotropic liquid crystals because they exhibit birefringence.²⁸⁵ Tiller et al. reported that a simple azo dye gels water at 5 wt % upon cooling from hot water. Using a glass slide coated with positive charge, the authors were able to induce hydrogelation on the surface when the solution concentration of 42 was as low as 0.10 wt %.²⁸⁶ The authors suggested that this significant reduction of CGC might be a useful concept for the design of drugs. This concept,²⁸⁷ indeed, is supported by the hydrogelators that inhibit bacteria¹⁸ or cancer cells.²⁸⁸ In a more detailed study, the authors found that the hydrogel prepared from 42 consisted of highly ordered and stable hierarchical structures. On the basis of nuclear magnetic resonance, rheology, X-ray scattering, birefringence, and microscopy, the authors suggested that 42 forms worm micelles as the matrixes of the hydrogel of 42.²⁸⁹ Kawai et al. reported hydrogelators 43 and 44, which consist of three amide moieties and one alkyl chain. At pH 9.0, the CGC values for 43 and 44 are 1.2 and 0.3 wt %, respectively. A decrease of the pH of the gels leads to a gel–sol transition due to the protonation of the ternary amine. On the basis of X-ray diffraction and FT-IR analyses, the authors concluded that 43 and 44 form lamellar-like aggregates in the hydrogels, presumably because the amide moieties form strong intermolecular hydrogen bonds. Despite the fact that there is a suspension phase between the pH-induced gel–sol transitions, the hydrogel of 43 or 44 exhibits high sensitivity to the pH change, which is needed for the phase transition.²⁹⁰

While redox chemistry is a fundamental process in nature, there are only limited numbers of reports on the electrochemical characterization of supramolecular hydrogels.^291,292 Yang et al. has proposed an electrochemical strategy to characterize the hydrophobic microenvironment of micellar hybridized supramolecular gels.²⁹³ As shown in Scheme 11, by using a gemini surfactant (45) and the classical gelator N,N-dibenzoyl-l-cystine (1) to form a micellar hybridized hydrogel, the authors quantitatively characterized the net positive shifts of the redox formal potential and the change of peak currents obtained from the cyclic voltammograms of methylene blue (46).²⁹³ According to the authors, by comparing the apparent diffusion coefficients of 46 in these different systems, it is feasible to characterize the hydrophobicity change of the hybrid supramolecular hydrogel made of 1 and 45.

Scheme 11. Representative Molecular Structures of Alkyl-Chain-Containing Hydrogelators.

Lee et al. demonstrated an ingenious way to combine a nonionic surfactant and an aromatic core to generate an innovative class of molecules that self-assemble in water.²⁹⁴ For example, the authors reported that a T-shaped aromatic amphiphile, consisting of tetrabranched oligo(ethylene oxide) chains, self-assembles to form nanofibers in water, which result in hydrogelation at a concentration of 0.5 wt %.²⁹⁵ Unlike most other hydrogelators, 48 forms a hydrogel when the temperature increases. This type of LCST, though being common for polymeric hydrogels, is less reported for supramolecular hydrogels.

Hao et al. investigated hydrogels formed by mixing alkyltrimethylammonium bromides 49 and sodium azobenzene-4,4′-dicarboxylic acid (50). In a typical example, 49 and 50 in a 2:1 ratio form a hydrogel at a concentration of about 4.0 wt %. The authors found that UV irradiation or the addition of a salt and an acid results in a gel–sol transition, while the addition of a base hardly changes the hydrogel, suggesting that it is important to maintain the ionic state of 50 for the hydrogelation.²⁹⁶ Ward et al. reported that amphiphilic guanidinium alkylbenzenesulfonates 51 exhibit lyotropic behavior in aqueous solvents. At a relatively high concentration, 10 wt %, 51 self-assembles to form a lamellar structure and results in hydrogels.²⁹⁷ Huang et al. demonstrated that the mixture of an imidazole-type surfactant, 1-hexadecyl-3-methylimidazolium bromide (52), and a sodium salicylate (53) produces a thermoresponsive hydrogel at a CGC of 2 wt %. The authors reported that, above the critical temperature, the sample exhibits viscoelastic properties of wormlike micelles, and the viscoelastic solution transforms into an elastic hydrogel accompanied by a remarkable increase of the elastic modulus.²⁹⁸ Hoffmann et al. studied the phase behavior and aggregation in the aqueous solutions of mixed 2-phenylbenzimidazole-5-sulfonic acid sodium (54), an anionic UV absorber, and cetyltrimethylammonium bromide (55), a cationic surfactant.²⁹⁹ The authors found that the morphologies (i.e., vesicles, tubules, or ribbons) of the self-assembled structures depend on the ratio of the two components in the mixture. For example, a hydrogel forms at 0.6 wt % with a molar ratio of 54 and 55 of 8:2. The authors found that the formation of very long stiff tubules about 14 nm in diameter leads to hydrogelation, and suggested that the stiffness of the bilayer of the vesicles and the stiffness of the tubules originate from the rigidity of 54.

4.1.4. Hydrogelators Containing Multi/Polyhydroxyl Groups

As shown in Scheme 12, Shan et al. reported an interesting small molecule (56) that self-assembles in 6 M KOH to form a hydrogel at a concentration as low as 0.3 wt %. When the concentration of 56 increases to 1.4 wt %, the gel–sol transition temperature almost reaches 100 °C. According to the authors, 56 is the first low-molecular-weight gel electrolyte having good electrochemical properties while solving the problem of solution leakage, which may find application in supercapacitors.³⁰⁰ Also, using sorbitol, Niu et al. developed a smart functional gelator containing a salen moiety (57). 57 self-assembles in a mixed solvent of DMSO/H₂O to form a gel at a CGC of 3 mM (0.13 wt %). The gel turns to a solution upon the addition of copper(II), and the solution reverses back to the gel state upon the addition of EDTA to competitively coordinate away the copper(II).³⁰¹ Song et al. also reported a d-sorbitol-based hydrogelator and the effect of salt on the hydrogelation of 2,4-(3,4-dichlorobenzylidene)-d-sorbitol (DCBS, 58), which forms a hydrogel at a concentration of 1 wt %. While SEM indicates that the hydrogels consist of globular aggregates, the addition of NaCl to the aqueous medium not only accelerates the gelation, but also results in networks of long fibers. Using UV/vis and fluorescence emission spectra to characterize the hydrogels, the authors concluded that extensive aggregation of the phenyl rings is responsible for the gelation. Variable-temperature ¹H NMR spectra further demonstrate that the addition of the salt NaCl enhances the π–π interactions. Wide-angle X-ray diffraction shows that the hydrogels have a layered structure that is independent of the addition of NaCl. The authors also used density functional theory (DFT) calculations to support the proposed molecular packing of the gelator in the nanofibers.³⁰²

Scheme 12. Hydrogelators Containing Multi/Polyhydroxyl Groups.

Griffiths et al. found that bis-α,β-dihydroxyl esters are able to gel thermoreversibly a wide range of solvents.³⁰³ As a gelator, 59 forms a gel in a water-rich (75%) ethanol/water mixture at a concentration of 0.18 wt %. On the basis of SANS, Ohsedo et al. suggested that in the gelation mechanism the bis-α,β-dihydroxyl ester motif forms rodlike structures.^304,305 On the basis of a well-known organogelator, dibenzylidenesorbitol, Smith et al. developed a simple condensation between sorbitol and 2 equiv of a benzaldehyde derivative to form a hydrogelator (60) which is functionalized with hydrazide (as replacements for carboxylic acids). 60 not only self-assembles to form hydrogels, at a CGC of 0.8 wt % in water across a wide pH range, with a small amount of DMSO, but also exhibits pH-switchable dye adsorption–desorption depending on the protonation of the target dyes.³⁰⁶ Kim et al. designed a hydrogelator (61a) derived from riboflavin (vitamin B₂).³⁰⁷ The authors found that 61a forms a hydrogel at a concentration of 1.6 wt %, but 61b and 61c are too soluble to form a hydrogel. One interesting observation reported by the authors is that the ability of hydrogelation apparently is beneficial for the delivery of vascular endothelial growth factor small interfering RNA (VEGF-siRNA) into human cells.³⁰⁷ Russo et al. reported that arborols, a type of dumbbell-shaped molecules acting as bolaamphiphiles, are able to assemble spontaneously into long fibers and to lead to thermally reversible gels. On the basis of wide-angle X-ray scattering, the authors concluded that the self-assembly of 62 at 0.2 wt % results in fibrils in solution and the formation of bundles of fibrils at 2 wt % is responsible for the hydrogelation.³⁰⁸ Harada et al. reported a chemical-responsive supramolecular hydrogel based on a derivative of β-cyclodextrin (63). After the hydrogelator 63 forms a hydrogel at a CGC of 2.9 wt %, the addition of 1-adamantanecarboxylic acid or a large amount of urea induced a gel-to-sol transition.³⁰⁹ On the basis of a detailed NMR study, the authors suggested that the host–guest and hydrogen-bonding interactions of cyclodextrins lead to the formation of supramolecular fibrils, which explains the chemoresponsiveness of the hydrogels.³⁰⁹ Instead of using β-cyclodextrin, Osakada et al. used α-cyclodextrin and an alkylpyridinium to generate a series of pseudorotaxanes that form hydrogels. According to the authors, the possible mechanism is that the host–guest interactions transform the micelles of an alkylpyridinium to nanofibers of the pseudorotaxanes and result in gelation at a concentration of about 12 wt %.³¹⁰

4.1.5. Hydrogelators Having C₃ Symmetry

As shown in Scheme 13, Xu et al. developed a supramolecular gel in a mixture of ethanol and water (1:1) based on N,N′,N″-tris(3-pyridyl)trimesamide (64a).³¹¹ The nitrogen and amide group in hydrogelator 64a can bind with phosphate and carbonate ions via H-bonding and act as biomineralization active sites for growing biominerals. The authors found that the calcium phosphate grew into curved platelike nanostructures along the fibers. In another work, Dastidar and Das et al. reported hydrogelators 64a and 64b derived from the pyridyl amide of trimesic acid³¹² and demonstrated that 64a and 64b are able to form gels in a mixed solvent of MeOH/H₂O at concentrations of 0.2 and 0.1 wt %, respectively. On the basis of the crystal structures of 64a and 64b, the authors also proposed intermolecular interactions among the gelators, which is reasonable if the matrixes of the gels are microcrystalline. Li and Xu et al. also studied the gel of 64a (0.55 wt %) in a 1:1 mixture of ethanol and water at pH 7.0. They found that the macroscopic viscoelastic properties of the gel of 64a depend on the microscopic hydrogen bonding between the amide N–H bond and the nitrogen on the pyridyl group (N–H···Py). One notable feature was the increase of the storage modulus of the gels upon a decrease of the pH to 5.0. The authors suggested that a partial break of the hydrogen bonds of N–H···Py leads to a highly branched and homogeneous fibrillar network in the gel, as revealed by XRD and field-emission scanning electron microscopy (FESEM) images.³¹³ Such highly branched fibrillar networks likely result from the reduction of the crystallinity of the fibrillar network, which is a useful insight for the design of supramolecular hydrogels. The same laboratory reported that another molecule of trimesic amide (64c) self-assembles to form hexagonal microtubes in a mixed solvent of H₂O/THF and is able to gel H₂O/THF at a concentration of 1.0 wt %.³¹⁴

Scheme 13. Hydrogelators Having C₃ Symmetry.

Replacing the pyridyl group in 64b by a benzoic acid in the trisamide to produce 64d, Schmidt et al. demonstrated that 64d acts as a hydrogelator and forms a photoluminescent hydrogel in water at a CGC of 0.2 wt %. Having carried out DFT calculations, the authors suggested that the photoluminescence originates from the formation of a supramolecular chromophore.¹⁰³ On the basis of the structure of 64d, researchers investigated a series of simple benzene-1,3,5-tricarboxamide (BTA)³¹⁵ aromatic carboxylic acid compounds. Lloyd et al.³¹⁶ found that the N-methylation of the amide bond in 64d results in a compound to give a precipitate upon a decrease in pH, implying the critical role of hydrogen bonding between the amides for hydrogelation. The authors also introduced a hydroxyl group or a naphthyl moiety to generate the hydrogelators 64e and 64f, respectively. Both 64e and 64f have a CGC of 0.1 wt %. Compared to the hydrogel of 64d, the hydrogel of 64e exhibits a 10-fold higher yield stress, and the hydrogel of 64f results in a 4-fold higher storage modulus. Nagarajan et al. reported that the replacement of carboxylic groups in 64d by alkyl chains also leads to gelators which largely gel organic solvents such as DMSO.³¹⁷

Bommel and van Esch et al. developed a class of effective hydrogelators based on cyclohexane-1,3,5-tricarboxylic acid. By capping the C-terminal phenylalanine with diethylene glycol, they obtained a hydrogelator (65) with a remarkably low CGC value (0.033 wt %).³¹⁸ The authors also obtained the crystal structure of an analogue (a nongelator) of 65 and provided useful insights for the molecular design of this type of hydrogelator.³¹⁸ In another illuminating study, Friggeri and van Esch et al. investigated the ability of several 1,3,5-cyclohexanetricarboxamide–phenylalanine derivatives 66 to form hydrogels. While they found that enantiomerically pure homochiral 1,3,5-cyclohexanetricarboxamide–l-phenylalanine crystallizes from water and fails to form gels, the heterochiral derivatives with either two l-phenylalanine moieties and one d-phenylalanine (LLD) or vice versa (DDL) are able to form hydrogels with a CGC value of 0.04 wt %. The authors also found that an increase of the concentration to 0.12 wt % LLD-66 or DDL-66 results in hydrogels with a remarkably high gel–sol transition temperature at 120 °C. The authors also demonstrated that the attachment of a second amino acid or a hydrophilic moiety to the C-terminal of the homochiral derivatives of 66 produces effective hydrogelators.³¹⁹ To align the amide bond in this type of 1,3,5-triamide cyclohexane derivatives, van Esch and Samori et al. used an electrical field to assist the alignment of the nanofibers and demonstrated that the application of a voltage bias, indeed, helps the directional orientation of the fibrils.³²⁰

Using the 1,3,5-triamide cyclohexane-based hydrogelators 67, van Esch et al. demonstrated an elegant system that forms well-defined nanostructures by the orthogonal self-assembly of hydrogelators and surfactants.³²¹ Taking advantage of the thermoreversibility of the hydrogels made of the 1,3,5-triamide cyclohexane-based hydrogelators (e.g., 67), the authors dissolved the hydrogelators in solutions of surfactants above the gel–sol transition temperature, followed by cooling the mixture and examining the hydrogelation. One of the most interesting results reported by the authors was that cryo-TEM studies revealed that, when lipids, DOPC, are used, the unilamellar DOPC vesicles encapsulate well-defined fibers with a diameter of 5 nm in the middle of their aqueous compartment (referred to as “gellosomes” ³²²) (Figure 2). Another remarkable feature was that the membrane wall is able to restrict the growth of the fibers to a few hundred nanometers. These observations imply that one should be able to use the mutual interactions between both of the self-assembled structures to design sophisticated soft materials, as recently suggested and further demonstrated by van Esch et al.^323,324 van Esch et al. recently reported another seminal study on the catalytic formation of the triamide cyclohexane-based hydrogelators.¹⁴⁴ Specifically, either an acid or a base can catalyze the formation of a trihydrazone hydrogelator (70) from the soluble building blocks 68 and 69. The authors demonstrated that the concentration of the catalyst used in the in situ formation of the hydrogelator controls the gelation time and mechanical stiffness of the final gel (Figure 3).^142,325 This work may lead to an elaborate way to form hydrogels via control of the reaction kinetics.³²⁶

Figure 2.

Cryo-TEM images of unilamellar dioleoylphosphocholine (DOPC) vesicles coexisting with a network of well-defined fibers of 67 with a high aspect ratio. Adapted with permission from ref (321). Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

4.1.6. Hydrogelators Derived from Rigid Aliphatics

As shown in Scheme 14, Terech et al. investigated hydrogels made of cationic bile acid derivatives (e.g., 71). They found that 71 (I^– as the anion) forms a robust hydrogel at 2.0 wt % to exhibit a storage modulus of 0.3 MPa. On the basis of X-ray crystallography of the single crystals and X-ray scattering experiments, the authors concluded that the gel state consists of a morphology different from that of the solid, which is supported by EM investigations of the xerogels to reveal the fibrous nature of the gel networks. On the basis of the structural difference of the derivatives and the morphology of the networks in the hydrogels, the authors suggested an interesting notion that more compact structures would develop at low concentrations.³²⁷ The same group of authors used SANS to study the self-assembled structures of the hydrogels formed by the hydrogelators 71–73. They found that 71 forms thick cylindrical fibers (R = 68 Å), the aggregates of 72 are ribbons with a bimolecular thickness of t = 37 Å and an anisotropy of the section of b/a ≈ 0.1, and 73 exhibits a remarkable transition from ribbons to thicker cylindrical fibers upon an increase of the concentration. The authors also suggested the existence of secondary aggregation mechanisms in the formation of bundles,³²⁸ differing from the behaviors of the hydrogels formed by sodium lithocholate.^329,330 Terech et al. also extensively investigated the effect of the electrolyte and counterions on the gelation of 73. They found that the addition of a monovalent salt (NaCl) favors the formation of gels. At larger salt concentrations, the gels become more heterogeneous with nodal zones on the micrometer scale.³³¹ These studies provide a rare case to compare the molecular arrangements of the gelators in water and in an organic solvent.³³²

Scheme 14. Bile Acid-Derived Hydrogelators.

To understand the remarkable ability of hydrogelators made of cationic derivatives of deoxycholic acid, Maitra et al. designed and synthesized a series of that class of hydrogelators and compared them with hydrogelators based on natural anionic bile salts.³³³ According to the authors, these cationic hydrogelators are pH independent and start to aggregate in water at a concentration an order of magnitude lower than those at which natural anionic bile salts aggregate.³³³ Recently, Maitra et al. reported tunable luminescent gels and xerogels formed by lanthanide(III) cholates, which, according to the authors, might find applications as luminescent coatings on a glass surface.³³⁴ Another type of innovative bile acid derivative reported by Maitra et al. is the perfluoroalkyl bile esters, which are efficient gelators in organic and aqueous–organic media.³³⁵ The requirement of an organic solvent as the cosolvent seems a quite common feature of the hydrogelators derived from bile acid, which occurs in several other bile acid derivatives reported.³³⁶ Maitra et al. also reported a hydrogelator based on tripodal cholamide (74). As a supergelator, 74 forms a hydrogel with a CGC of 0.02 wt % in water containing 0.01% acetic acid. Using fluorescent probes, 8-anilinonaphthalene-1-sulfonic acid and pyrene, the authors found two critical aggregation concentrations and suggested a progressive increase in aggregate size and the microviscosity of the aqueous pool encompassed by the self-assembled fibrillar network during the gelation. One noteworthy result from this work is that the microviscosity of the aqueous phase around the network of nanofibers is far less than the bulk viscosity of the gel.³³⁷ The authors also used the tripodal cholamide-based hydrogel to synthesize semiconducting nanostructures and obtained nanotubes and nanorods of CdS, ZnS, and CuS.³³⁸

Galantini and Tato et al. reported an interesting hydrogelator of a bile salt derivative that forms a hydrogel in bicarbonate buffer (pH 10) at a concentration of 0.18 wt %.³³⁹ The authors used a range of techniques (static light scattering (SLS), CD, SAXS, TEM, and optical microscopies) to establish the details of the self-assembly of 75, which occurs at 1.8 wt % in the buffer and forms supramolecular nanotubes. According to the authors, the tubule formation starts with the aggregation of the fibrils, followed by a slow transformation and ordering of the tubule walls in well-spaced layers. One interesting feature is that the final elongation of the tubules proceeds without a further aggregation of fibrils.^340,341 By introducing a diamine or a dicarboxylic aromatic residue on the lateral of a natural bile acid, Galantini et al. obtained compounds 76 and 77. While 77 forms a hydrogel at a CGC of 0.16 wt %, a mixture of 76 and 77 results in a hydrogel at a CGC of 0.05 wt %. This work, thus, illustrates that the presence of the electrostatic interaction promotes the hydrogelation from more dilute samples, suggesting that cationic and anionic mixtures enhance the efficiency of the gelators.³⁴² Since there is greater understanding of the structures of the hydrogelators based on bile acids, the exploration of their applications is emerging as well. For example, Shen and Zhang et al. reported the use of the hydrogels of bile acid derivatives for creating gold and silver nanoparticles in situ.³⁴³ Xin and Xu et al. reported that sodium deoxycholate (78) forms hydrogels at a concentration of 2 wt % in the presence of NaCl or NaBr.³⁴⁴ They made an interesting observation that the addition of l-lysine or l-arginine turns the hydrogels to solutions. The authors suggested that the addition of amino acids competes with the hydrogen bonds needed for hydrogelation, thus causing a gel–sol transition.

As shown in Scheme 15, Shinkai et al. reported a versatile gelator (79) that is able to gel more than 10 different solvents.³⁴⁵ Although 79 is unable to form a hydrogel, Sierra et al. used “click” chemistry to connect this type of estradiol-based gelator to form another effective gelator (80) that gels a DMSO and water mixture at concentrations as low as 0.04 wt %.³⁴⁶ Using a cholesteryl derivative, Fang et al. developed another gelator (81) that gels a 1:1 mixture of acetone and water at 0.06 wt %.³⁴⁷ Also employing a cholesteryl group, Ji et al. developed a series of phospholipid hydrogelators (82). Besides exhibiting polymorphism of the networks in the hydrogels, this type of hydrogelator forms hydrogels at CGC values as low as 0.05 wt % (82, m = 0, n = 2).³⁴⁸ Ju et al. reported a conjugate of oleanolic acid with adenine (83) which forms a gel in mixed solvents of THF and water (2:3) at a CGC of 2 wt %. One feature of this gel is that the addition of uracil decreases the stability of the gel due to the disruption of hydrogen bonding between the hydrogelators.^54,349,350 Lu et al. recently reported that sodium glycyrrhetinate (84) is able to form a hydrogel with a CGC of 5.6 wt %, and the authors suggested the dipole–dipole interaction of sodium carboxylates as the main driving force for the hydrogelation of 84.³⁵¹ Another related interesting work is the hydrolysis of succinated triamcinolone acetonide which forms triamcinolone acetonide and results in hydrogelation.³⁵²

Scheme 15. Some Bile Acid- or Cholesterol-Derived Hydrogelators.

Dastidar and Shibayama et al. took a combinatorial library approach to generate 60 organic salts by reacting 5 bile acids (e.g., 85) with 12 secondary amines (e.g., 86). After the gelation test with various aqueous and organic solvents, they found that 16 salts are supramolecular gelators, 6 of which are able to form gels in aqueous as well as organic solvents. The salt didodecylammonium cholate (85 + 86) is the most versatile gelator, forming a gel in a 1:1 mixed solvent of DMSO and H₂O at a CGC of 1 wt %. The authors used dynamic light scattering (DLS) and SANS to infer the fibrous network formed via flexible clusters of a few tens of nanometers in length, followed by the immobilization of the network in the gel.⁶⁸ Bhattacharya et al. demonstrated the formation of a supramolecular hydrogel by simply mixing lithocholic acid (87) with dimeric or oligomeric amines (e.g., 88), at a total concentration of 5 wt %. However, the replacement of lithocholic acid (LCA) by cholic acid or deoxycholic acid results in no hydrogelation. On the basis of the single-crystal X-ray diffraction analysis with one of the amine–LCA complexes, the authors suggested that the electrostatic forces and hydrogen bonding between the amines and the carboxylate and hydroxyl moieties result in the formation of fibers as the matrixes of the hydrogels.³⁵³ Song et al. used zwitterionic alkyldimethylamine oxide 89 to interact with litholic acid (87) to form hydrogels. When n = 12 in 89, the two-component system exhibits a high gelation capability (CGC = 0.08 wt %). One notable feature is that an increase in the temperature results in a transition from helical fibrils to vesicles with an intermediate mesophase.³⁵⁴ Tian et al. also reported an interesting photoswitchable cholesterol derivative as a gelator,³⁵⁵ though it is too hydrophobic to act as a hydrogelator.

4.1.7. Bolaamphiphilic Hydrogelators

As shown in Scheme 16, Blume et al. reported a class of symmetric long-chain bolaamphiphiles that are efficient hydrogelators (90 and 91).^{199,356−366} Among them, dotriacontane-1,19-diylbis[2-(dimethylammonio)ethyl phosphate] (90) forms a clear hydrogel at 0.1 wt % and pH 5. TEM reveals the hydrogelator to form a dense network of helically structured nanofibrils with a diameter of 3–4 nm. At pH 5, 90 self-assembles to form nanofibrils that are stable up to at least 75 °C. Although there is no gelation at pH 10, nanofibrils form, but they become fragmented at 75 °C.³⁵⁸ The authors reported that SANS data support the significantly higher stability of the hydrogel of 90.¹⁹⁹ While the formation of nanofibrils of 90 or 91 in the hydrogels agrees with these hydrogelators self-assembling to form worm micelles, the interfibrillar interactions depend on the kinetics of the self-assembly, thus resulting in rich polymorphism. For example, the cryo-TEM of the hydrogels of 91 (n = 17 or 18) reveals the formation of square lamellae.³⁶¹ Besides using the pH to control the morphology of the assemblies of those hydrogelators, the authors also demonstrated that the changes of the symmetry of the head groups are able to tune the self-assembly behavior of single-chain bolaamphiphiles in an aqueous suspension.³⁶⁴

Scheme 16. Bolaamphiphiles as Hydrogelators.

Dobner et al. designed and synthesized a series of polymethylene-1,ω-bis(phosphocholine) (PC-C_n-PC) analogues,³⁶⁷ and found that the even-numbered ones form nanofibers composed of stretched molecules with an all-trans-alkyl chain conformation.³⁶⁷ Meanwhile, they synthesized the odd-numbered analogues to study a possible even–odd effect of these bolaamphiphiles during their aggregation in water. In addition to these bolaamphiphiles with phosphocholine head groups, they designed a series of polymethylene-1,ω-bis(phosphodimethylethanolamine)s (Me₂PE-C_n-Me₂PE) with smaller sizes of the head group. These bolaamphiphiles show an additional fiber–fiber transition when the alkyl chain length exceeds 26 carbon atoms. The behavior of the mixed bolaamphiphiles indicates that the fiber structure allows differences in the alkyl chains of up to six carbon atoms long. The mixing of two Me₂PE-C_n-Me₂PE- or PC-C_n-PC-type bolaamphiphiles with different alkyl chain lengths offers the possibility to adjust the temperature of the gel–sol transition, at which the cross-linking of the fibers breaks and the fibers dissociate. On the basis of this feature, the authors obtained thermally switchable hydrogels, which may allow fine-tuning for drug delivery applications. The comparison with dotriacontane-1,32-diylbis[2-(methylammonio)ethyl phosphate] (MePE-C₃₂-MePE, 92), a bolaamphiphile with an even smaller phosphomonomethylammonium head group, illustrates the importance of the size of the head group for self-assembly. This bolaamphiphile self-assembles exclusively into lamellar structures, a type of assembly that persists in mixtures containing the fiber-forming molecules (90).³⁵⁹

Using different methods, researchers already have characterized the supramolecular behavior of the bolaamphiphilic hydrogelators dotriacontane-1,32-diylbis[2-(trimethylammonio)ethyl phosphate] (PC-C₃₂-PC, 91) and the pH-sensitive dotriacontane-1,32-diylbis[2-(dimethylammonio)ethyl phosphate] (Me₂PE-C₃₂-Me₂PE, 90).^357,359 Depending on the temperature, pH, and concentration, these bolaamphiphiles self-assemble into long nanofibers or other assemblies, such as short rods or micelles. To obtain information about the motional dynamics and microscopic order inside these assemblies, Blume et al. carried out a systematic electron spin resonance (ESR) spin probe study and reported that the spectra obtained with the spin probes 5-, 12-, and 16-doxylstearic acid (n-DSA) are highly sensitive to the changes in the bolaamphiphilic arrangement. The authors obtained rotational correlation times and order parameters from full ESR line shape simulations and found that the transition temperatures, determined by the maximum hyperfine splitting, agree with the differential scanning calorimetry (DSC) data. By comparimg 5-DSA and 12-DSA, which reside at different positions in the alkyl chain region of the assemblies, the authors found that trans–gauche isomerization predominantly occurs in the outer region of the assemblies. For Me₂PE-C₃₂-Me₂PE (90) at pH 10, the authors reported that ESR data indicate the micelles to be short rods rather than spherical in shape and that an increase of the concentration from 1 to 10 mg/mL leads only to a one-dimensional growth of these micelles.³⁶⁰

On the basis of the structure of 90, Drescher and Meister et al. developed two unique bolalipids (93 and 94) which not only self-assemble at a concentration of 0.01 wt %, but also are able to modulate the viscoelastic properties of the hydrogel made of 90 or 91.³⁶⁸ Generally, these bolaamphiphiles are much more effective hydrogelators than the hydrogelators made of diacylphosphatidylcholine.³⁶⁹ Zhang et al. developed a bolaamphiphile (95) that has two carboxylic acid ends and a diketopyrrolopyrrole chromophore in the center. On the basi of the color change associated with the self-assembly process, the authors concluded that the π–π stacking of the central parts and the hydrogen bonding between the ends are responsible for the formation of the nanofibrils of 95 in water. Although 95 self-assembles at a concentration as low as 0.06 wt % in water, the formation of a hydrogel has not been reported by the authors.³⁷⁰ Similarly, another bolaamphiphile (96) bearing a bipyridine moiety at the central part, though forming nanofibers at 0.15 wt % by self-assembly, was not reported to form a hydrogel by Zhang et al.³⁷¹ Benvegnu et al. reported unsymmetrical diacetylenic bolaamphiphiles 97, which bear a carbohydrate residue and a cationic glycine betaine moiety. When m = 13, 97 forms a hydrogel at a CGC of 1.7 wt %. TEM studies by the authors revealed the polymorphism of these bolalipids and the dense filament of the hydrogelators in the hydrogels.³⁷² Patnaik et al. developed a series of two-component hydrogels based on cetyltrimethylammonium bromide (55) and bis(decyloxy)succinic acid (98) to study the effect of the chirality of the amphiphile on gelation. Besides finding that 55 and 98 form vesicles and hydrogels that are pH and temperature responsive, the authors concluded that molecular chirality is responsible for the formation of supertwisted fibrils in the hydrogels at a 98:55 molar ratio of 1:2 with 31% water.⁷¹ Zakharova et al. reported a macrocyclic bolaamphiphile (99) consisting of thiocytosine fragments. 99 forms a gel in water–DMF (20 vol %) at a concentration of about 1 wt %. One interesting feature is the observation of two break points in the surface tension isotherms, which correspond to the critical micelle concentration (CMC) and critical gelation concentration. The authors also observed that the pH of the solution decreases with an increase of the concentration of the hydrogelators.³⁷³

4.1.8. Hydrogelators Bearing a Cavity

On the basis of cyclotriveratrylene (CTV), Jiang et al. developed a class of supramolecular hydrogelators having a cavity. By introducing deprotonable COOH or protonable NH₂ as the terminal groups into the rigid and hydrophobic CTV backbones, the authors successfully used 100 and 101 (Scheme 17) to form supramolecular hydrogels with CGCs of 1.0 and 1.5 wt %, respectively. The obtained hydrogels of 100 and 101 are luminescent and exhibit pH-responsive, reversible gel–sol transitions. The work also illustrates that the skeleton of an organogelator is a promising starting point for designing a hydrogelator.³⁷⁴ Mocerino and Ogden et al. reported a proline-functionalized calix[4]arene (102) that forms hydrogels in the presence of specific anions such as nitrate, bromide, iodide, and perchlorate. However, it requires a considerable amount of the hydrogelators (over 18 wt %) to form the hydrogels. Since acidic conditions and the presence of a lanthanide drastically reduce the amount of anions needed for hydrogelation, the hydrogelation likely depends on more than just the presence of the anions.³⁷⁵ The later report by the same group, indeed, confirmed that the lanthanum cations connect two supramolecular helices to form a 2D network for hydrogelation.³⁷⁶ Escuder, Miravet, and Ballester et al. developed an aryl-extended calix[4]pyrrole that acts as a receptor for tetramethylammonium. The authors found the formation of hydrogels in basic conditions and at neutral pH, thus suggesting that, as a guest molecule, tetramethylammonium interacts with the calix[4]pyrrole to form noncovalent polymers, resulting in hydrogelation.³⁷⁷ Lee and Park also reported a similar structure–property relationship for another calix[4]arene-based hydrogelator, in which large alkali-metal cations (K⁺ or Rb⁺) trigger the hydrogelation.³⁷⁸

Scheme 17. Some Hydrogelators Containing a Cavity.

Marletta and Cunsolo et al. reported a type of pH-responsive hydrogelator based on calix[8]arene.³⁷⁹ After capping the lower rim of calix[8]arene with an isopropyl group and attaching alkyl amino groups at the upper rim, the authors obtained a series of hydrogelators, the most effective one of which exhibits a CGC of 0.2 wt %. Kim et al. reported that cucurbit[7]uril (CB[7], 103) is able to form a hydrogel in acidic conditions at a concentration of 3 wt %.³⁸⁰ Although the optical appearance of the hydrogel is opaque, the authors demonstrated a unique guest-induced stimulus-responsive behavior of the gel of CB[7] by using 4,4′-diaminostilbene dihydrochoride as a guest. Besides the observation by Kim et al. that CB[7] forms a hydrogel,³⁸⁰ Tan et al. reported a thermoresponsive supramolecular hydrogel consisting of cucurbit[6]uril (CB[6]) and butan-1-aminium 4-methylbenzenesulfonate (BAMB). However, the formation of the hydrogel requires relatively high concentrations of CB[6] (30 mM, 3 wt %) and BAMB (2.5 M).³⁸¹ Kazakova et al. reported an octaamino amide resorcin[4]arene (104)³⁸² acting as a hydrogelator that starts to aggregate at 0.1 wt % and forms a hydrogel at 1.2 wt %. SEM reveals a unique cell-like micrometer size feature when the concentration of 104 reaches 5%. The authors suggested that the fusion of aggregates (micelles) leads to the network for gelation, and found that the walls of the “cell-like” aggregates possess a multilayer structure consisting of 100–400 molecules of 104.³⁸³ Using surfactants (e.g., 10% N,N,N-trimethylhexadecan-1-ammonium bromide and 5% N-(3-((3-(3-((3-(3-(hexadecyldimethylammonio)-2-hydroxypropoxy)-3-oxopropanoyl)oxy)propoxy)-3-oxopropanoyl)oxy)-2-hydroxypropyl)-N,N-dimethylhexadecan-1-ammonium chloride) as the cogelators, Tian et al. reported the photoisomerization of two pseudorotaxanes (consisting of cucurbit[7]uril or being composed of α-cyclodextrin and cucurbit[7]uril) in the hydrogels.^384,385 Recently, the same laboratory and co-workers successfully achieved photoactivated sol–gel conversion using α-cyclodextrin^386,387 without using the surfactants. In addition, they also extended a similar interaction into polymeric hydrogels.³⁸⁸

4.1.9. Hydrogelators Containing a Polyaromatic Core

As shown in Scheme 18, Banerjee et al. introduced l-tyrosine into the perylenebisimide core to generate a hydrogelator (105) that forms stable, semiconducting, photoresponsive, and pH-sensitive hydrogels. The authors found that the CGC value of 105 is about 0.27 wt % at pH 5. TEM indicates that the self-assembly of 105 starts at 8.8 μM (6.3 μg/mL), which is exceptionally low. On the basis of the impressive photoswitching behavior of this hydrogel, the authors suggested that such a high photoresponse value could lead to soft photodetectors.³⁸⁹ Malik et al. reported an interesting case that perylene diimide derivatives with melamine form fluorescent hydrogels.³⁹⁰ Employing the concept of bolaamphiphiles, Banerjee et al. reported another hydrogelator (106) based on perylenebisimide. They found that 106 self-assembles in water at physiological pH and forms a hydrogel at a CGC of about 1.3 wt %. The authors demonstrated that the incorporation of graphene oxide or reduced graphene oxide into the hydrogels enhances the photoresponsiveness of the hydrogel of 106.³⁹¹ Zang et al. also reported that a perylenebisimide derivative (107) forms a hydrogel via pH triggering. The authors found that the addition of hydrochloric acid in the 4.4 mM (about 0.2 wt %) solution of 107 (in the presence of 26.4 mM triethylamine) results in a dark red hydrogel,³⁹² which agrees with the formation of a charge transfer complex.³⁹³

Scheme 18. Some Hydrogelators Containing a Polyaromatic Core.

George et al. reported an amphiphile (108) that consists of coronenebisimide at the core of the molecule and self-assembles in THF/water through aromatic–aromatic interactions. Despite the observation of self-assembled nanotubes by TEM, hydrogelation was not reported.³⁹⁴ However, the authors introduced the coronene motif to a donor–acceptor pair (109/110), which is able to self-assemble to form a hydrogel with a CGC of 0.65 wt %. On the basis of UV–vis, NMR, and XRD, the authors suggested a molecular packing to explain the formation of the nanofibrils from cylindrical micelles.³⁹⁵ Using a naphthalenediimide as the core and ethylene glycol as the side chains, Ghosh et al. developed a nonionic bolaamphiphile (111) that starts to aggregate at 0.05 wt % and forms vesicles. As an electron-deficient core-based bolaamphiphile, 111 forms donor–acceptor (DA) charge-transfer (CT) interactions with pyrene, a water-insoluble electron-rich donor. This interaction ruptures the membrane vesicles to form 1D fibers, thus producing CT-mediated hydrogels with a CGC of 0.3 wt %.³⁹⁶ George et al. reported a two-component hydrogel that consists of an oligo(phenylenevinylene) derivative (112) and a perylenebisimide derivative (113). These two molecules form a strong donor and acceptor interaction in water to result in a hydrogel at a concentration of 0.4 wt %. TEM reveals that aggregation starts at much lower concentration (0.012 wt %). One impressive feature is the critical strain of the hydrogel is over 10%, which is unusual for a supramolecular hydrogel at such a low concentration.³⁹⁷ Zhang et al. developed a bolaamphiphile (114) consisting of a naphthalenediimide as the rigid core and a viologen derivative as the hydrophilic head. The authors demonstrated that the addition of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (115) turns the two-dimensional nanosheets of 114 into ultralong nanofibers. However, the hydrogelation of this interesting two-component system remains to be tested.³⁹⁸

4.1.10. Other Homotypic Hydrogelators

As shown in Scheme 19, the current record of the smallest hydrogelator likely is a squaric acid derivative containing a phenyl group (116) reported by Ohsedo et al.³⁹⁹ According to the authors, the CGC of 116, in 1 M HCl, is about 1 wt %. A slight increase of the pH (from 0 to 1.68) shifts the CGC to 10 wt %. SEM and XRD reveal that 116 forms microcrystals as the matrix of the hydrogel. This microcrystalline morphology explains that it requires 25 wt % 116 to obtain a reliable rheological measurement because the weak interactions between the microcrystals likely only offer a fragile hydrogel that fails to maintain integrity during oscillatory rheometry. The authors also proposed that, at a certain concentration, 116 forms fibrils with hydrophilic porous cavities which have diameters and lengths on the order of micrometers and submillimeters, respectively.³⁹⁹ It would be very interesting to have more insight into the intermolecular interactions within these fibrils. Chi and Xu et al. reported an N-(4-carboxyphenyl)trimellitimide (117) that forms a gel in a mixed solvent of DMF and water with a CGC of 0.4 wt %. Containing microcrystals as its matrix, this hydrogel exhibits a thermoirreversible property and precipitates upon heating, which indicates the hydrogelation likely is an entropy-driven process.⁴⁰⁰

Scheme 19. Some Hydrogelators of Homotypic Hydrogels.

On the basis of the earlier works of bis(amino acid) oxalamides,^401,402 Zinic reported gelators composed of bis(amino alcohol) oxalamides. For example, the authors found that 10 mg of (S,S)-bis(tyrosinol) oxalamide (118) forms a metastable hydrogel in 7.5 mL upon rapid cooling.⁴⁰³ The structure–property relationship study carried out by Feng et al., in fact, suggests that the carbonyl groups in amino acids play a critical role in the formation of hydrogels. Feng et al. developed two C₂-symmetric benzene-based hydrogelators (119 and 120).⁴⁰⁴119 forms a hydrogel at pH 2 with a CGC of 0.25 wt %. With ethylene glycol to cap the C-terminal of 119, the authors obtained a more effective hydrogelator (120) which forms a hydrogel at 0.1 wt %. The authors found unique layered porous structures in the hydrogel of 119 and fibrous structures in the hydrogel of 120. Wang et al. developed a one-pot Ugi reaction from simple starting materials for the synthesis of tripeptoids as hydrogelators, which lead to gelation in a mixed solvent of DMSO and H₂O (1:1) with CGCs of 0.5 wt % (121) and 0.2 wt % (122). This result also reflects that the sufficient unsubstituted amide moiety (−CONH−) is crucial for the formation of supramolecular hydrogels without a cosolvent.⁴⁰⁵ Szymanski and Feringa reported the design of a dichromonyl compound (123) that bears an azobenzene photoswitch and forms a hydrogel in its trans conformation (with a CGC of 1.5 wt %). Compared to other reported photoswitchable hydrogelators,^{406,408−410}123 seems to exhibit much faster kinetics and is able to form a gel within 1 min upon the cis form of 123 being irradiated.⁴⁰⁷

4.1.11. Hydrogelators Composed of Two Components

A hydrogelator composed of two components usually means that the two constituents are nongelators by themselves, but together they can act as a gelator via intermolecular interactions. The two-component hydrogels have certain benefits over one-component small molecule hydrogels because the tunability of the individual components allows more versatile and dynamic reversibility, which may result in greater diversity in morphology and greater variation in mechanical and optical properties. Moreover, the gelation process and the properties of the gels can be easily tuned by changing the components or the compositions of the components or by functional modifications in one of the components, which should be beneficial for the applications of these hydrogels.

As shown in Scheme 20, Tang et al. reported a supramolecular hydrogel consisting of two types of building blocks, 1,2,4,5-benzenetetracarboxylic acid (124) and 4-hydroxypyridine (128), at a concentration of 2.5 wt %. On the basis of XRD of powders and other characterization methods, the authors derived the interactions of the building blocks. One interesting feature is that the hydrogen bonding between the carboxylic acid and pyridine units is strong enough to allow the fibers to be drawn from the melted building blocks.⁴¹¹ The authors used m-hydroxypyridinium (129) to interact with 124 at a molar ratio of 1:2 to form hydrogels. One striking feature of these hydrogels is the rare gel-to-crystal transition, which agrees with microcrystalline particles constituting the networks of the hydrogels and the hydrogels being metastable. The solved crystal structure also provides confirmation that water mediates the hydrogen bonding between the pyromellitic acid and the 4-hydroxypyridine.⁴¹² The authors used 126 to interact with 128, 129, or 130 to form hydrogels with a CGC of about 0.7 wt %.⁴¹³ Using a branched gelator consisting of 1,2,4-benzenetricarboxylic acid (125) and 4-hydroxypyridine (128), the same laboratory shows a two-component hydrogel with a CGC of 4 wt %. By analyzing the single-crystal structures of the complex formed from 128 and o-phthalic acid, and 125, the authors suggested that the molecules assemble into branched fibers via different hydrogen bondings. Interestingly, the melting of the gelators also allows the supramolecular fibers to be pulled to a length of centimeters.⁴¹⁴ When the concentration of the gelator (i.e., the mixture of 125 and 128) is 2.5 wt % and below the CGC, the authors found that the gelator self-assembles in water to form macrospheres with diameters of millimeters.⁴¹⁵

Scheme 20. Some Hydrogelators Composed of Two Components.

Tang et al. developed another type of two-component hydrogelator by mixing 3,3′,4.4′-benzophenonetetracarboxylic acid (127) with 128 or 129 at molar ratios of 1:2 and 1:4, respectively. The authors reported that the self-assembled fibers act as the matrixes of the hydrogels.⁴¹⁶ Using 2-amino-3-hydroxypyridine (131) to interact with 1,2,4,5-benzenetetracarboxylic acid (124), the authors obtained a hydrogel that exhibits a higher T_gel than that of the hydrogel made of 124 and 129, which likely originates from the formation of stronger hydrogen bonding enhanced by the o-amino group of 131.⁴¹⁷ Tang et al. reported the use of ultrasound to promote the mixture of 1,3,5-benzenetricarboxylic acid (126) and 4-hydroxypyridine (128) to gel water at a concentration of 1.5 wt %. The authors observed that the width of the nanofibrils in the hydrogels depends on the power of the ultrasound,⁴¹⁸ and demonstrated that a higher power of the ultrasound results in nanofibrils with a smaller fiber width. On the basis of the works of Tang et al.,^412,419 Yang and Shen et al. employed microfluidics to generate microgels made of 1,2,4,5-benzenetetracarboxylic acid and 4-hydroxypyridine. On the basis of thermal analysis, the authors concluded that, due to the entangled three-dimensional network structures crowded in a small volume, the supramolecular hydrogel microspheres are more thermally stable and can immobilize more water molecules.⁴²⁰

Using a similar approach, Feng et al. reported a hydrogel made of 2,6-pyridinedicarboxylic acid (132) and 4-hydroxypyridine (128) at a concentration of 5 wt %. On the basis of a range of techniques of characterization, the authors suggested that the interaction between 132 and 128 is highly directional.⁴²¹ Nandi et al. reported a two-component hydrogel of melamine (133) and gallic acid (134).⁴²² The authors mixed 133 and 134 in different ratios and demonstrated that hydrogels form at 2 wt %. The optical appearance of the hydrogel suggests the formation of microcrystalline networks or micrometer-sized fibrillar bundles, agreeing with SEM imaging. The authors also observed the enhancement of photoluminescence (PL) at the gel state, which is consistent with the enhanced fluorescence in the colloidal state.^64,423,424 Nandi et al. also used positional isomers of hydroxybenzoic acid (135–137) to interact with melamine (133) in a 1:1 molar ratio to form two-component hydrogels.⁶⁵ The CGC values are 0.5, 1.0, and 0.1 wt % for the hydrogels containing 135, 136, and 137, respectively. On the basis of the upfield shift of the aromatic protons in the gels, the authors suggested that the π–π stacking in the gels follows the order 137 (para) > 135 (ortho) ≈ 136 (meta). The authors found that the thermal stability, the storage moduli, and the critical strain of the two-component hydrogels follow the order 137 > 135 > 136.

Tantishaiyakul et al. reported the thermoreversible gelling systems consisting of melamine (133) and three positional isomers of aminobenzoic acid (138–140) at a concentration of 3 wt %. The authors found that the gel strengths at lower temperatures follow the order 140 > 139 > 138. It is interesting that the gel–sol transition temperatures follow the order 140 > 139 > 138.⁴²⁵ Ballabh et al. also used melamine to interact with maleic acid (141) to form a hydrogel at a CGC of 15 wt %, and applied this two-component hydrogel as a template for making silver nanoparticles.⁴²⁶ Song et al. reported a hydrogel formed by mixing melamine and bis(2-ethylhexyl)phosphoric acid (142)⁴²⁷ at a CGC of 6 wt %. One notable feature of this study is that the authors used a well-established polymer solubility theory (the Fedors method) to estimate Flory–Huggins interaction parameters for predicting the gelation behavior. Later, the same authors also calculated Hansen solubility parameters and Flory–Huggins parameters to estimate the gelator–solvent interaction in a mixed solvent of methanol and water.⁴²⁸ Recently, Wang and Liu et al. reported a series of supramolecular nanotubes formed by combining melamine-based⁴²⁹ and l-glutamic acid-based bolaamphiphiles.⁴³⁰ The authors demonstrated that the ratio of the components dictates the final nanostructures formed by the self-assembly of the constituents. However, how these nanostructures influence hydrogelation remains to be determined.

As shown in Scheme 21, Douliez et al. reported that the ethanolamine salt of 12-hydroxystearic acid (143) forms tubes several tens of micrometers in length with a temperature-tunable diameter at a concentration of 1 wt %. Despite the observation of a gel–sol transition via DSC and SANS measurement, it is unknown whether these salts result in a hydrogel at 1 wt %.⁴³¹ To pair melamine (133) with more complicated molecules, such as lumichrome (144), Nandi et al. produced thermoreversible hydrogels that consist of 144 and 133 at a molar ratio of 3:1 or 1:1.⁶³ The hydrogels, formed at a concentration of 0.2 wt %, are thermoreversible and exhibit higher intensities of photoluminescence than that of pure 144. On the basis of the red shift of the emission peak, the authors suggested a transformation from H-aggregates to J-aggregates.⁶³ Steed et al. reported hydrogelation by a mixed system comprising two entirely rigid, insoluble, mutually complementary small organic molecules, melamine (133) and uric acid (145), which act as a planar multifunctional hydrogen bond donor/acceptor and result in a hydrogel at a CGC of 0.8 wt %.¹⁹⁶ Combining molecular dynamic calculation and the data from ¹³C MAS NMR spectroscopy and the powder XRD pattern of the xerogels, the authors proposed plausible intermolecular interactions to explain the hydrogelation of these two components.

Scheme 21. Some Representative Hydrogelators Composed of Two Components.

Zhang et al. found that the addition of oxoanions (e.g., NO₃^–, PO₄^3–, ATP, and SO₄^2–) to a solution of melamine is able to trigger the formation of hydrogels. SEM reveals that microcrystals act as the matrixes of the hydrogels. The total amount of salts and melamine needed for gelation is from 3.1 to 10 wt %, apparently depending on the anions.⁴³² Nandi et al. reported a hydrogel of melamine (133) containing 6,7-dimethoxy[1H,3H]quinazoline-2,4-dione, riboflavin,⁴³³ and rhodamine B in a proper proportion. The authors suggested that this type of gel might find application in generating white light.⁴³⁴ Hud et al. reported an elegant design that uses cyanuric acid (146) and a modified triaminopyrimidine (147) to form noncovalent interactions that result in a hydrogel at pH 7 and a concentration of 5 mM (0.18 wt %). The authors suggested that the formation of a hexameric rosette can serve as a functional architecture to generate the hydrogels.^435,436 Tang et al. synthesized two isomeric building units, 4-oxo-4-(2-pyridinylamino)butanoic acid (148) and 4-oxo-4-(3-pyridinylamino)butanoic acid (149). While 148 and 149 form fiber- and treelike crystals in aqueous solutions, respectively, cooling the aqueous solutions of their mixtures over a wide range of molar ratios (7:1 to 1:3) yields a series of supramolecular hydrogels at a total concentration of 4 wt %.⁷²

Bhattacharya et al. reported two-component hydrogels consisting of stearic acid (150) or eicosanoic acid with di- or oligomeric amines (151). The authors demonstrated that 150 and 151 at a molar ratio of 2:7, with a total concentration of 5 wt %, result in a hydrogel containing a three-dimensional network formed by the self-assembling nanofibers made of 150 and 151. Since two of these hydrogelator salts are able to crystallize, the authors obtained very useful crystal structures which provide insights into the molecular packing in the condensed phase (Figure 4).⁵⁵ Taking advantage of the interaction of β-cyclodextrin (β-CD, 155) with a ditopic molecule (152) having adamantane at one end and a pyromellitic diimide moiety, Gopidas reported a two-component hydrogel that is stable even with a 10^–6 M concentration each of 155 and 152. Considering that isothermal titration calorimetry (ITC) shows the association constant K between 155 and 152 is about 6 × 10⁴ M^–1, it is unusual (and probably needs confirmation) that the hydrogel forms at such a surprisingly low CGC value (about 2 mg/L).⁴³⁷ Harada used the interaction between 155 and adamantane to cross-link acrylamide to form self-healing hydrogels.⁴³⁸ Hao et al. found that alkyltriphenylphosphonium bromide 153, an ionic liquid-based surfactant, and 155 are able to form a supramolecular complex which further aggregates to result in vesicles in an aqueous solution. The authors found that the addition of inorganic salts (e.g., KCl, NaCl, CuCl, and K₂CO₃) induces the formation of sheetlike hydrogels.⁴³⁹ Using diimidazolium salts with different alkyl chain lengths to interact with α-CD and 155, D’Anna and Noto et al. developed another series of two-component hydrogels, with CGC values from 1.0 to 4.7 wt %, formed by host–guest interactions based on cyclodextrins and cationic imidazoliums 154.⁴⁴⁰ They demonstrated that the nature of the cyclodextrin, the salts, and the host:guest ratio are effective tools for tuning the properties of these two-component hydrogels.

Figure 4.

ORTEP diagrams of 150 and 151 with the atom numbering scheme for the asymmetric unit, and the molecular packing of 150 and 151 showing the columnar supramolecular architectures, characterized by a lipophilic exterior and a polar interior. Adapted with permission from ref (55). Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

4.2. Inorganic–Organic Hybrid Hydrogels

The incorporation of inorganic components into the hydrogels constitutes an irreplaceable way to introduce unique properties of the inorganic components or metal complexes, such as redox, catalytic, conductive, photoresponsive, photochemical, and other properties related to coordination complexes in soft materials. Similar to the development of hydrogels of small organic molecules, the development of metallogels^441,442 largely begins with organogels. Readers who are interested in organogels of metal complexes^443,444 are encouraged to consult several excellent reviews.^25,445 In this section we focus on the hydrogels of metal complexes (Table S2). Although the coordination between organic and metal ions is common in nature, the organic molecules used in these complexes for generating hydrogelators are largely centered on several functional groups that serve as the ligands for metal ions. Therefore, we arrange these relevant hydrogelators according to the following classifications: (i) carboxylic groups as the ligands, (ii) ligands containing nitrogen as electron donors to a metal ion, (iii) binding via phosphate groups, (iv) ligands comprised of thiol groups, and (v) others.

4.2.1. Hydrogelators Containing Carboxylic Groups as the Ligands

Although some amphiphiles with carboxylic groups themselves self-assemble in water to form nanostructures as the matrixes of hydrogels, organic–inorganic hybrid hydrogels still have attracted considerable interest due to the specific functions conferred by the metal ion or inorganic elements. As shown in Scheme 22, Kogiso et al.⁴⁴⁶ reported that a dicarboxylic valylvaline bolaamphiphile (156) self-assembles in water to form nanofibers in the presence of divalent transition-metal cations (e.g., Cu²⁺ and Zn²⁺) at a concentration of 1.6 wt %. The resulting nanofibers have widths of 15–20 nm and lengths of several micrometers. Suzuki et al.⁴⁴⁷ designed molecules 157 and 158 containing a carboxylic group, which form a thermally sensitive hydrogel. The addition of the inorganic salts (e.g., K⁺, Ca²⁺) to the hydrogel affords improved mechanical strength at a CGC concentration of about 1.2 wt %. On the bsais of this result, they also developed an l-lysine derivative (159) and its alkali-metal salts. Compound 159 is insoluble in water, but 159 with an alkali metal is readily water soluble.⁷⁶ All these compounds alone fail to form a hydrogel. Surprisingly, the mixtures of these compounds (e.g., the mixture of 159 and its lithium salt) form hydrogels (at CGC values from 0.5 to 1.7 wt %) via hydrogen-bonding and van der Waals interactions. While 159 forms spherical micelles, the mixtures of the hydrogelators self-assemble to form nanofibers. Shen and Zhang et al.⁴⁴⁸ reported an intriguing example in which the chirality of the ligand controls supramolecular hydrogelation via the coordination of phenylalanine (Phe, 160) to Cu(II) at a CGC of 0.35 wt %. According to the authors, a decrease of the enantiomer excess of ligand l-Phe (or d-Phe) weakens the gelation ability of the Phe–Cu(II) complex. On the basis of this interesting observation, the authors suggested that this hydrogelator may open up a new window for developing promising chiral sensing and recognition platforms.

Scheme 22. Ligands That Bind Metal Ions via Carboxylic Group To Form Supramolecular Hydrogels.

The photoluminescence of lanthanide ions has constantly attracted research interest⁴⁴⁹ because of the long lifetimes, narrow-band emission, and robust photochemical stability. Maitra et al.⁴⁵⁰ demonstrated a lanthanide-based luminescent hydrogel by mixing sodium cholate (85) and europium acetate, at a concentration of about 0.7 wt %, with the addition of pyrene at extremely low concentration (e.g., 10^–6 M) as a sensitizer. In addition, the authors also synthesized nanoparticles (metal sulfides) in a calcium cholate hydrogel using the matrixes of the hydrogel as the template.⁴⁵¹ Huang et al.^452,453 used 85 as the ligand to bind lanthanide to generate a “superhydrogelator”. At 0.04 wt %, 85 forms a hydrogel in which the hierarchical nanostructures are thermoresponsive (e.g., twisted nanohelices and nanotubes at low temperature (4 °C) and untwisted ribbons at a higher temperature (50 °C)). Huang et al. also found that the color of the emission of a lanthanide–cholate hydrogel depends on different lanthanide ions or codoping ions.⁴⁵⁴ Utilizing the iminodiacetic acid (161) as a ligand to coordinate with lanthanides, Alves et al.⁴⁵⁵ prepared a series of hydrogels with the lowest CGC value at 0.04 wt %. Banerjee et al.⁴⁵⁶ designed a series of tyrosine-based amphiphiles (162) which bind with Ni²⁺ ion selectively and result in hydrogels at CGC values of 0.78, 0.75, and 0.63 wt % when the chain length is 10, 12, and 14, respectively. Yang et al.⁴⁵⁷ reported a supramolecular hydrogel based on N,N-dibenzoyl-l-cystine (1) with a CGC of 0.2 wt %. Owing to the interaction between Eu(III) ions and the hydrogelators, immobilized Eu in the hydrogel exhibits enhanced luminescence.^457,458

4.2.2. Hydrogelators Coordinating via Nitrogen(s)

Due to its versatile catalytic properties, palladium is of great interest in synthetic chemistry.^459,460 It has also been explored in the context of supramolecular hydrogels. Escuder et al.⁵⁸ reported a supramolecular hydrogel (4) containing pyridine which forms a hydrogel at 0.5 wt %. Also acting as an organogelator, 4 binds with Pd(II) to create a functional material to catalyze the oxidation of benzyl alcohol, similar to the case reported by Xu et al.⁴⁴¹ As shown in Scheme 23, Takaya and Nakamura et al.⁴⁶¹ designed a supramolecular gel (163) containing Pd(II) for catalysis. The authors made the xerogel of 163 and used it to act as a highly efficient catalyst for the intramolecular addition–cyclization of alkynoic acid in water. Jung et al.⁴⁶² designed a molecule of 1,4-bis(dimethyl-4-pyridylsilyl)benzene (164), which binds with (tmeda)Pd(NO₃)₂ (tmeda = N,N,N′,N′-tetramethylethylenediamine) to afford a hydrogel containing 98.5% water below 2 °C. According to the authors, one interesting feature of these hydrogels is the formation of dynamic catenated cyclotrimers in water. Andrew et al.⁴⁶³ reported that the addition of water to the mixture of 1H-5-(2-pyridyl)tetrazole (165) with LaCl₃ results in a hydrogel. Surprised by this result, the authors also obtained the crystal structure of the complex, which appears to be consistent with the microcrystalline nature of the hydrogel.

Scheme 23. Some of the Ligands That Bind Metal Ions via Nitrogen in the Supramolecular Hydrogels and Some Representative Complexes.

Considering that the understanding of the relationship between molecular structure and gelation ability is still poor and the design of a gelator remains a significant challenge, McNeil et al.^464,465 proposed an approach that selects the molecules according to the intermolecular interaction revealed by the Cambridge Structural Database (CSD) for the development of hydrogels. On the basis of this principle, the authors selected 166, which specifically binds with Hg(OAc)₂ to afford a gel in 90:10 MeOH/H₂O with a CGC value at 1.6 wt %. Mal and Rissanen et al.⁴⁶⁶ also found that the ligand of 40-[4-(4-aminophenyl)phenyl]-2,2′:6′,2″-terpyridine (167) selectively binds with Hg²⁺ to afford a hydrogel. The interaction between benzo-18-crown-6 ether and the ammonium group on 167 is able to disrupt the hydrogelation, which can be partially recovered by the addition of K⁺ to bind competitively with the crown ether. Employing the ligand 5-(mercaptomethyl)tetrazole (168) as a surface coating to stabilize the CdTe nanocrystal, Voitekhovich and Eychmuller et al.⁴⁶⁷ prepared a hybrid gel in the presence of Cd(II) cations. This work illustrated an interesting way to use colloids directly as part of 3D networks for generating hydrogels.

Utilizing N,N′-bis(3-pyridyl)butylenebisurea (169) to coordinate with Cu²⁺, Dastidar et al.⁴⁶⁸ made a gel in a DMSO/H₂O or DMF/H₂O mixture with CGCs of 8–10 wt %. EM reveals that the matrixes of these gels are largely microcrystalline. As shown in Scheme 24, MacGillivray et al.⁴⁶⁹ reported a hydrogel via coordination of copper with rctt-1,2-bis(3-pyridyl)-3,4-bis(4-pyridyl)cyclobutane (170). Being composed of nanoscale metal–organic particles, this hydrogel exhibits thixotropic properties with a yield value of 8.33 Pa. It is worth noting that Tu et al.^470,471 also prepared a thixotropic hydrogel via simple mixing of 171 and Cu(II) at a concentration of 0.25 wt %. Furthermore, they also developed a photoswitchable metallohydrogel by utilizing the photoresponsive 2,2′-azopyridine as the ligand.⁴⁷² In addition, Bhattacharya et al. also prepared a thixotropic hydrogel based on the interaction of pyridylenevinylene and copper(II).⁴⁷³

Scheme 24. Some of the Ligands That Bind Metal Ions via Nitrogen in the Supramolecular Hydrogels and Some Representative Complexes.

Being designed and reported by Che et al.,⁴⁷⁴ a terpyridylplatinum(II) complex with biphenylacetylide ligands (172) self-assembles to form nanofibers with diameters of 20–40 nm and results in a hydrogel with a CGC of 3.7 wt %. Yu et al.⁴⁷⁵ designed a pair of decapeptides, formyl-EFEAEAEAEW-carboxyl and formyl-OFOAOAOAOW-amide (O = ornithine), which are able to form a hydrogel at pH 7. Moreover, the peptide conjugated with a Gd(III) chelate (173) speeds the gelation process and increases the cross-sectional area of the peptide fibers. Further study found that the conjugates 173 are incorporated into the peptide fibers and aggregate toward the center of the peptide fibers. One notable feature of this study is that the authors correlated SAXS and T2-weight magnetic resonance imaging (MRI) to provide insights into the self-assembly process. Zhang and Ye et al.⁴⁷⁶ prepared a metallogel in water by using 4,6-bis(2-pyridyl)-1,3,5-triazin-2-ol (174) to coordinate with copper acetate at a molar ratio of 4 (Cu:L, 5.6 wt %). It is interesting that the hydrogel is stable at room and high temperature (e.g., 60 °C), but flows upon cooling to 4 °C. This kind of LCST behavior is uncommon for supramolecular metallohydrogels.

Upon mixing melamine (133) with zinc(II) orotate at a 1:1 molar ratio, Nandi et al.⁴⁷⁷ developed a bicomponent hydrogel at a CGC of 0.7 wt %. The authors demonstrated that this hydrogel is thixotropic and undergoes a gel–sol transition when being treated with formic acid or sodium borohydride. Employing a dendritic ligand (175) to coordinate diplatinum(II), Yang et al.⁴⁷⁸ developed a gel in a mixed solvent of acetone and water (5:3, v/v) with a CGC of 0.23 wt %. Moreover, the addition of bromide ions causes the gel–sol transition, which can be reversed by the addition of silver. Upon addition of α-cyclodextrin to the solution of the Pd(II) complex with a bipyridinium ligand (176), Osakada et al.⁴⁷⁹ prepared a hydrogel with a T_gel of 47 °C. The authors suggested that the complex self-assembles to form polyrotaxane-like fibrous architectures and further aggregates to afford nanoparticles with a size around 20–40 nm to trap the water and to result in the hydrogel. Fernandez et al.⁴⁸⁰ designed and synthesized a ligand (177) consisting of a pyridyl group, an oligo(phenyleneethynylene) motif, and three ethylene glycol chains. 177 coordinates with Pt(II) metal ions to result in a hydrogel. On the basis of ROESY (rotating-frame Overhauser effect spectroscopy) NMR, the authors elucidated that π–π interactions and unconventional C–H···X hydrogen bonding drive the complex to form a hydrogel at a concentration of 1.4 wt %. The most impressive result in this work is correlation of UV/vis spectroscopy, NMR spectroscopy, and X-ray crystal structure analysis to infer the molecular arrangement in the hydrogel. Diaz and Banerjee et al.⁴⁸¹ reported a complex of l-3-methyl-2-[(pyridin-4-ylmethyl)amino]butanoic acid (178) and Zn(II) to form a metallohydrogel with a CGC value of 8.4 wt % (Scheme 25). According to the authors, this hydrogel undergoes a gel–sol transition upon application of different stimuli (e.g., EDTA, ammonia solution, trifluoroacetic acid, sodium sulfide, shaking, or heating), which likely originates from the unique amphoteric property of zinc ions.

Scheme 25. Some of the Ligands That Bind Metal Ions via Nitrogen in the Supramolecular Hydrogels and Some Representative Complexes.

4.2.3. Hydrogelators Containing Thiol Groups as the Ligands

Ballabh et al.⁴⁸² designed 2-aminothiazole and its derivatives (179) that bind with mercury acetate to result in hydrogels with thixotropic behavior. One interesting feature of this work is the dependency of the thixotropic property on the substitution groups in the C-4 and C-5 positions of the aminothiazole. Jiang et al.⁴⁸³ have demonstrated an intriguing case of a highly selective anion-responsive supramolecular hydrogel, based on silver(I) glutathione (GSH) (180), with a CGC value of 0.20 wt %. Compared with other anions (F^–, Cl^–, Br^–, and H₂PO₄^–), I^– is capable of triggering the gel–sol transition, likely due to the higher affinity of iodide to silver ion than that of other halogen anions. Odriozola et al.⁴⁸⁴ used a drug, N-acetyl-l-cysteine (NAC, 181),⁴⁸⁵ to generate the supramolecular hydrogel (0.82 wt %, 50 mM) in the presence of Au(III), Ag(I), and Cu(II) salts. Although the hydrogelation of these metal complexes occurs at a relatively low pH (<4), this work still provides new insights into creating supramolecular metallogels. Pakhomov et al.⁴⁸⁶ reported a hybrid hydrogel based on the complex (182) of l-cysteine and silver nitrate (Scheme 26). Via sulfur–silver bonds, complex 182 self-assembles to form nanofibers and results in hydrogelation at a rather low concentration (0.01 wt %). Further studies found that the hydrogel exhibits antibacterial properties.⁴⁸⁷ Odriozola et al. designed two N-terminal-capped tripeptides, Ac-RGC and Ac-RCG, which have an isoelectric point close to 7. Because these two peptides contain the cysteine residue, they form hydrogels upon the introduction of Au(I) to Ac-RGC and Ag(I) to Ac-RCG, at final peptide concentrations of about ∼3 wt %.⁴⁸⁸ Arachchige et al.⁴⁸⁹ reported a highly efficient method to prepare a monolithic Ag hydrogel by using a large amount of oxidant C(NO₂)₄ to remove the surface thiolate in Ag nanoshells. Moreover, the resulting aerogel exhibits extremely high surface areas (40–160 m²/g) and interconnected mesoporous structures. Pal et al.^490,491 reported a Cu(I) metallogel at a concentration of 0.15 wt % via simple mixing of CuCl₂ and thiourea (183). The coordination of Cu(I)–thiourea and extensive hydrogen-bonding interactions result in a hydrogel which is redox responsive, and the oxidation of Cu(I) to Cu(II) by FeCl₃ leads to a gel–sol transition. Moreover, the metallogel shows high selectivity for picric acid, and the addition of picric acid causes a color change of the gel from white to yellow.

Scheme 26. Some of the Ligands Bearing Thiol Groups.

4.2.4. Hydrogelators Utilizing Phosphates as the Ligands

The phosphate group also has strong affinity with many kinds of metal ions.⁴⁴ Yeh et al.⁴⁹² developed a facile method to prepare hydrogel-like GdPO₄·H₂O nanorods. The authors reported that the nanorods are cell compatible. On the basis of the high affinity between phosphate groups and certain metal ions, Takahara et al.⁴⁹³ reported an imogolite-based hydrogel which is able to immobilize an enzyme containing phosphoric groups. The authors found the enzyme is easily recovered from the reaction system and reused. Patil and Mann et al.⁴⁹⁴ reported that cerium oxide nanoparticles act as a catalyst to dephosphorylate Fmoc-Tyr-P (14), thus resulting in hybrid supramolecular hydrogels. The most interesting part of this study is the observation of Maltese cross-patterns (spherulites) or nematic birefringence of the hydrogels, which suggests directional orders. As shown in Scheme 27, Kasuga and co-workers^495−499 have prepared a metaphosphate hydrogel (184) via hydration of the metaphosphate glass powders. The authors have also performed extensive studies on the conductive properties of this viscous gel. The resulting hydrogel exhibits a low self-discharge rate and fast charge–discharge capability, and shows a high electrical conductivity of 5 mS cm^–1 at room temperature because of proton hopping between P–OH groups and water molecules. The authors suggested that this hydrogel promises an alternative electrolyte.

Scheme 27. Chemical Structure of a Ligand Containing Phosphate Groups.

4.2.5. Others

Besides the electron donors discussed above, other types of nucleophiles may coordinate with metal centers to generate hydrogelators. As shown in Scheme 28, Kulkarni et al.⁵⁰⁰ reported that copper(II) binds with inositol and 2,2′-bipyridine to form a trinuclear complex (185). The self-assembly of the complex affords a supramolecular hydrogel at pH 12.4 via inter- and intramolecular π–π stacking between the bipyridyl groups. In addition, Rodriguez et al.⁵⁰¹ designed a discrete molecule [(PTA)Au(4-pyridylethynyl)] (PTA = 1,3,5-triaza-7-phosphaadamantane) (186); this complex forms a hydrogel immediately in water even at a concentration as low as 0.015 wt %. Che et al.⁵⁰² designed a dinuclear platinum(II) complex (187) having a [Pt(6-phenyl-2,2′-bipyridyl)((2,6-dimethylphenyl)isocyanide)] motif connected by an oligo(oxyethylene) chain which is able to form hydrogels driven by intra- and intermolecular Pt···Pt and π–π interactions. Moreover, 187 acts as a supramolecular cross-linker to trigger the gelation of [Pt(6-phenyl-2,2′-bipyridyl)((2,6-dimethylphenyl)isocyanide)] at a molar ratio of 1:30. Wu et al.⁵⁰³ designed a hydrogelator (188) based on hydrazide. 188 is able to afford a hydrogel with a CGC of 0.6 wt %. Upon the addition of Tb³⁺, the resulting hydrogel exhibits enhanced green luminescence due to the coordination between the gelator and the metal ion. Lima and Rodriguez et al.⁵⁰⁴ developed a hydrogelator of [Au{7-(prop-2-yn-1-yloxy)-1-benzopyran-2-one}(DAPTA)] (DAPTA = 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) (189) which self-assembles to form long luminescent fibers and induces a hydrogel with a CGC of 0.01 wt %. Other cases in this category include the polyoxometalate (POM) reported by Bonchio et al.⁵⁰⁵ as well as others,⁵⁰⁶ which forms a gel at a specific condition (e.g., 90:10 MeOH/H₂O).

Scheme 28. Metal Complexes As Hydrogelators.

4.3. Hydrogels Based on Amino Acids and Peptides

Besides the small organic and inorganic hydrogelators, the hydrogelators derived from amino acids or peptides are of great importance due to their inherent biocompatibility and biological activities. The utilization of amino acids as the molecular building blocks of hydrogelators provides unique opportunities for generating supramolecular hydrogels that are not readily available from traditional organic/inorganic molecules. Peptide-based hydrogelators self-assemble via various noncovalent interactions, including hydrogen bonding, electrostatic interactions, aromatic–aromatic interactions, or hydrophobic interactions.⁵⁰⁷ These intermolecular interactions lead to the formation of organized supramolecular structures that entrap water under appropriate stimuli. In addition, due to the well-established protocol of solid-phase peptide synthesis (SPPS),⁵⁰⁸ it is easy to produce and to chemically and biologically modify peptides in large quantities and at a relatively fast turnaround. These merits have attracted considerable research activities focusing on the exploration of supramolecular hydrogels made of peptides. In this section we give a summary of different types of peptide-based hydrogelators in the past decade. We hope that the introduction of the recent progress will contribute to the understanding of the relationship between the peptide structures, the resulting conformation of the hydrogelators, and the morphologies of the matrixes of the supramolecular hydrogels. By presenting an updated report on peptidic hydrogelators, we intend to provide molecular information for researchers to uncover the hypotheses for the rational design of supramolecular hydrogelators.

We start with a brief description of the supramolecular hydrogels formed by amino acid derivatives. Strictly speaking, amino acid derivatives are not exactly “peptides”, because the shortest peptides are dipeptides. However, because the building blocks of peptides are amino acids, amino acid-based hydrogelators share considerable similarities with peptide-based hydrogelators, making them relevant to be discussed in this section. We categorize the amino acid derivatives into three groups (i.e., those containing an alkyl chain, those that are salt-based, and those containing an aryl group (i.e., ferrocenyl, fluorenyl, naphthyl, pyrenyl, etc.)) and discuss several representative molecules in each group. Then we introduce the hydrogels formed by native peptides (consisting of amino acids only and without any chemical modification). We summarize most of the peptidic hydrogelators according to their molecular size (Table 1). In such an arrangement, we give an overall review of the design strategies of peptide-based hydrogelators and highlight the notable advances made recently. After identifying key features of the native peptides, we discuss the chemically/biologically modified peptides (i.e., peptide derivatives) that are the representative hydrogelators from six subgroups. The six subgroups are the peptides with terminal acetylation (or formylation) and amidation, peptide derivatives containing an alkyl chain, peptide derivatives containing an aromatic group (e.g., fluorene-based, naphthalene-based, and pyrene-based peptide derivatives), peptide derivatives containing a photosensitive group, bolaamphiphiles, and dendritic peptides.

Table 1. Peptide Sequences and Their Reported Hydrogelation Concentrations.

peptide length (no. of amino acids)	peptide sequence	reported hydrogelation concn (wt %)	refs
2	FF (208)	0.1–10.0	(555), (565−570)
2	IF (209)	1.0–2.0	(564)
2	VF (210)	1.0–2.0	(564)
2	cyclic YK (211)	0.6–1.5	(84)
3	cyclic tripeptide (212)	0.5	(577)
3	cyclic tri-β-peptide (213)	0.006	(578)
3	DVFF (214)	0.67	(89, 579)
3	DFFV (215)	0.67	(89, 579)
3	DLFF (216)	0.67	(89)
3	LFF (217)		(89)
3	FFY (218)	0.1	(580)
4	FEFK (220)	10	(163, 582)
4	PWWP (221)	0.25	(583)
4	RWDW (222)		(584)
5	KLVFF (223)	2.5	(187)
7	(2-Thi)(2-Thi)VLKAA (224)	2.0	(585)
8	FKFEFKFE (225)	0.1	(588)
8	AEAEAKAK (226)	0.8	(591, 635)
	AEAKAEAK (227)
	FEFEFKFK (228)
	FEFKFEFK (229)
8	VKVKVEVK (230)	10.0	(592)
9	FEFEFKFKK (231)	2.0	(594)
10	GPGGDGPGGD (232)	1.0	(595)
11	QQRFEWEFEQQ (233)	0.016	(597)
	Ac-QQRFEWEFEQQ (234)
12	RRRRGSWWWWSG (235)		(599)
	cyclic RRRRGSWWWWSG (236)
13	PELELELELELEP (237)	3.6	(600)
13	PEFEFEFEFEFEP (238)	4.0	(600)
14	EIAQLEYEISQLEQ (239)	1.0	(601)
	KIAQLKYKISQLKQ (240)
16	KKQLQLQLQLQLQLKK (241)	1.0	(602)
16	KKSLSLSLSLSLSLKK (242)	2.0	(603)
	EESLSLSLSLSLSLSLEE (243)
	KKQLQLQLQLQLQLKK (244)
16	LELELELELELELELE (245)	0.67	(91)
	VEVSVKVSVEVSVKVS (246)
16	AAKAAAKAAAKAAAKA (247)	0.5	(604, 605)
16	RADARADARADARADA (248a)		(181,606)
	RADARADARADARADAKKKK (248b)
	RADARADARADARADASSSSS (248c)
16	FEFEFKFKFEFEFKFK (249)	0.007	(607)
20	VKVKVKVKVDPPTKVKVKV (250)	0.5–2.0	(134, 200), (610−625), (627), (636−638)
	Max1: VKVKVKVKVDPPTKVKVKVKV-NH2 (251)
	Max2: VKVKVKVKVDPPTKVKTKVKV-NH2 (252)
	Max4: KVKVKVKVKDPPSVKVKVKVK-NH2 (253)
	Max5: VKVKVKVKVDPPSKVKVKVKV-NH2 (254)
	Max6: VKVKVKVKVDPPTKVKEKVKV-NH2 (255)
	Max7: VKVKVKVKVDPPTKVKCKVKV-NH2 (256)
	Max8: VKVKVKVKVDPPTKVEVKVKV-NH2 (257)
	MLD: KVKVXVKVKVDPPTKVKVXVKVK (258)
	SSP1: VKVKVDPPTKVKVKVKVKVKV-NH2 (259)
	SSP2: VKVKVKVDPPTKVKVKVKVKV-NH2 (260)
	SPP3: VKVKVKVKVDPPTKVKVKVKV-NH2 (261)
	VKVKVKVKVPPTKVKVKVKV (262)
	VKVKVKVKVPPTKVKVKV (263)
20	VKVKVKVCGPKECVKVKVKV (264)	2.0	(639)
28	KIAALKQKIASLKQEIDALEYENDALEQ (265)		(633)
	KIRRLKQKNARLKQEIAALEYEIAALEQ (266)
30	CKQLEDKIEELLSKAACKQLEDKIEELLSK (267)		(634)

4.3.1. Amino Acid Derivatives

4.3.1.1. Amino Acid Derivatives Containing Alkyl Chain(s)

Most amino acid derivatives containing alkyl chains are conventional amphiphiles, consisting of a polar head group and one or two hydrophobic tails.⁵⁰⁹ The amino acids make the polar head, while the alkyl chains are the hydrophobic tails with many variations in the length and flexibility. These molecules are quite common in nature and are well-known to form a variety of nanostructures in water.^510−513 For example, Bhattacharya et al. reported the gelation of enantiomerically pure N-alkanoyl-l-alanine amphiphiles in a series of organic solvents.⁵¹⁴ Recently, Dey et al. designed and developed a series of amino acid-based gelators, N-(n-alkylcarbamoyl)-l-alanine (e.g., 190) (Scheme 29), all of which form stable organogels in solvents in the presence of water at a concentration of 1% (w/v). The authors concluded that water-mediated intermolecular hydrogen bonds between amphiphiles result in the formation of supramolecular self-assemblies. The urea linkage of this hydrogelator, as the simplest motif that provides two hydrogen bond donors and one acceptor, is a common and useful hydrogen-bonding building block for supramolecular hydrogels.⁵¹⁵ Similarly, N^α-[4-(n-alkyloxy)benzoyl]-l-histidine (191) with a hydrocarbon chain, reported by the same group, affords thermoreversible hydrogels in a wide range of pH at room temperature. They also reported that the CGC value of 191 at different pH values varies from 2.5% to 5.0% (w/v).⁵¹⁶ Liu et al. reported a multiple-H-bonding amphiphile, N-stearoyl-l-glutaminc acid (C₁₈-Glu, 192), which forms disk- and fiberlike nanostructures in both hydrophilic and hydrophobic environments, respectively, due to intra- and intermolecular hydrogen bonds.⁵¹⁷ Maruyama et al. reported that the attachment of a carbohydrate motif to the N-terminal of the alkyl chain–amino acid structure affords another hydrogelator, 193, which is able to harden a broad range of solvents, in addition to gel water over a wide pH range at gelation concentrations of 0.1–2 wt %.⁵¹⁸ Using a similar synthetic strategy, Cai et al. produced zwitterionic supramolecular gel lubrucants.⁵¹⁹ Interestingly, l-glutamic acid, through both α- and ω-aminoalkylation, affords a double-long-chain-terminated hydrogelator, 194. This amphiphile disperses in various solvents ranging from water to hexane, and forms nanofibrils or aggregates. It also has a relatively good solubility, which results in a CGC value higher than 20 mM.⁵²⁰ Dey et al. designed a zwitterionic hydrogelator of sodium N-(n-dodecyl-2-aminoethanoyl)-l-valinate (195) that is able to afford a supramolecular hydrogel with a CGC of 0.05 wt % in the presence of sodium dodecyl sulfate (SDS). In addition, the resulting hydrogel is thermoreversible and sensitive to a change of pH.⁵²¹

Scheme 29. Representative Molecular Structures of Amino Acid Derivative Hydrogelators Containing an Alkyl Chain.

4.3.1.2. Ionic Amino Acid Derivatives

While they augment the self-assembly ability of hydrogelators, long alkyl chains or aromatic rings are the hydrophobic segments that decrease the solubility of the hydrogelators in water. One of the simplest strategies to achieve relatively better solubility required for forming a hydrogel is to introduce charge(s) into the hydrogelators.^522−524 Suzuki et al. reported the synthesis and properties of l-lysine alkali-metal salt 159. In water, 159 forms a supramolecular hydrogel, within which the self-assembled nanofibers entangle to result in a 3D network.²³⁰158 is another l-lysine-based hydrogelator, with a pyridinium bromide salt, developed by Suzuki and Liu et al., independently.^525−527 It forms a hydrogel at a wide range of pH and has a CGC as low as 0.3 wt % at neutral pH. Yang et al. developed two chiral l-phenylalanine-based salts (196^528,529 and 197(530)) as hydrogelators. As shown in Scheme 30, 196 has an ammonium salt, and 197 has a pyridinium salt. They both self-assemble in aqueous media at different pH values to form supramolecular hydrogels with CGC values of 1.2–2.0 wt % (for 196) and <1.0 wt % (for 197).

Scheme 30. Representative Hydrogelators of Ionic Amino Acid Derivatives.

4.3.1.3. Amino Acid Derivatives Containing Aromatic Group(s)

Although ionic force and the overlap between alkyl chains offer interfiber interactions, they are intrinsically weak and inefficient. Aromatic–aromatic interaction, extensively used by nature as a stabilizing force for generating ordered structures in proteins,¹³ not only exceeds the Van der Waals interaction between the alkyl chains, but also leads to more predictable and efficient self-assembly of the molecules in the aqueous phase for the formation of mechanically strong and stable supramolecular hydrogels due to its compact volume. Thus, aryl motifs (e.g., ferrocenyl, fluorenyl, naphthyl, and pyrenyl)^531−533 and phenylalanine (or its derivatives) become prevalent and effective residues used in generating supramolecular hydrogels.

As shown in Scheme 31, Zhang et al. serendipitously discovered that ferrocenoylphenylalanine (198) aggregates in water via a rapid self-assembly to form a stable hydrogel which exhibits a sharp phase transition in response to multiple stimuli (i.e., oxidation–reduction reaction, guest–host interaction, and pH changes). Though being exceptionally simple and small, this hydrogelator is electrochemically active, which illustrates a remarkably facile approach to introduce an organometallic moiety into a supramolecular hydrogel.¹¹⁴ Inspired by this work, He et al. also investigated the self-assembly properties of ferrocenoyldiphenylalanine.⁵³⁴ Since the serendipitous observation of an effective hydrogelator made of [(fluorenylmethyl)oxy]carbonyl (Fmoc)-protected dialanine,¹¹ Fmoc, widely used as the protecting group in SPPS, has become a popular N-terminal capping motif in peptidic hydrogelators because the Fmoc-protected amino acids or peptides are commercially available or relatively easy to make. The intensive use of Fmoc in making supramolecular hydrogelators also originates from its advantages, such as ease of being incorporated into peptide/amino acid derivatives (i.e., derived directly from SPPS), low cost, and excellent capacity to promote self-assembly. Changing the pH is a common way to make supramolecular hydrogels from Fmoc-protected amino acids. For example, Nilsson et al. reported supramolecular hydrogels generated by fluorinated Fmoc-phenylalanine derivatives.^535−537 Suspensions of Fmoc-pentafluorophenylalanine (199) in water undergo rapid self-assembly, which gives rise to rigid supramolecular hydrogels even at concentrations as low as 0.1 wt %. To better understand the electronic and steric contributions of the benzyl side chain to the hydrophobic and aromatic–aromatic interactions during self-assembly, the authors also changed the halogen substituents (i.e., F, Cl, Br) or the position of substitution (e.g., ortho, meta, or para) on the aromatic side chain of Fmoc-Phe (200). The authors reported that the position of the halogen and the size of the halogen atom significantly affect the rate of self-assembly and the bulk rheological properties of the resulting hydrogels. For example, the rate of self-assembly increases in the order ortho < meta < para, and the hydrogel rigidity increases in the order Br < Cl < F.^536,537

Scheme 31. Representative Molecular Structures of Hydrogelators Containing Aromatic Groups.

Xu et al. also reported the first case of the use of Fmoc-amino acid derivatives to generate multicomponent supramolecular hydrogels.^153,538,539 Equal moles of 14 and 201 and 2 equiv of Na₂CO₃ afford a clear hydrogel after heating and addition of alkaline phosphatase.¹⁵³ Similarly, mixing and then heating 1 equiv of 200, 1 equiv of 201, and 2 equiv of Na₂CO₃ in water eventually leads to the formation of a clear solution, which turns into a hydrogel after being cooled back to room temperature. The subsequent hydrogel exhibits exceptionally high storage moduli and a rapid recovery of its original mechanical strength after removal of the external forces.^538,539 In another study, Xu et al. prepared a supramolecular hydrogel by mixing 200 and Fmoc-Lys (202). The resulting hydrogel acts as a reaction medium to mimick a bioluminescence environment with a more than 10-fold enhanced quantum yield of chemiluminescence.⁵⁴⁰ Banerjee et al. also reported that supramolecular hydrogels were formed by coassembling two oppositely charged Fmoc-amino acids (i.e., 202 and Fmoc-Glu, 203) with a CGC value of 50 mM (1.8 wt %).⁵⁴¹ The authors reported that molecular chirality is translated into the supramolecular helicity and the handedness of these fibers because the latter depends on the corresponding molecular chirality of the two-component system.

Fluorescent spectroscopy is a useful tool to study the interactions among the fluorophore-bearing hydrogelators in the gel state. Besides providing strong and efficient aromatic–aromatic interaction, pyrene is a suitable motif for fluorescent spectroscopy as its emission changes due to aromatic–aromatic interaction (e.g., the formation of an excimer⁵⁴²). Thus, pyrene has become a popular moiety to be incorporated into supramolecular hydrogelators.^18,543 Banerjee et al. designed and developed a hydrogelator (204) consisting of a single amino acid and a pyrene group. 204 forms hydrogels in a wide range of pH (7.5–14) in the aqueous phase. With an increase of the pH, a distinct morphological change of the nanofibers of the hydrogel occurs, and the morphology transforms from helical to tapelike.¹⁰⁴ Although the aromatic–aromatic interaction between two phenyl groups is weaker than that between two fluorenyl or two pyrenyl groups, phenylalanine is often incorporated into peptides for increasing their ability to self-assemble. Kim et al. reported a biotin-based small molecule (biotin-l-Phe, 205) that displays remarkable gelation properties in aqueous media, including buffer solutions with different pH values.⁵⁴⁶ Although biotin-based organogelators have been reported,⁵⁴⁴ the study of supramolecular hydrogelators of biotin is rare.

Bile acids are among the most investigated rigid molecular motifs due to their ability to self-organize into many different supramolecular structures. Galantini et al. reported the synthesis and self-assembly behavior of cholic acid-connected amino acid derivatives⁵⁴⁵ (e.g., 206). The molecules of 206 aggregate in globular micelles at high pH, whereas they form tubular superstructures under acidic conditions and result in hydrogels at a concentration of 18 mM (1.2 wt %). These two representative cases imply that the conjugation of l-phenylalanine to natural products (i.e., biotin (205)⁵⁴⁶ and cholic acid (206)⁵⁴⁷) likely will result in a rich class of supramolecular hydrogelators.

4.3.1.4. Amino Acid-Based Bolaamphiphiles

Bolaamphiphiles are a class of hydrogelators composed of two terminal hydrophilic groups linked by a hydrophobic backbone/chain. Variation of the structures of the “head group” and the linker of bolaamphiphiles has great influence on their ability to aggregate and the properties of the hydrogels. On the basis of l-valine- and l-proline-based peptide bolaamphiphiles, a class of efficient organogelators, Miravet et al. designed and developed one bolaform amino acid derivative (4) which self-assembles in water to form hydrogels.^59,549−551 The CGCs for these small molecules range from 4 to 26 mM (from 0.18 to 1.2 wt %).^59,548−551 As shown in Scheme 32, Liu et al. reported another series of bolaamphiphiles with head groups made of l-glutamic acid and a hybrid linker composed of two rigid benzene rings and a butyl segment (207). They controlled the hierarchical self-assemblies via changing the solution pH. For example, at pH 3, these molecules form a hydrogel in water at a concentration as low as 0.5 wt % (CGC); at pH 12, these hydrogelators then form vesicle-like aggregates.⁵⁵²

Scheme 32. Representative Molecular Structure of a Hydrogelator Based on a Bolaamphiphile.

4.3.2. Peptides

As a consequence of evolution, proteins (e.g., insulin¹⁹) form hydrogels, implying that peptides, as the component of proteins, should be able to form supramolecular hydrogels. This notion turns out to be valid. For example, Namba et al. have demonstrated that the terminal regions of flagellin (consisting of 25 amino acids) form hydrogels at concentrations >15 mg/mL.²¹ Surprisingly, much smaller peptides,^553,554 such as dipeptides, are able to self-assemble in water to generate supramolecular hydrogels. Besides the easy synthesis of peptides, several seminal works on peptide-based hydrogels^{18,188,555−557} at the beginning of the century have stimulated the development of supramolecular hydrogels based on small peptides. In this section on supramolecular hydrogels formed by peptides, we put the emphasis on small peptides (20 or less amino acid residues) because there are already several excellent reviews on medium-sized oligopeptides and their derivatives.^558−563 We arrange the discussion of peptide-based hydrogelators according to the length of the peptides and start with the shortest one.

The report by Gazit et al. on the nanofibers formed by the self-assembly of diphenylalanine (FF, 208)^{555,565−568} has stimulated intensive investigations of the self-assembly of small peptides, including dipeptides.^569−572 Ventura et al. examined the dipeptides Ile-Phe (IF, 209) and Val-Phe (VF, 210) and found that 209, at 1.5 wt %, forms a hydrogel consisting of persistent nanofibers with a diameter of about 55 nm. The nanofibers appear to be crystalline and melt upon heating. Unlike 209, 210 cannot self-assemble. Besides the fact that 209 is probably the smallest hydrogelator, it is worth noting that the difference of one methyl group between 210 and 209 has a profound impact on their capabilities to self-assemble.⁵⁶⁴ The authors also proposed the self-assembled structure of 209 (Figure 5), which is quite similar to the recent structure of Aβ_1–40 determined by solid-state NMR.²¹⁴ Since the report of Gazit et al. on diphenylalanine, several other groups have explored the properties of 208 in great detail. Park et al. have demonstrated that 208 self-assembles to form nanowires and nanotubes with high thermal, chemical, and proteolytic stability. An XRD study of these nanowires and nanotubes reveals their crystallinity.⁵⁶⁹ Qi et al. reasoned the use of hexafluoroisopropyl alcohol (HFIP) to dissolve 208 would introduce a solvent effect, so they used CH₃CN/H₂O as the solvent to process 208. They found that cooling the solution of 208 results in microtubes and the evaporation of the solution of 208 on a glass surface affords nanofibers. The higher content of CH₃CN leads to bigger diameters of the nanofibers.⁵⁷⁰ Krishnan et al. also investigated the effect of HFIP and found that the amount of HFIP significantly affects the morphologies of the nanotubes formed by the self-assembly of 208.⁵⁷¹ Considering the morphology of nanoscale assemblies dictates their interactions with proteins,^573,884 this work, indeed, provided a useful insight for understanding the discrepancy⁵⁷⁴ in the reports on the cytotoxicities of Aβ because HFIP is the most common solvent used to dissolve commercially available Aβ.

Figure 5.

Molecular model of 209 self-assembled structures. The model is based on the crystal X-ray structure of the diphenylalanine peptide. The dipeptide backbone and hydrophobic side chains are shown as stick representations. Adapted with permission from ref (564). Copyright 2007 Biophysical Society.

As shown in Scheme 33, Feng et al. reported that an innovative cyclodipeptide (211), made of tyrosine and lysine, acts as a hydrogelator and forms a hydrogel at a CGC of 0.6 wt %.⁸⁴ The hydrogel, having a relatively low mechanical strength, only forms during cooling with the assistance of ultrasound for breaking up the intermolecular interactions. Interestingly, after the attachment of an alkyl chain at the ε-amino group of 211, the CGC in water becomes 1.5 wt %. Due to the difficulty of synthesis, reports on the self-assembly of cyclic peptides are rather scarce.^575,576 Zhao and Dory et al. reported a cyclic tripeptide (212) forming micrometer-sized tubes by self-assembly in a liquid crystal. On the basis of molecular dynamics, the authors suggested a hierarchical hexagonal assembly to generate the observed hollow macrotubes of 212.⁵⁷⁷ In a similar study, Kimura et al. reported the self-assembly of a unique tri-β-peptide (213) that consists of two β-glucosamino acids and one trans-2-aminocyclohexanecarboxylic acid. 213 self-assembles in a mixture of formic acid and water to form nanofibers at a concentration of about 0.006 wt %.⁵⁷⁸ It would be interesting to obtain more information about the biological properties of 213.

Scheme 33. Representative Molecular Structures of Cyclopeptide Hydrogelators.

Marchesan et al. systematically investigated tripeptides containing one d-amino acid residue at the N-terminal and found that chirality plays a critical role in the morphology of the nanofiber networks of the resulting hydrogels. The authors observed a very interesting and unexpected result by modulating the chirality of the tripeptides: while the tripeptides VFF and FFV fail to self-assemble at physiological pH, ^DVFF (214) and ^DFFV (215) are able to self-assemble at a concentration of about 0.67 wt %. Both 214 and 215 self-assemble to form distinct nanostructures: the former results in nanotapes and the latter twisted nanofibers.⁵⁷⁹ The authors also observed similar behavior for ^DLFF (216), which forms a hydrogel at 0.67 wt %. While the self-assembly of 216 results in mainly nanofiber networks, the self-assembly of LFF (217) is rather polymorphic and fails to form a self-supporting hydrogel.⁸⁹ Cao et al. examined the self-assembly of FFY (218) and Fmoc-FFY (219) for photo-cross-linking after the self-assembly. They found 218 self-assembles to form nanoparticles which turn into nanorods after photo-cross-linking tyrosine residues. 219 forms a weak hydrogel at 0.1 wt % which consists of nanofibers that turn into nanoparticles after photo-cross-linking. This result provides a useful insight into the effect of cross-linking to the self-assembled nanostructures.⁵⁸⁰ Moreover, Ulijn and Tuttle et al. have presented a coarse-grain molecular dynamics (MD) protocol for screening tripeptides for their aggregation behavior and applied this to a set of 8000 gene-encoded tripeptides.⁵⁸¹ It would be highly valuable to verify if these self-assembly tripeptides exist endogenously and at concentrations high enough to self-assemble.

Miller et al. designed a tetrapeptide (FEFK, 220) consisting of two hydrophobic residues, one positive residue, and one negative residue. While 220 itself hardly forms a hydrogel, 220 can serve as a substrate for thermolysin to undergo reverse hydrolysis for enzymatic hydrogelation by the formation of octapeptides as the final products. Although the hydrogelation concentration of the tetrapeptide concentration is 10 wt %, this unique reversible hydrolysis of the amide bond represents an evolutionary approach to screen hydrogelators from a dynamic library of peptides.¹⁶³ Saiani et al. further studied this tetrapeptide for enzymatic hydrogelation and reported that the initial concentration of the tetrapeptide dictates the morphology of the peptide nanofiber network, and the concentration of the enzyme has little effect on the final morphology of the nanostructures formed by the self-assembly of the peptides.⁵⁸² Verma et al. reported the self-assembly of a tetrapeptide, PWWP (221), which forms vesicular microstructures. The author reported that the vesicles can entrap dye molecules and the addition of KCl (0.25 mM) disrupts the vesicles.⁵⁸³ Tine et al. examined the effect of temperature on the self-assembly of RWDW (222) and found that an increase of temperature produces nanofibers with smaller diameters.⁵⁸⁴ According to the AFM experiment, the authors also found that 222 self-assembles to form a dense network of entangled nanofibers at 15 °C, but assembles into sparse globular and fibrillar structures at 35 °C.

Hamley et al. used an array of techniques to investigate the self-assembly of the pentapeptide KLVFF (223), which is the core motif of β-amyloid peptide. The authors reported that 223 forms a hydrogel at 2.5 wt % in water, but at 3.0 wt % in phophate-buffered saline (PBS). The nanofiber dimensions determined by cryo-TEM and SAXS confirm that fibrils (10 nm in diameter) are formed in aqueous solution, which helps to address some contradictions in the literature, as the authors pointed out.¹⁸⁷ On the basis of the motif AAKLVFF, Hamley et al. replaced phenylalanine with β-2-thienylalanine (2-Thi) to generate (2-Thi)(2-Thi)VLKAA (224). 224 starts to self-assemble at a concentration of 0.03 wt % and forms a hydrogel in water at 2.0 wt %. Although the self-assembly of 224 occurs as designed, the conductivity of the nanofibrils has yet to be demonstrated.⁵⁸⁵

Doyle et al. studied the kinetics of the hydrogelation of an octapeptide (FKFEFKFE, 225) and found that 225 forms a hydrogel at about 0.1 wt %. One notable feature of the hydrogelation of 225 is that an increase of the pH from 3.5 to 4.0 speeds the formation of the nanofiber networks by almost 2 orders of magnitude, from hours to minutes, but the gelation follows the same mechanism. The authors proposed a simple model of self-assembly based on Derjaguin–Landau–Verwey–Overbeek (DLVO) theory.^586,587 This detail kinetic study offers a useful insight for the design of fast responsive hydrogelators.⁵⁸⁸ Saiani et al. investigated a set of four octapeptides, AEAEAKAK (226), AEAKAEAK (227), FEFEFKFK (228),^589,590 and FEFKFEFK (229), which consist of oppositely charged residues and hydrophobic residues with slightly different positionings. The authors found the phenylalanine in the octapeptides to promote β-sheet conformations in solution and the alanine to favor α-helices. According to the authors, 227 is unable to self-assemble in solution, but 226 self-assembles to form thick, rigid nanofibers (6 nm in diameter). However, 226 fails to form a hydrogel at concentrations up to 10 wt %. On the contrary, both phenylalanine-based peptides 228 and 229 self-assemble in solution and form hydrogels at a CGC of 0.8 wt %. Both hydrogels contain a dense network of semiflexible nanofibers 4 nm in diameter. Besides further confirming the importance of aromatic–aromatic interactions for the self-assembly of peptides in water, the detailed SANS measurement in this work has provided invaluable information on the morphologies of the nanofibers in solutions and in hydrogels.⁵⁹¹ Miller et al. developed an innovative way to stabilize enzymes by covalently attaching a self-assembling peptide (230) to pentaerythritol tetranitrate reductase, which co-self-assemble to form hydrogels. The stability of the entrapped enzyme increases significantly, and the activity of the enzyme is retained even after exposure to a high temperature (90 °C) and 12 months of storage,⁵⁹² which is quite remarkable.

Cavalli et al. synthesized a nonapeptide, FEFEFKFKK (231), which forms a hydrogel at 2.0 wt %. On the basis of this result, the authors conjugated 231 with a DNA-recognizing protein (Tus) by a sortase A methodology.⁵⁹³ The authors demonstrated that the hydrogel containing Tus binds to DNAs.⁵⁹⁴ Sun et al. designed and synthesized a decapeptide, GPGGDGPGGD (232) based on the spider flagelliform silk protein and the Ca²⁺ binding domain of lipase Lip A. The authors suggested that such a design can avoid redundancy and preserve the properties of both sequences. At 1.0 wt %, 232 largely forms nanospheres which become nanofibers upon the addition of Ca²⁺. On the basis of CD data, the authors suggested a β-spiral structure as the secondary structure of the peptide.⁵⁹⁵ McPherson et al. reported a new method to synthesize peptide QQRFEWEFEQQ (233) based on the protein expression in Escherichia coli.⁵⁹⁶ The recombinant peptide self-assembles to form nanofibers and results in a hydrogel at a concentration of 1.5 wt % and pH 2.0.

McPherson et al. used a SUMO (small ubiquitin-related modifier)–peptide fusion approach for efficient production and purification of β-structured recombinant self-assembling peptides with native N- and C-terminals (233) to compare with the chemically synthesized peptide that has an acetylated N-terminal (234). The authors reported that the chemically synthesized peptide 234 forms a hydrogel at 1.0 wt % and pH 2.0,⁵⁹⁷ which are essentially the same conditions as those needed for SUMO–233.⁵⁹⁸ Intending to investigate the effect of the topology of the peptides on self-assembly, Lim et al. designed and synthesized two dodecapeptides that have the same sequence (RRRRGSWWWWSG) but different topologies: one (235) is linear, and the other (236) is cyclic. Their study revealed that the cyclic and linear peptides exhibit significantly different self-assembled nanostructures and thermal stabilities, but share similar critical aggregation concentrations and cytotoxicity profiles.⁵⁹⁹ This study offers an important topological approach to tailor the nanostructures formed by the self-assembling peptides. Rapaport et al. reported four tridecapeptides (e.g., PELELELELELEP (237) and PEFEFEFEFEFEP (238)) consisting of negatively charged residues and hydrophobic residues. As an amphiphilic and acidic peptide, 237 self-assembles to form β-sheet structures and results in a hydrogel at pH 7 and a concentration of 4 wt %. The addition of calcium ion can increase the storage modulus of the hydrogel by almost 10-fold. Furthermore, the authors also demonstrated that the hydrogel of 238 could provide scaffolds for cell adhesion and spreading of Saos-2 cells.⁶⁰⁰ Hartgerink et al. reported a tetradecapeptide (EIAQLEYEISQLEQ, 239) consisting of two heptads for adopting coiled-coil structures. At a concentration of 1 wt %, the mixture of 239 with another tetradecapeptide (KIAQLKYKISQLKQ, 240) containing a positively charged residue results in the formation of nanofibers having β-sheet features. The authors suggested that these short peptides illustrate the minimum requirements necessary to form dimeric coiled coils.⁶⁰¹

Hartgerink et al. developed a hexadecapeptide (KKQLQLQLQLQLQLKK, 241) which fits into an ABA triblock motif (termed “multidomain peptides” (MDPs) by the authors). The authors reported that 241, at 1 wt % and with 10 mM Tris at pH 7, self-assembles to form nanofibers and results in a hydrogel. One impressive result in this work is the control of the length of the nanofibers, as demonstrated by the fact that the aging of the solution of 241 hardly changes the maximal length of the nanofibers.⁶⁰² On the basis of the same principle, the authors also developed several other MDPs (KKSLSLSLSLSLSLKK (242), EESLSLSLSLSLSLSLEE (243), KKQLQLQLQLQLQLKK (244)) and used them to form hydrogels at 2 wt %. Moreover, they demonstrated the use of lysyl oxidase or plasma amine oxidase to oxidize primary amines to aldehydes so the reactive aldehydes react further to cross-link the nanofibers in the hydrogels. However, the efficiency of the oxidation process remains to be improved.⁶⁰³ Kinoshita et al. designed two kinds of β-peptide, (LE)₈ (245) and (VEVSVKVS)₂ (246). Both peptides self-assembled to form nanofibers and afford hydrogels upon addition of calcium ion. Owing to the interaction between the carboxylic acid in the peptide and the calcium ions, the resulting hydrogel exhibited enhanced mechanical properties with an increase of the concentration of the calcium ions. The authors suggested that this mineralization approach provides a new way to prepare bone-filling materials.⁹¹

After Schweitzer-Stenner et al. observed that the hexadecapeptide AAKAAAKAAAKAAAKA (247) forms a hydrogel at low concentration (0.5 wt %),⁶⁰⁴ Li et al. analyzed the initial phase of the aggregation process by molecular dynamics. They reported that the peptide aggregates into stable antiparallel β-sheets. In addition, the authors suggested that the formation of trimers is very sensitive to the concentration of the peptides,⁶⁰⁵ which provides a useful insight to explain the conformation transition of 247 from an α-helix to a β-sheet.⁶⁰⁴ After Zhang et al. reported the hydrogels of RADARADARADARADA (248a), Unsworth et al. carried out a detailed study to provide a mechanistic understanding of the self-assembly process by designing and synthesizing two peptides (RADARADARADARADAKKKK (248b) and RADARADARADARADASSSSS (248c)) that are related to 248a. The authors observed that 248b is unable to self-assemble, suggesting the role of the charged residues. The authors also found that self-assembly of 248a and 248c is entropy driven, with hydrophobic force as the main factor for 248a and hydrogen bonding as the main factor for 248c.⁶⁰⁶ The authors also confirmed that counterions contribute little to self-assembly, which differs from the current conceptual understanding of the self-assembly of 248a. Muller et al. designed a library of peptides having alternating hydrophobic and polar amino acids by substituting phenylalanine in FEFEFKFKFEFEFKFK (249) with glycine, alanine, valine, leucine, or isoleucine. The authors found that an increase of the number of glycines eventually prevents the hydrogelation of the peptide, and concluded that the sequence and steric size of the nonpolar residues dictate the secondary structure and morphology of the nanofibers of the self-assembled peptides.⁶⁰⁷ As supramolecular hydrogelators, one of the most impressive classes of oligopeptides made of 20 amino acids is the de novo β-hairpin peptides (250–264) pioneered by Schneider and Pochan et al.^188,608 These synthetic peptides undergo intramolecular folding at a proper condition, and the subsequent intermolecular self-assembly generates β-sheets in water to result in hydrogels.⁶⁰⁹ The authors ingeniously used d-Pro-l-Pro to define a type II′ β turn. At physiological pH, the peptides exist as random coils and exhibit excellent solubility in water, but a stimulus (e.g., ionic strength, pH, temperature, light, or shear forces) switches the peptide to a β-hairpin conformation so these β-hairpin peptides self-assemble into a highly cross-linked, but semiflexible, network of nanofibers⁶¹⁰ to afford mechanically rigid hydrogels. A notable application of these peptides is that they act as antibacterial hydrogels. Since there are several excellent reviews on the β-hairpin hydrogelators and their applications, we only list some representative sequences^{134,200,610−627} in Table 1, and interested readers are encouraged to read the excellent reviews written by Schneider et al.^28,628−630

When the peptides get longer, the intermolecular interactions certainly can increase and favor self-assembly and physical cross-linking to form a hydrogel.⁶³¹ However, the synthesis of long peptides becomes more difficult or more expensive so the peptides should have unique functions or aims to address important and general problems to justify the high cost. After reporting the first example of peptide hydrogels consisting of purely helical structures,⁶³² Woolfson et al. recently designed a self-assembling fiber system consisting of a dimeric coiled-coil peptide (e.g., KIAALKQKIASLKQEIDALEYENDALEQ (265) and KIRRLKQKNARLKQEIAALEYEIAALEQ (266)) that assemble in a controlled manner. The self-assembly of 265 and 266 results in fibers that are tens of nanometers wide and tens of micrometers long. Using cryo-TEM, the authors also obtained an ultrastructure for interpreting the packing of individual α-helices within the fibers. On the basis of the electron density map, the authors derived a model for elucidating how these α-helical fibrils pack into larger fibers.⁶³³ To understand how to use local interactions between proteins for creating materials that have well-determined microstructures, Fairman et al. designed and synthesized an oligopeptide (CKQLEDKIEELLSKAACKQLEDKIEELLSK, 267) that relies on hydrophobic interactions to drive self-assembly. As pointed out by the authors, the hydrophobic effect between 267 molecules favors axial assembly and their electrostatic forces modulate lateral assembly. At a concentration of 0.05 wt %, the peptide self-assembles to form a filament consisting of about 120 molecules of 267. The authors also reported that various environmental factors (e.g., pH, salt, molecular crowding reagents, and “‘capping’” peptides) can regulate the self-assembled filaments in an assembly of predictable manner,⁶³⁴ which provides useful insights for developing coiled coils as peptide-based materials. It would be interesting to know the proteolytic stability of these self-assembled filaments.

4.3.3. Peptide Derivatives

4.3.3.1. Peptides with Capped N- and C-Terminals

Besides native peptides acting as hydrogelators, peptide derivatives can also self-assemble in water to form hydrogels. The most common way to modify a peptide is to cap the N-terminal by an acetyl or the C-terminal by an amide or to cap both ends.^640−642 As shown in Scheme 34, Solaro et al. reported a capped tetrapeptide, Ac-RWDW-NH₂ (268), containing oppositely charged residues and hydrophobic residues, to form a soft hydrogel at 0.026 wt % in PBS buffer. They also observed an increase of the concentration of 268 and the addition of CaCl₂ to result in a thick and transparent hydrogel. Moreover, the authors demonstrated the use of the hydrogels for culturing cells (mouse embryo fibroblasts balb/3T3 clone A31 and human hepatoblastoma HepG2).⁶⁴³ Xu and Lu et al. reported a capped hexapeptide, Ac-IIICGK-NH₂ (269, Table 2), which self-assembles in water to form long nanofibers and results in a hydrogel at 0.53 wt %. It is worth noting that oxidation of the Cys residue in 269 greatly increases the storage modulus of the hydrogels (from 200 to ∼10000 Pa). Moreover, the authors found that oxidation reduces the CGC to as low as 0.034 wt %. Replacing the Cys by a Met residue allows the formation of nanofibers at 1.7 mM, but fails to generate any hydrogels.¹¹⁵ Nilsson et al. synthesized a series of capped octapeptides (Ac-XKXKXKXK-NH₂; X = Val (270a), Ile (270b), Phe (270c), ^F5Phe (270d), and cyclohexylalanine (Cha) (270e)) that are composed of alternating hydrophobic residues (Val) and positively charged residues (Lys). At 8 mM, 270a is too hydrophilic to form a hydrogel, but 270b, 270c, 270d, or 270e self-assembles in water to form a hydrogel. On the basis of CD, the authors concluded that these peptides adopt β-sheet structures in the nanofibers formed by self-assembly. The authors also found that 270c and 270d, containing aromatic residues, form rigid hydrogels, but 270b and 270e form weak gels. One notable feature is that the self-assembly of 270b–270e requires the addition of NaCl to the solution.⁶⁴⁴

Scheme 34. Representative Molecular Structure of a Peptidic Hydrogelator with Capped N- and C-Terminals.

Table 2. Peptide Sequences and Their Reported Hydrogelation Concentrations.

peptide length (no. of amino acids)	peptide sequence	reported hydrogelation concn (wt %)	refs
4	Ac-RWDW-NH2 (268)	0.026	(643)
6	Ac-IIICGK-NH2 (269)	0.53	(115)
8	Ac-VKVKVKVK-NH2 (270a)	∼0.8	(644)
	Ac-IKIKIKIK-NH2 (270b)
	Ac-FKFKFKFK-NH2 (270c)
	Ac-XKXKXKXK-NH2, X = F5Phe (270d)
	Ac-XKXKXKXK-NH2, X = cyclohexylalanine (270e)
8	Ac-FKFEFKFE-NH2 (271a)	∼0.4	(646, 647)
	Ac-AKAEAKAE-NH2 (271b)
	Ac-VKVEVKVE-NH2 (271c)
	Ac-LKLELKLE-NH2 (271d)
	Ac-FKFKFEFE-NH2 (271e)
	Ac-KEFFFFKE-NH2 (271f)
	Ac-KFFEKFFE-NH2 (271g)
	Ac-FFKEKEFF-NH2 (271h)
8	Ac-LIVTQTMK (272a)	1.0	(648)
	LIVTQTMK-NH2 (272b)
9	Ac-PSFNFKFEP-NH2 (273)	1.0	(650)
11	Ac-KWKAKAKAKWK-NH 2 (274)	2.3	(651)
	Ac-EWEAEAEAEWE-NH2 (275)
11	Ac-QQRFQWQFEQQ-NH2 (276a)	0.01–0.1	(652−654)
	Ac-QQRFQWQFQQQ-NH2 (276b)
	Ac-SSRFSWSFESS-NH2 (276c)
	Ac-QQRFOWOFEQQ-NH2 (276d)
	Ac-SSRFEWEFESS-NH2 (276e)
	Ac-SSRFOWOFESS -NH2, O = ornithine (276f)
11	Ac-CFKFEFKFECG-NH2 (with S–S bond) (277)	0.9	(116)
11	Ac-QQKFQFQFEQQ-NH2 (278a)	0.76	(656)
	Cys-QQKFQFQFFEQQ-Gly-thioester (278b)
21	Ac-GGRGDSGGGQQKFQFQFFEQQ-NH2 (279)		(656)
16	Ac-AAKAAAKAAAKAAAKA-NH2 (280)	0.5	(657)
16	Ac-RADARADARADARADA-NH2 (281)	0.5	(181, 648, 659, 660)
	Ac- RASARADARADARADA-NH2 (282)
	Ac- RASARASARASARADA-NH2 (283)
16	Ac-KKQLQLQLQLQLQLKK-NH2 (284a)	1.0	(661, 662)
	Ac-EQLQLQLQLQLQLE-NH2 (284b)
	Ac-KKSLSLSLSLSLSLKK-NH2 (284c)
	Ac-ECLSLCLSLCLSLE-NH2 (284d)
	Ac-KKQFQLQFQLQFQLKK-NH2 (284e)
	Ac-KKQFQFQFQFQFQFKK-NH2 (284f)
	Ac-KKQWQWQWQWQWQWKK-NH2 (284g)
	KKQYQYQYQYQYQYKK-NH2 (284h)
13	Ac-GTAGLIGQERGDS (285)	1.0	(663)
21	Ac-LKELAKVLHELAKLVSEALHA-NH2 (286)	0.1	(93)
10	Ac-WKVKVKVKVK-NH2 (287a)	0.25	(664, 666)
	Ac-EWEVEVEVEV-NH2 (287b)
	formyl-WOAOAOAOAO-NH2 (287c)
	formyl-WEAEAEAEAE-NH2 (287d)
	formyl-WOAOAO-NH2 (287e)
	formyl-WEAEAE-NH2 (287f)
	formyl-WOAOAOAOAOAOAO-NH2 (287g)
	formyl-WEAEAEAEAEAEAE-NH2 (287h)
16	SASLSASLSASLSASL-NH2 (288)	1.0	(667)

To understand the relative contributions of different hydrophobic groups (e.g., aromatic vs nonaromatic hydrophobic groups), Nilsson et al. designed another series of octapeptides (Ac-FKFEFKFE-NH₂ (271a),⁶⁴⁵ Ac-AKAEAKAE-NH₂ (271b), Ac-VKVEVKVE-NH₂ (271c), and Ac-LKLELKLE-NH₂ (271d)). They found that only 271a formed a hydrogel at a concentration of 0.46 wt % in a NaCl (40 mM) solution, suggesting that aromatic–aromatic interactions, though less essential for the self-assembly of this type of peptide (Ac-XKXEXKXE-NH₂), are necessary for hydrogelation. Moreover, the authors also reported that the cosolvent HFIP exerts a significant influence on the stabilization of the helical morphology of the self-assembled nanofibers.⁶⁴⁶ The authors also varied the sequence of 271a to Ac-FKFKFEFE-NH₂ (271e), Ac-KEFFFFKE-NH₂ (271f), Ac-KFFEKFFE-NH₂ (271g), and Ac-FFKEKEFF-NH₂ (271h). The authors found that the self-assembly of these peptides is pH dependent. At pH 3–4, 271e self-assembles to form β-sheet nanoribbons and 271f forms distinct nanotapes with a width of ∼20 nm, but 271g and 271h fail to self-assemble to form fibrils/tapes; however, 271h does form micelle-like aggregates. At neutral pH, 271h forms 20 nm nanotapes and the other peptides behave similarly to the way they do at pH 3–4.⁶⁴⁷ These results underscore that the amino acid sequence plays a key role in the self-assembly of the oligopeptides, which is quite reasonable. Voyer et al. designed a series of octapeptides based on milk proteins (lactoglobulin). They found that both the N-terminal-capped octapeptide Ac-LIVTQTMK (272a) and the C-terminal-capped octapeptide LIVTQTMK-NH₂ (272b) adopt a β-sheet conformation and self-assemble to form hydrogels at a concentration of 0.1 wt %. However, 272a forms hydrogels instantly at neutral pH and acidic pH, and 272b forms hydrogels only at basic pH. This study also provided useful insight into the transition from a random coil to a β-sheet conformation upon a change of the pH.⁶⁴⁸

By introducing four phenylalanine residues into a capped nonapeptide (Ac-Pro-Ser-Phe-Asn-Phe-Lys-Phe-GIu-Pro-NH₂, 273), Zhao et al., found that 273 forms a hydrogel at 1 wt %. The authors reported that the β-turn and β-sheet structures result in a concentration-dependent self-assembly to afford hierarchically arranged nanostructures.^649,650 Yu et al. investigated the effect of the ionic strength on the mechanical, structural, and transport properties of the hydrogels made of two capped undecapeptides, Ac-KWKAKAKAKWK-NH₂ (274) and Ac-EWEAEAEAEWE-NH₂ (275). At a peptide concentration of about 2.3 wt %, the authors changed the ionic strength of the solution and found that an increase of the ionic strength results in higher final storage moduli, slows the rate of hydrogelation, decreases the cross-section of the nanofibers in the hydrogels, and reduces the diffusion coefficients of water in the hydrogels.⁶⁵¹ Waigh et al. used photon correlation spectroscopy to study the internal dynamics of self-assembled fibrils of charged peptides, such as Ac-QQRFQWQFEQQ-NH₂ (276a), Ac-QQRFQWQFQQQ-NH₂ (276b), Ac-QQRFEWEFEQQ-NH₂ (234), and Ac-SSRFSWSFESS-NH₂ (276c). The authors found that these peptides form hydrogels at 0.1 wt %, but they have different critical concentrations for self-assembly, which are 0.05, 0.03, 0.01, and 0.1 mM for 276a, 276b, 234, and 276c, respectively.⁶⁵² Aggeli et al. studied the effect of the ionic strength on the self-assembly, morphology, and gelation of charged peptides, such as 234, Ac-QQRFOWOFEQQ-NH₂ (276d), Ac-SSRFEWEFESS-NH₂ (276e), and Ac-SSRFOWOFESS-NH₂ (276f) (O = ornithine). According to their study, at a concentration of 0.7 wt %, 234 and 276e, in water, exhibit a sharp transition from antipaprallel β-sheets at pH < 6.5 to random coils at pH > 8. This transition results in a change of the properties of the solutions from a nematic solution (pH < 8) to an isotropic fluid (pH > 8). Bearing charges opposite those of 234 and 276e, 276d and 276f undergo a transition from antiparallel β-sheets (pH > 10) to random coils (pH < 8) at 8 < pH < 10. The most insightful observation is that, in the presence of NaCl (130 mM), 234, 276d, 276e, and 276f all form nematic hydrogels at physiological pH.⁶⁵³ Nelson et al. used electrochemical impedance spectroscopy to examine the interaction of this class of peptides with phospholipid monolayers. They found that peptides with side chains of serine and threonine interact with DOPC layers more strongly compared to peptides with side chains of glutamine and asparagine.⁶⁵⁴ These insights should be useful for designing the hydrogels to interact with cells.

By introducing cysteine residues into 271a, Nilsson et al. designed a novel cyclic peptide, Ac-CFKFEFKFECG-NH₂ (277). Upon reduction by DTT, cyclic-277 becomes a linear peptide, linear-277, which forms a hydrogel at 0.9 wt %.¹¹⁶ This excellent strategy also has been applied by Yang et al. to design redox-trigged hydrogelation of small peptides.⁶⁵⁵ To improve the mechanical properties of the self-assembled peptide hydrogels, Collier et al. devised an innovative approach to perform native chemical ligation (NCL) of the peptides after the self-assembly. On the basis of the self-assembling peptide Ac-QQKFQFQFEQQ-NH₂ (278a), they designed and synthesized the sequence Cys-QQKFQFQFFEQQ-Gly-thioester (278b). The self-assembling 278b forms a hydrogel at 0.76 wt %, and NCL of the hydrogel results in a 6-fold increase of the storage modulus. The authors reported that ligation also leads to a significant enhancement of HUVEC cell proliferation on the surface of the hydrogel. Moreover, they demonstrated that NCL is orthogonal to the inclusion of an RGD-functionalized peptide (e.g., Ac-GGRGDSGGGQQKFQFQFFEQQ-NH₂, 279), which further increases the cell proliferation.⁶⁵⁶ Schweitzer-Stenner et al. investigated the self-assembly of a hexadecapeptide (Ac-AAKAAAKAAAKAAAKA-NH₂, 280) and found that the presence of a salt can stabilize the self-assembly of 280 to result in a hydrogel at a CGC of 0.5 wt %. It was found that 280 starts self-assembling at about 0.001 wt % and forms a network of filaments at 1 wt %, and the filaments turn into a nanoweb after the addition of 1.0 M NaCl. The authors also showed that the hydrogel can encapsulate and slowly release proteins.⁶⁵⁷

Yokoi, Arosio, and Zhang et al. further evaluated the Ac-RADARADARADARADA-NH₂ peptide (281)¹⁸¹ by reassembling the peptides after disassembling the hydrogel of 281 by sonication or by changing the ionic strength.⁶⁵⁸ After examining the kinetics of the reassembly, the authors proposed a sliding diffusion model for the reassembly of 281.¹⁸¹ Zhao et al. further investigated the temperature and pH effects on the self-assembly of 281 and found that the extent of β-sheet conformation decreases at low or high pH and an increase of the temperature to 70 °C results in smaller globular aggregates.⁶⁵⁹ Later, Yokoi et al. mutated 281 to change the number and position of the net charges on the peptide. For example, they found that, at a concentration of 0.5 wt % and pH of 1.0, while Ac-RASARADARADARADA-NH₂ (282) fails to form a hydrogel, Ac-RASARASARASARADA-NH₂ (283) does form a hydrogel. Their results confirm that the number of charges and the sequence are critical for the formation of the antiparallel β-sheets for self-assembly.⁶⁶⁰ On the basis of their design of MDPs, Hartgerink et al. varied the sequence of the MDP motif to generate a series of hexadecapeptides (Ac-KKQLQLQLQLQLQLKK-NH₂ (284a), Ac-EQLQLQLQLQLQLE-NH₂ (284b), Ac-KKSLSLSLSLSLSLKK-NH₂ (284c), and Ac-ECLSLCLSLCLSLE-NH₂ (284d)) for enhancing the viscoelasticity of the hydrogels formed by these peptides at 1 wt %. The authors found that 284a–284d all self-assemble to form nanofibers with diameters of 6 nm and lengths on the order of hundreds of nanometers. The addition of Mg²⁺ to the solutions of 284b and 284d and PO₄^3– to the solution of 284c increases the length of the nanofibers to result in entangled networks and hydrogelation. The authors also demonstrated that the addition of Mg²⁺ to the hydrogels of 284b and 284d and PO₄^3– to the hydrogels of 284a and 284c or the oxidation of the hydrogel of 284d significantly increase the storage moduli of the hydrogels, by up to about 60-fold.⁶⁶¹ In another study, Hartgerink et al. introduced aromatic residues into the MDP peptides to obtain Ac-KKQFQLQFQLQFQLKK-NH₂ (284e), Ac-KKQFQFQFQFQFQFKK-NH₂ (284f), Ac-KKQWQWQWQWQWQWKK-NH₂ (284g), and KKQYQYQYQYQYQYKK-NH₂ (284h).⁶⁶² The authors reported that the peptides 284e–284h self-assemble to form nanofibers with intertape spaces larger than that of 284a. At 1 wt %, 284e–284h all form hydrogels at pH 7.4, and 284e and 284f exhibit higher storage moduli than those of 284g and 284h.⁶⁶² In addition, Hartgerink et al. designed a peptide amphiphile containing a tridecapeptide (Ac-GTAGLIGQERGDS, 285). While the sequence RGDS promotes cell adhesion, Ac-GTAGLIGQ serves as a substrate of MMP-2. The authors demonstrated the enzymatic degradation (by type IV collagenase) of the hydrogels made of the peptide amphiphile, and suggested that the degradation of the nanofiber networks mimics a key property of the natural extracellular matrix (ECM).⁶⁶³

Dexter et al. developed a 21-residue peptide (Ac-LKELAKVLHELAKLVSEALHA-NH₂, (286)) that forms a hydrogel at 0.1 wt % upon a change of the pH. On the basis of TEM, DLS, and electronic circular dichroism (ECD) studies, the authors reported that 286, due to hydrophobic interactions, self-assembles to form hexameric coiled coils, which promote vertical alignment for rearrangement to fibril networks for hydrogelation.⁹³ Tseng and Yu et al. designed a pair of self-repulsive peptides (Ac-WKVKVKVKVK-NH₂ (287a) and Ac-EWEVEVEVEV-NH₂ (287b)) consisting of alternating charged/neutral amino acid sequences. The authors reported that the simple mixing of the two peptides, at a total concentration of 0.25 wt %, affords a hydrogel. It was found that the coassembled hydrogel exhibits rapid recoveries from repeated shear-induced breakdowns. The authors also found that the hydrophobicity of the neutral amino acids dictates the viscoelastic properties of the hydrogels.⁶⁶⁴ By designing two similar peptides, formyl-WOAOAOAOAO-NH₂ (287c) and formyl-WEAEAEAEAE-NH₂ (287d), Yu et al. found that the temperature significantly affects the structure and mechanical properties of hydrogels formed by mixing 287c and 287d at a total concentration of 1 wt %. The authors found that the peptide nanofibers assemble faster to result in mechanically stronger hydrogels at 25 °C than at 5 °C,⁶⁶⁵ suggesting entropy-driven self-assembly. Later, Yu et al. compared 287c and 287d with other similar peptides that have the same alternative amino acid sequences but different lengths of the peptides (i.e., formyl-WOAOAO-NH₂ (287e), formyl-WEAEAE-NH₂ (287f), formyl-WOAOAOAOAOAOAO-NH₂ (287g), and formyl-WEAEAEAEAEAEAE-NH₂ (287h)). They found that, upon mixing, 287c + 287d and 287g + 287h form hydrogels at 1 wt %, but the mixture of 287d + 287e does not. In addition, the hydrogel formed by 287g + 287h is mechanically weaker than that of 287c + 287d, likely due to the tighter packing of the amino acid side chains in the nanofibers formed by 287c + 287d.⁶⁶⁶ Kinoshita et al. designed a C-terminal-capped hexadecapeptide, SASLSASLSASLSASL-NH₂ (288). They found that 288 adopts a β-sheet structure and forms a hydrogel at 1 wt %. One interesting feature of 288 is that it also forms organgels in polar organic solvents (e.g, DMF, DMSO, N-methyl-2-pyrrolidone (NMP), and 2,2,2-rifluoroethanol (TFE)).⁶⁶⁷

4.3.3.2. Peptide Derivatives Containing Alkyl/Lipid Chain(s)

Alkyl-chain-containing peptide derivatives,^668−672 sometimes termed “surfactant-like peptides”,⁶⁷³ essentially consisting of hydrophilic and hydrophobic domains,^674−679 are some of the most representative peptide amphiphiles.⁶⁸⁰ The alkyl or lipid chains, usually attaching to the N-terminal or the C-terminal of peptides, not only drive self-assembly through van der Waals force, but also allow the functional peptides to be presented on the surface of nanofibrils at an exceedingly high density.⁶⁸¹ Moreover, the modular nature of peptide chemistry facilitates the variation of the amino acids or chain length, thus easily tuning both the mechanical properties and biological activities of the subsequent self-assembled nanofibrils or hydrogels.⁶⁸² As shown in Scheme 35, Miravet and Escuder et al. reported an l-proline-based supramolecular hydrogelator (289) which affords a supramolecular hydrogel at a concentration of 0.25 wt %. This l-proline-based supramolecular hydrogel has a remarkable efficiency as a heterogeneous organocatalyst for a direct aldol reaction.^683,684 Another dipeptide derivative (290), consisting of two amino acids (i.e., l-histidine and β-alanine) and a lipid chain, is able to form a hydrogel in a variety of conditions (e.g., at both alkaline and acidic conditions and in the presence of additives, such as NaCl or alcohol) with a CGC value of 3.4–6.8 wt %.⁶⁸⁵291 (β-Ala-His-(EO)₂-alkyl chain), with one more ethylene glycol group compared with 290, can also self-assemble to form a hydrogel at a concentration of 2.0 wt %.⁶⁸⁶ Goto et al. reported a tetrapeptide amphiphile (292) which has a simple molecular structure but results in a hydrogel at a remarkably low concentration (0.03 wt %).⁶⁸⁷ Hamley et al. designed and developed an enzymatically cleavable peptide amphiphile (C₁₆-KKFFVLK, 293)). The peptide part of this small molecule is cleaved by α-chymotrypsin at two sites, leading to several products: C₁₆-KKF, FVLK, C₁₆-KKFF, and VLK. 293 molecules form nanotubes and helical ribbons at room temperature, and both C₁₆-KKF and C₁₆-KKFF self-assemble to form spherical micelles. FVLK and VLK are unable to adopt well-defined aggregated structures. In this way, the enzyme can modulate the self-assembly of the systems and tune the resulting nanostructure.⁶⁸⁸ It would be more interesting to determine the biological activities of 293 in a cellular environment. Stupp et al. reported a peptide amphiphile (294) with more than 10 amino acids. The addition of soluble metal ions or an adjustment of the pH triggers the hydrogelation of this molecule at a concentration of 20 mM (2.4 wt %), indicating the self-assembly is mediated by a screening counterion and stabilized by van der Waals and hydrophobic forces, ionic bridging, and hydrogen bonding.⁶⁸⁹ To elucidate the self-assembly behavior of the peptide amphiphiles, which tend to form cylindrical nanostructures that imply worm micelles, the same group used a pair of compounds (295 and 296), containing chromophores (i.e., tryptophan), to study the aqueous solvation within the self-assembled structures. Self-assembly constrains the chromophores to a defined location within the aggregate, which leads to different fluorescence changes of the chormophores after the addition of aqueous acrylamide (a quencher of fluorescence). The authors found that, at lower pH, 295 and 296 have a tendency to form a cylindrical structure with the alkyl chain inside the nanostructure as a hydrophobic core.⁶⁹⁰297 represents a class of peptide derivatives, called gemini peptides, formed by the complexation of cationic gemini surfactants and anionic oligoglycine-aspartate. By studying the aggregation behaviors of these molecules (the hydrophobic chain length (C₁₀ to C₂₂) and the length of the oligoglycine (0–4)), Oda et al. found that the hydrogels of 297 only form below a certain temperature (e.g., the Krafft temperature^691,692) when either the hydrophobic chains or the peptides are long enough.⁶⁹³ Besides one-component hydrogels, Stupp et al. developed hydrogels formed by coassembly of amphiphiles with opposite peptide polarities (i.e., peptide amphiphiles with free N-terminals or C-terminals), for example, 298 and 299. The mixture of these molecules with complementary polarities results in coassembled structures which show unusual thermal stability compared to the assemblies composed by only one of the constituents.⁶⁹⁴

Scheme 35. Representative Molecular Structures of Hydrogelators Containing Alkyl/Lipid Chains.

4.3.3.3. Peptide Derivatives Containing Aromatic Group(s)

Peptides functionalized at the N-terminal with large aromatic groups have recently emerged as an exciting class of small molecular hydrogelators.^695−698 The most used aromatic group is Fmoc.^699−701 As shown in Scheme 36, shortly after the report of an unexpected small peptidic hydrogelator made of Fmoc-l-Ala-l-Ala (300) and Fmoc-d-Ala-d-Ala (22),¹¹ Ulijn et al. reported the development of Fmoc-diphenylalanine (Fmoc-FF or Fmoc-Phe-Phe (6)), one of the most investigated low molecular weight hydrogelators.^703−705 The molecules of 6 form hydrogels by adjusting the pH of the aqueous solution of 6,^97,703 by applying 6 to a silica wafer surface,⁷⁰⁶ or by the addition of water to a DMSO solution of 6.^707,708 Despite considerable studies on 6,^{566,709−715} it was unclear why the mechanical properties reported for the hydrogels of 6 vary significantly, up to 4 orders of magnitude. To address this inconsistency, Adams et al. have systematically studied the mechanical properties of hydrogels of 6 prepared using different protocols. They demonstrated that, independently of the method of gel formation, the final pH of the hydrogels is the principal determinant of the mechanical properties, which is quite reasonable due to the C-terminal carboxylic group in 6. Besides, additional variability arises from experimental factors such as the fraction of DMSO or the nature of the buffers used in the selected systems.⁷¹⁶ In addition to Fmoc-FF (6), a variety of Fmoc-dipeptide derivatives have been reported in the past several decades.^717−731 Adams and Firth et al. investigated the influence of the molecular structure on the gelation behaviors of a range of Fmoc-dipeptides. In general, they found that the overall hydrophobicity of the Fmoc-dipeptide determines the ability to form a stable hydrogel.⁷¹⁸ Interestingly, Gazit et al. designed a 3,4-dihydroxy-l-phenylalanine (DOPA)-containing Fmoc-dipeptide (301) by using DOPA to substitute for the phenylalanines in 6. The resulting hydrogelator self-assembles at a concentration of 0.5 wt % and forms supramolecular nanostructures, which can be used as multifunctional platforms for various technological applications, such as glass glue.⁷³² In addition to Fmoc-dipeptide, Fmoc-peptide derivatives with more than two amino acids are also excellent candidates for self-assembly to form hydrogels.^733,734 These small molecules are obtained by both chemical approaches^{674,735−737} and enzymatic reactions.^{161,162,738−741} Parquette et al. further functionalized Fmoc-dipeptide Fmoc-KK (302) at the ε-amino position with a naphthalenediimide (NDI) chromophore to afford Fmoc-KK(NDI) (303). The resulting molecule forms a self-supporting hydrogel at a concentration as low as 1.5 wt % which exhibits high thermal stability up to 75 °C.⁷⁴²

Scheme 36. Representative Molecular Structures of Hydrogelators Containing Aromatic Groups.

Like Fmoc, naphthalene^743,744 is another aromatic group frequently used in peptide derivatives to achieve strong intermolecular interaction for self-assembly.⁷⁴⁵ Among them, 2-(naphthalen-2-yl)acetic acid is a convenient motif for constructing naphthalene-based peptide derivatives because of its ease of being directly used in SPPS.^{169,238,655,746−759} For example, compound 3, reported by Xu et al., is a dipeptide derivative made by conjugating 2-(naphthalen-2-yl)acetic acid with Phe-Phe. In the molecule of 3, the naphthalenyl group provides the hydrophobic force to enhance self-assembly in an aqueous environment, while the dipeptide backbone acts as both hydrogen bond acceptors and hydrogen bond donors. Xu et al. demonstrated 3 as an effective hydrogelator that forms a hydrogel at a concentration of 0.8 wt %, with the gel–sol transition temperature at about 323 K.⁷⁶⁰ The crystal structure of 3 (Figure 6),¹⁴ though obtained from single crystals grown from a mixed solvent of ethanol and water, reveals that the aromatic–aromatic and hydrogen-bonding interactions apparently reinforce each other. Moreover, 3 has found applications in enabling other molecules to be hydrogelators.^761,762 Xu et al. also designed and synthesized a similar small molecule (304) with an exposed N-terminal and a naphthalene-blocked C-terminal. This N-terminated hydrogelator affords a stable hydrogel even below a concentration of 0.8 wt %, but unlike its C-terminal analogues 3, 304 forms hydrogels only within a narrow pH range (5–6) (Scheme 37).⁷⁶³ Adams et al. used 2-naphthol to construct naphthalene-based dipeptide derivatives^764,765 with an ether bond,^{88,99,100,205,766−770} such as 305(101) and 306.⁷⁶⁷ They reported that the hydrogelation of 305 and 306 could be controlled by reducing the pH value with the hydrolysis of GdL at a concentration of 2.2 mM. They chose GdL for pH control because its hydrolysis to gluconic acid allows a slow, uniform pH change. Das et al. also developed a series of hydrogelators (e.g., 307) with 2-naphthol as a terminal, but with an ester bond.^119,771

Figure 6.

Molecular packing and hydrogen bonds in the crystal of 3 (recrystallized from ethanol): views from the (A) a, (B) b, and (C) c axes and (D) view from the c axis to show the hydrogen bonding (green dotted lines) of one molecule with four other molecules and some aromatic–aromatic interactions (yellow lines). Adapted from ref (14). Copyright 2011 American Chemical Society.

Scheme 37. Representative Molecular Structures of Hydrogelators Containing Naphthalenyl Groups.

As mentioned before, the pyrene motif not only enhances intermolecular interaction to promote self-assembly, but also exhibits fluorescence that acts as a useful tool for studying the aggregation behaviors of the hydrogelators.^772,773 Atkins et al. replaced the disulfide bond in the oxidized disulfide form of glutathione (γ-glutamylcysteinylglycine (GSH)) (GSSG) to increase the self-assembly ability of GSSG in aqueous solution, which otherwise only self-assembles to generate fibrillar aggregates and gels in organic solvents. The resulting GSH–pyrene (308; Scheme 38) then forms a gel in a mixed solvent (95% H₂O and 5% DMSO) at a concentration of 1 mM (0.056 wt %).⁷⁷⁴ Xu et al. developed a pyrene-terminated dipeptide (309) which affords a weak hydrogel at a concentration of 30 mM (1.3 wt %). However, the addition of vancomycin (310) at a 1:1 ratio remolds the self-assembly of hydrogelators and drastically increases the elasticity of the hydrogel by 10⁶-fold due to the ligand–receptor interaction between vancomycin and 309. A spectroscopic analysis confirms the aromatic–aromatic interactions between the pyrene groups in the hydrogel.²³⁷ EM also reveals the formation of highly cross-linked networks in the hydrogels, likely contributing to the significant increase of the storage modulus. Stupp et al. developed several molecules (311, 312, and 313) with pyrene chromophores in the backbone to investigate the aqueous solvation within the self-assembled structure formed by these peptide amphiphiles. They found that, as the chromophore is placed closer to the exterior of the aggregates, the Stern–Volmer quenching constants and the fractional accessibility of covalently bound pyrene progressively increase. Their study also demonstrated that covalently bound fluorophores within an aggregate can interact with the external environment.⁶⁹⁰

Scheme 38. Representative Molecular Structures of Hydrogelators Containing Pyrene Groups.

In addition to the exploration of Fmoc, naphthalene, and pyrene, Ladouceur et al. have synthesized novel self-assembling hydrogelators that contain an electroactive aromatic group, anthraquinone. As shown in Scheme 39, they used the well-known redox couple of anthraquinone/anthrahydroquinone as the hydrophobic component for a series of hydrogelators, such as 314. The molecules of 314 undergo two separate processes: a reversible redox reaction and a reversible self-assembly at ta concentration of 4 wt %.⁷⁷² Dynamic combinatorial chemistry was originally a method for developing synthetic receptors and ligands for biomolecules by linking building blocks together using a reversible reaction, resulting in a thermodynamically controlled product distribution. The studies of Otto et al. proved that dynamic covalent disulfide linkages are not only instrumental in dynamic combinatorial discovery of self-assembling materials, but also further stabilize the consequent self-assembly. They designed a building block (315) equipped with a short peptide sequence capped by a dithiol terminal and reasoned that self-assembly would become feasible for a macrocycle (315₆) that reached a critical size. They discovered that photoirradiation of the solution containing 315₆ (0.6 mM) results in the formation of a hydrogel.⁷⁷⁵ Miravet and Escuder et al. prepared a pH-sensitive complex molecular hydrogel from oppositely charged tetrapeptide components (e.g., 316 and 317). They have shown that small peptides bearing alternating phenylalanine and aspartic acid residues such as 316 are able to form hydrogels at low concentration, and 317, designed as a charge complementary analogue bearing alternating phenylalanine and lysine, is also able to aggregate at low concentration. Then they obtained a pH-sensitive coassembled network from these two oppositely charged small self-assembling peptides. By changing the pH of this system, they were able to switch between two-component networks at neutral pH and one-component networks at either basic or acidic pH.⁷⁷⁶ Wang et al. described the design and synthesis of molecular hydrogelators composed of peptides and peptoids (i.e., one type of unnatural peptide with the side chain attached to the amide nitrogen as well), such as 318. They tested the hydrogelation ability of 318 by the inverted-tube method and found that 318 and its analogues afford hydrogels with CGC values of 0.5–0.8 wt %.⁷⁷⁷ Yang et al. developed a reversible hydrogelation system using a redox system with selenium-containing peptides (319 and its reduced version). They achieved the reversible transformation between the solution and hydrogel of the peptide derivative 319 at a concentration of 1 wt %, which is accompanied by the reversible transformation between selenide and selenoxide, by triggering with vitamin C and H₂O₂ (0.1 wt %).⁷⁷⁸ Hamachi et al. reported a series of dipeptide derivatives, for example, Bhcmoc-FF (320), in which FF is tethered with [(6-bromo-7-hydroxycoumarin-4-yl)methoxy]carbonyl (Bhcmoc). They found that compound 320 forms a hydrogel above 0.35 wt % and the resulting hydrogel collapses upon application of stimuli such as UV irradiation.⁷⁷⁹ Liang et al. developed a salt-responsive peptide (321) as a luminescent hydrogelator with a CGC of 0.3 wt %. They found that only the presence of salt rather than the temperature, pH, or solvent caused the dispersed hydrogelators to self-assemble to form a hydrogel network to turn on bright emission.⁷⁸⁰

Scheme 39. Representative Molecular Structures of Hydrogelators Containing Aromatic Groups.

4.3.3.4. Peptide Derivatives Containing a Photoresponsive Group

In addition to enzyme-responsive hydrogelation,^781−783 supramolecular hydrogelators containing a photoresponsive group are of great interest since light can act as an external stimulus to modulate the properties of the hydrogels.⁷⁸⁴ For example, upon photoirradiation, a solution can transform into a hydrogel, and vice versa. A variety of photoresponsive groups have served as the photochemical module in peptides for the design of photoresponsive hydrogelators. UV and visible light can regulate the geometry of spiropyran (between the nonplanar spiropyran form and the planar merocyanine) to control the hydrogelation process because the planar merocyanine isomer favors the formation of aggregates (due to stronger intermolecular π–π stacking) while the nonplanar spiropyran form disfavors π–π interaction. As shown in Scheme 40, Zhang et al. reported that the spiropyran-conjugated dipeptide 322 forms a hydrogel upon photoisomerization to a merocyanine form. Besides responding light, this hydrogel undergoes a gel–sol transition upon the addition of vancomycin because of the strong interaction of vancomycin with the peptide unit d-Ala-d-Ala.⁷⁸⁵ Azobenzene-group-containing peptides are another typical class of photoresponsive hydrogelators.^786−789 The reversible photoregulated trans to cis isomerization of azobenzene significantly influences the intermolecular interaction among the hydrogelators, thus changing the morphologies of the aggregates or controlling the gel–sol transition. Huang et al. reported a dipeptide amphiphile incorporated with an azobenzene moiety (323) which self-assembles to form well-defined nanoribbons that result in a macroscopic hydrogel. After UV irradiation, the authors found a dramatic decrease of the viscosity of the sample, accompanied by a transition from laminated ribbons to short fibers, as revealed by EM.⁷⁹⁰ Tamaoki et al. studied the mechanism by which azobenzene isomerization induces the breaking and reorganization of the assemblies of N-(l-valyl-l-valyl-l-valyl)azobenzene-4-carboxamide (azo(l-Val)₃, 324). As a hydrogelator, 324 forms photoresponsive nanofibrils, and undergoes dispersion/reorganization upon trans to cis photoisomerization that breaks and re-forms the hydrogen bonds to induce reversible gel–sol transitions.⁷⁹¹ Zhang et al. reported an azobenzene-linked symmetrical gemini α-helical peptide (325) that undergoes light-switched self-assembly. With the reversible molecular structure transition between trans and cis (U-shape), the morphology of the self-assembled gemini α-helical peptide can reversibly change between nanofibers and nanospheres in acidic conditions, and between nanospheres and vesicles in basic conditions.⁷⁹²

Scheme 40. Representative Molecular Structures of Hydrogelators Containing a Photoresponsive Group.

4.3.3.5. Peptide Bolaamphiphiles

Many different types of amphiphilic molecules are able to form organogels or hydrogels, but they usually gel either water or an organic solvent. In other words, few of them have the ability to form gels in both water and an organic solvent, except certain peptide-based bolaamphiphiles.⁷⁹³ Because of the versatile functional groups on peptides, such as carboxyl, amine, thiol, hydroxyl, and other hydrophobic groups, peptide-based bolaamphiphiles exhibit diverse self-assembly behaviors in responding to different environments.⁷⁹⁴ As shown in Scheme 41, Das et al. developed a library of bolaamphiphiles by varying the amino acids (e.g., Phe, Tyr, Leu, or Gly) as the head groups, and reported that sequential pH changes trigger the hydrogelation of these peptide bolaamphiphiles (e.g., 326 with a gelation concentration of 0.003 wt %). One interesting observation reported by them is that the addition of dimethyl sulfate, a reagent used for the methylation of phenols and esterification of acids via an S_N2 reaction, can dramatically change the morphologies of the hydrogels.⁷⁹⁵ Zinic et al. systematically studied the gelation properties, self-assembly motifs, chirality effects, and morphological characteristics of the gels formed by retro-dipeptidic bolaamphiphiles similar to 326, and the dimethyl ester and dicarboxamide derivatives of these bolaamphiphiles.⁷⁹⁶327, comprising a dibenzofuran template and two peptide strands made up of alternating hydrophilic and hydrophobic residues and a blocked carboxyl terminal, represents another class of bolaamphiphiles that have a collapsed U-shaped structure. In the bolaamphiphiles 327, the dibenzofuran template positions the strands about 10 Å apart. These molecules afford wide nanofibers, having a cross-β-sheet structure, in water via intermolecular hydrogen-bonding and hydrophobic interactions.⁷⁹⁷ Self-assembly of π-conjugated small molecules has attracted a lot of attention for potential use in organic electronic devices, such as photovoltaic cells. Recently, several laboratories have introduced π-conjugated nanostructures into peptide-based bolaamphiphiles to replace the lipid chain in the linker segment.^798,799 For example, Stupp et al. designed a peptide-based bolaamphiphile (328) that has three segments (e.g., polar amino acids for solubility, β-sheet-forming amino acids for self-assembly, and an oligothiophene core for conductivity). The self-assembly of this molecule results in a self-supporting hydrogel at low concentrations (1 wt %). The authors envisioned that, in combination with biological epitopes, the 1D nanostructure formed in the hydrogelation process may be used to simultaneously signal cells with electrical currents and epitope–receptor interactions.⁸⁰⁰

Scheme 41. Representative Molecular Structures of Hydrogelators Based on Bolaamphiphiles.

4.3.3.6. Dendrimers or Dendrons Made of Peptides

In the early 1990s, Newkome et al. pioneered the development of dendritic bolaamphiphiles as effective hydrogelators.⁸⁰¹ Since then, there has been considerable interest in exploring the dendritic hydrogelators for self-assembly to form hydrogels due to their highly controllable sizes, topologies, and surface properties. One particular appealing attribute of peptide-based dendrons or dendrimers is their extremely broad structural diversities by varying the α-amino acids used in their construction. As shown in Scheme 42, Woolfson et al. described an approach that utilizes nonlinear or dendritic peptides, such as 329, to direct the self-assembly of two complementary linear peptides. The two peptides, which combine to form exclusively linear fibers, coassemble with 329 (at 100 μM) to form specific structures, such as hyperbranched networks, polygonal matrixes, and regularly segmented and terminated fibers.⁸⁰³ Dendritic peptide 330, with repeating hydrophobic and hydrophilic residues as well as lysine terminals, self-assembles to give a uniform toroid structure. Lee et al., who developed this small molecule, also found that removing the electrostatic repulsions or increasing the hydrophobic interactions of this peptide drives the β-sheet peptides to form 1D nanostrucutres.⁸⁰⁴ Liu et al. reported an amphiphilic dendron containing three dendrite l-glutamic acid units and a long alkyl chain (331). The dendron can form hydrogels over a wide pH range (from 2 to 13). The lowest CGC is 2.2 mM at pH 2, and when the pH value is increased, the CGC also increases. At pH 13, nearly 10 mM 331 is needed to form a hydrogel.⁸⁰⁵ Chau et al. developed a two-component self-assembling system (332 and 333) using the interaction of aromatic groups (Fmoc) to construct nanoparticles. The triskelion Fmoc conjugate 333 can quickly self-assemble to form spherical particles around 70 nm in diameter at physiological pH and at a concentration of 100 μM. The Fmoc-dipeptide 332 can wrap up the resulting nanoparticles and stabilize them.⁸⁰⁶

Scheme 42. Representative Molecular Structures of Hydrogelators Based on Dendrimers or Dendrons.

4.3.3.7. Others

Some self-assembling peptides have a (tert-butyloxy)carbonyl (Boc) group on their N-terminal.^807−811 As shown in Scheme 43, Reches et al. describe the formation of complex nanostructures by the coassembly of two simple peptides, Boc-FF (334) and FF (208). They found that 334 itself self-assembles to form nanospheres and 208 self-assembles to give tubular structures, but being combined together, the two peptides coassemble into a construction of beaded strings at concentrations of each higher than 0.3 wt %.⁸¹² Feng et al. developed cyclic dipeptide 335, conjugated with a carbohydrate, and found that the solution of 335 transforms into a transparent hydrogel with the assistance of shear force at a concentration of 5.0 wt %.⁸¹³ According to the authors, cyclic dipeptides are a group of special peptides with unique properties, and most of them afford hydrogels after shearing.^813−815 Liskamp et al. studied the incorporation of a single β-aminoethane sulfonyl amide moiety in highly amyloidogenic peptide sequences (e.g., 336) and observed that this incorporation results in a complete loss of amyloid fibril formation. Instead, they found 336 affords supramolecular nanofibers at a concentration of 0.1 wt %.⁸¹⁶ Similarly, Maggini et al. inserted an oligo(p-phenylenevinylene) into a peptide to afford 337. They found that pH changes trigger a reversible self-assembly of 337, which has a CGC of 13 mM (2.3 wt %).⁸¹⁷ Tian et al. recently reviewed the complexion between metal ions and a series of interesting peptides,⁸¹⁸ which can also lead to hydrogelation.

Scheme 43. Representative Molecular Structures of Hydrogelators.

4.4. Hydrogels Based on Nucleobase Derivatives

Since the discovery of the DNA double helix structure over 60 years ago, the interactions between base pairs have been a subject of interest in the fields of cell biology and supramolecular chemistry. Because of their exceptional abilities for forming intermolecular interactions in water, nucleobases are able to serve as the building blocks of hydrogelators and have received considerable research attention. Because Araki et al.²⁶ reviewed the development of nucleobase-containing gelators in 2005, in this section we mainly focus on supramolecular hydrogelators made of nucleobases over the past decade (Table S3). Considering that the most attractive feature of nucleobases is the intermolecular interactions between base pairs, we arrange these nucleobase hydrogelators according to the classification of homotypic and multicomponent hydrogels.

4.4.1. Homotypic Hydrogels Based on Nucleobases

As shown in Scheme 44, Barthelemy et al.¹⁹⁵ prepared a family of new uridine phosphocholine hydrogelators (338) which self-assemble in water to form DNA-like helical nanofibers and result in hydrogels at a concentration around 6 wt %. One interesting feature of this hydrogel is that the hydrogelators, below the phase transition temperature (T_m), self-assemble to form helical fibers which are transformed to compact bilayers above the T_m. In addition, they also designed and synthesized a series of glycosyl nucleoside lipids (GNLs; 339, 340) by using a convenient “double-click” chemistry. 339 and 340 are able to form nanofibers and result in hydrogels. Particularly, 340 self-assembles to form circular nanofibers (Figure 7) that afford a hydrogel with a CGC of 0.1 wt %.⁸¹⁹ It would be interesting to determine the mechanism of the formation of those circular nanostructures. In another study, Barthelemy et al. prepared a hydrogel of the GNLs for trapping nanoparticles or nanomaterial from an aqueous suspension, and suggested the use of hydrogelators as an additive for decontamination.⁸²⁰

Scheme 44. Hydrogelators Containing Nucleobases.

Figure 7.

TEM images of gel 340 (scale bar 50 nm). Adapted from ref (819). Copyright 2009 American Chemical Society.

To compare the effect of hydrophilic/hydrophobic balance on hydrogelation, Kim et al.⁸²¹ designed and synthesized four nucleosides (341, 342) by modifying the 5-position of the uracil base with an (alkylbenzyl)triazole unit. Unlike 341, which forms metastable partial gels in water when the concentrations are higher than 2.5 wt %, 342 affords a stable hydrogel with a CGC of 1.0 wt %, likely due to the hydrophobicity of the butylbenzyl group. Kim et al.⁸²² reported an intriguing example in which a 2′-deoxyladenosine derivative (343) forms aggregates in the process of heating to cooling, but only forms a hydrogel under ultrasonic radiation. The authors suggested a very interesting explanation: that the production of oxidized species of 2′-deoxyladenosine during sonication might contribute to tuning the hydrophilic/hydrophobic balance to result in a hydrogel. Yang et al.⁸²³ developed an aminonucleoside phospholipid (344) which self-assembles to form superhelical strands and results in a hydrogel at a concentration of 6 wt %. They found that 344 binds with double-stranded DNA on the basis of π–π stacking and H-bonding, so they suggested that this work has potential application in gene delivery. Marlow et al.⁸²⁴ designed and synthesized a new cytidine-derived gelator (345) that forms a gel in a mixture of methanol and water (MeOH:H₂O = 1:1) at a concentration of 0.5 wt %. Sleiman et al.⁸²⁵ prepared a series of nucleobase peptide amphiphiles in which 346 self-assembles to form nanofibers and results in a hydrogel in water (5% DMSO) with a CGC of 0.5 wt %. On the basis of the base pair interaction, the authors suggested this work provides new avenues for nucleobase-specific electrophoresis and oligonucleotide delivery.

4.4.2. Multicomponent Hydrogels Based on Nucleobases

As shown in Scheme 45, Lehn et al.⁸²⁶ reported an interesting example in which guanosine hydrazide (347) affords a stable hydrogel (0.46 wt %) on the basis of the formation of a guanine quartet (G-quartet) in the presence of various metal cations (e.g., Na⁺, K⁺). The authors used various spectroscopies (electronic and vibrational circular dichroism) to reveal that 347 forms long-range chiral aggregates consisting of G-quartets which result in columns due to the binding of metal cations between G-quartet species.⁸²⁷ In addition, the supramolecular structure is sensitive to the cations. Further studies by the authors prove that 347 in solution confers a pseudo-four-stranded helix with guanine–guanine hydrogen bonding to form a continuous helical strand rather than the usually planar G-quartet.⁸²⁸ Moreover, 347 is capable of forming reversible acylhydrazone bonds with various aldehydes so that the nature of the aldehyde can tailor the macroscopic properties of the resulting materials. For example, 347 reacts with pyridoxal monophosphate to afford 348, which forms a hydrogel (0.8 wt %) in the presence of K⁺.⁸²⁸This seminal work illustrates a dynamic combinatorial library, based on the proper aldehyde, for the selection of the strongest hydrogel within a pool of certain building blocks. Using DFT calculations, Urbanova et al.⁸²⁹ predicted and elucidated the molecular arrangement of 347 in the gel state, and reported that the predictions are in good agreement with the experimental data.

Scheme 45. Nucleobase Derivatives for Multicomponent Hydrogels.

Using nucleotides 349 and 350 as counterions to interact with cationic gemini surfactants, Oda and co-workers⁸³⁰ designed and synthesized nucleobase–gemini hybrids which are able to form hydrogels with a proper hydrophobic chain length (e.g., 349 forms a hydrogel at a CGC of 0.64 wt %). Intriguingly, the addition of complementary nucleosides to the solution of 349 affords a stable hydrogel at an even lower CGC (e.g., the addition of adenosine to 349 reduces the CGC to 0.32 wt %). By simply mixing a nongelator, 2′,3′,5′-tri-O-acetylguanosine (351), with a guanosine gelator (352), Rowan et al.⁸³¹ prepared a hydrogel in the presence of potassium. The resulting hydrogel exhibits an extended lifetime and enhanced thermal stability compared with that of 352 alone, likely due to the incorporation of the more hydrophobic 351 into G-quartets. Besides demonstrating the ratio of the two components as a tool to tune the mechanical and thermal properties of the hydrogels, the authors used a combination of light scattering, small-angle neutron and X-ray scattering, and viscometric experiments to study the mechanism of hydrogelation and found that an increase in the volume fraction of microgel domains ultimately leads to macroscopic gelation.⁸³² By mixing guanosine (352) with 0.5 equiv of KB(OH)₄, Davis et al.⁸³³ developed a guanosine–borate hydrogel. Further studies found that the resulting hydrogel is able to selectively absorb a cationic dye and nucleosides via electrostatic interaction and hydrogen bonding. Employing Ag⁺ as the metal ion to coordinate with 5′-guanosine monophosphate (353), Mann et al.⁸³⁴ prepared a hydrogel based on the Ag–GMP (guanosine monophosphate) nanofilaments in water. This hydrogel binds a cationic dye and protein (e.g., cytochrome c) without the loss of biological activity, suggesting possible use in controlled drug release and molecular recognition. In another experiment, Kumar et al.⁸³⁵ reported a porous hydrogel based on a mixture of 350 and β-FeOOH. The freeze-dried gel shows a high swelling ratio of 326% and loading capacity for methylene blue, suggesting that this hydrogel has potential applications in drug delivery and other biological applications. Li et al.⁸³⁶ reported a new two-component hydrogel based on thymidine (354) and melamine at a CGC of 0.1 wt %. Using FT-IR and X-ray diffraction, the authors confirmed that the thymidine and melamine, via intermolecular hydrogen bonding, form supramolecular complexes. Utilizing click chemistry to connect benzyl azide and 8-aza-7-deaza-2′-deoxyadenosine, Seela et al. prepared a nucleoside hydrogel of 355 at a concentration of 0.3 wt % in water. SEM reveals that 355 self-assembles to form nanotubes.⁸³⁷ Abet et al. recently reviewed guanosine and isoguanosine derivatives (356 and 357) that self-assemble in water.⁸³⁸ This review provides useful information on different self-assembled architectures generated by guanosine and isoguanosine scaffolds, including recent examples of their use in the preparation of functional devices. In another work, Nachtsheim et al. reported N-(uracil-5-ylmethyl)urea (358) as a minimalistic hydrogelator which undergoes phosphate-induced self-assembly, as evidenced by IR, UV/vis, and NMR spectroscopy, electron microscopy, and rheological experiments. According to the authors, it is a rare example of an anion-triggered self-assembly in aqueous solution without additional aromatic or lipophilic groups. The macroscopic appearance of the hydrogels implies the formation of microcrystals as the gel matrixes.²⁵⁶ Adhikari and Kraatz et al. designed and synthesized a hydrogelator of deoxylguanosine (359) that affords a hydrogel with a CGC of 0.57 wt % in the presence of Ag⁺. One interesting feature of this work is that the cogel of 352 and 359, being injectable, exhibits enhanced stability, an extended lifetime, and self-healing properties.⁸³⁹ Xu et al. designed two nucleopeptides (360 and 361) that alone fail to form hydrogels, while the mixture of the two nucleopeptides self-assembles to form nanofibers and result in a hydrogel. One intriguing feature of this work is that the resulting heterodimer dramatically enhances the proteolytic stability of these nucleopeptides.⁸⁴⁰

4.5. Hydrogels Based on Saccharides

Bearing multiple hydroxyl groups, saccharides provide hydrogen bond donors and acceptors for intermolecular interactions that are critical for molecular self-assembly in water to result in hydrogelation. Moreover, the inherent hydrophilicity of saccharides allows the easy dissolution of the hydrogelators prior to the triggering of the hydrogelation by changing the pH, temperature, or ionic strength. In this section, we categorize the saccharide-based hydrogelators into monosaccharide-based and oligosaccharide-based hydrogelators (Table S4).

4.5.1. Monosaccharide-Based Hydrogelators

As shown in Scheme 46, Lu et al. designed and synthesized a hydrogelator (362) containing a phenyl β-d-glucopyranoside which self-assembles to form a tubular structure and results in a hydrogel at a concentration of 0.2 wt %.⁸⁴¹ They also prepared hydrogels based on β-d-glucopyranoside substituent thiosemicarbazide derivatives 363,^842,843 and used the hydrogels as templates for fabricating netlike CdS nanofibers. John and Shimizu et al. reported a series of low molecular weight gelators (364, 365) based on simple glycolipids which are capable of forming gels in a water/alcohol mixture at a concentration of 0.15 wt %. They found that the T_gel of 364 is higher than that of 365.⁸⁴⁴ Hamachi et al. developed a supramolecular hydrogelator (366) composed of N-acetyl-d-glucosamine which self-assembles to form nanofibers and results in a hydrogel with a CGC of 0.1 wt %.⁸⁴⁵ This work pioneered the use of a supramolecular hydrogel array to monitor enzymatic reactions. According to the authors, this work has potential applications in pharmaceutical research and diagnosis. Meanwhile, Hamachi et al. also developed a photoresponsive gel droplet based on the hydrogelator 367 and demonstrated the use of light to trigger mass transport in gels. The authors suggested this type of hydrogel as an intelligent delivery system.⁸⁴⁶ Moreover, their further studies found that 366, mixed with an appropriate amount of 368 (e.g., 366:368 = 1:1), affords a pH-responsive shrinkage/swelling supramolecular hydrogel at a concentration of 0.5 wt %.⁸⁴⁷ Particularly, the authors envisioned that this approach is useful for triggering the release of hydrophilic drugs.

Scheme 46. Some Monosaccharide-Based Hydrogelators.

Bhattacharya et al. reported an intriguing example in which a tetrameric d-xylofuranuronic acid derivative containing an azobenzene core (369), assisted by initial dissolution in a small amount of DMSO, exhibits pronounced hydrogelation at 0.1 wt %. Since the hydrophobic azobenzene group is packed inside the gel state, the resulting hydrogel displays a remarkable photostability under UV irradiation. Moreover, the addition of a salt (e.g., CaCl₂) to the hydrogel changes the morphology of the networks in the hydrogel from globular spongy to “rodlike” fibers.⁸⁴⁸ Jung et al.⁸⁴⁹ reported a d-glucopyranoside-based hydrogelator (370) by incorporation of an unsaturated diacetylene unit as the hydrophobic group. After its self-assembly in water, 370 forms well-defined helical ribbons with diameters of 20–150 nm, which results in a hydrogel with a CGC of 0.5 wt %. Upon UV irradiation at 254 nm, 370 forms typical nanofibers. On the basis of CD spectra and other measurements, the authors suggested that photopolymerization turns the well-ordered bilayer structure into disordered molecular packing. Jung et al.⁸⁵⁰ also designed other hydrogelators (e.g., 371) which self-assemble to form fibrils with diameters of 10–38 nm. Meanwhile, the hydrogel based on 371 with crown ether can serve as a template to prepare various structures of silica nanomaterials.⁸⁵¹

By functionalization of a commercially available glucose derivative, Wang et al.^852,853 designed and prepared a series of d-glucose-based hydrogelators (372) (Scheme 47). Among them, 372 (n = 5, 12) exhibits an excellent gelation ability and affords a hydrogel at a concentration of 0.4 wt %. Further studies⁸⁵⁴ found that the conjugates containing a terminal acetylene and an aryl group exhibit an enhanced ability of gelation. One interesting feature of this work is that these gelators form birefringent fibers⁸⁵⁵ and tubules. By connecting the d-glucamine derivatives and hydrophobic unit via ureido or bis(ureido) moieties, Cintas et al.⁸⁵⁶ prepared a family of carbohydrate amphiphiles and bolaamphiphiles. The resulting molecules form hydrogels upon application of thermal or ultrasound stimuli. For example, 373 molecules afford a gel at room temperature under sonication. Wan et al.⁸⁵⁷ reported a new saccharide-appended hydrogelator of 4-(4′-butoxyphenyl)phenyl β-O-d-glucoside (374) which self-assembles to form planar ribbons with widths ranging from tens to thousands of nanometers and results in a hydrogel in water with a CGC of 0.5 wt %. Yamanaka et al.⁸⁵⁸ designed and synthesized a d-glucose-based hydrogelator (375) which self-assembles to form a hydrogel with a CGC of 0.25 wt % in the presence of Tris–glycine buffer. The intriguing feature of this work is that the hydrogel can serve as an electrophoresis matrix to separate the native protein. This underexplored application appears to have much potential.

Scheme 47. Some Monosaccharide-Based Hydrogelators.

As shown in Scheme 48, Takaguchi et al.⁸⁵⁹ reported a unique example of an anthracene-based photoresponsive hydrogelator (376) which self-assembles in water to afford a hydrogel with a CGC of 2.9 wt % and a T_gel of 46 °C. Upon photoirradiation, the hydrogel is transformed to a solution due to the dimerization of the anthryl moieties. Kameta and Shimizu et al.⁸⁶⁰ reported an intriguing example in which N-(β-d-glucopyranosyl)-N′-[2-(glycylglycylglycinamido)ethyl]octadecanediamide (377) self-assembles to form nanotubes with a 9 nm inner diameter and results in a hydrogel at a concentration of 0.5 wt % and pH 8.0. Notably, the fixed green fluorescent protein (GFP) and myoglobin (Mb) in the hollow cylinders of the nanotubes exhibit remarkable resistance to denaturants such as guanidinium chloride and urea at high concentration. Furthermore, Kameta et al.⁸⁶¹ also found that the nanotubes of this hydrogelator (377) are able to act as artificial chaperones to assist the transformation of encapsulated proteins into their refolded states via simply changing the pH values. The authors suggested that the modification of the diameter and inner surface of the nanotubes enhances the efficiency of encapsulation and refolding of the proteins.

Scheme 48. Monosaccharide-Based Hydrogelators.

Tritt-Goc et al.⁸⁶² prepared an α-d-glucopyranoside-based hydrogel (378) at a concentration of 1.5 wt %. The authors measured the thermal properties of the resulting hydrogel and determined the gel–sol transition enthalpy as 43 kJ/mol. By conjugating pyrene and a glucose derivative, Fang et al.⁸⁶³ reported a superhydrogelator (379) which affords a hydrogel in water with a CGC of 0.07 wt %. Interestingly, 379 also gels many of the organic solvents tested. Zhang et al.⁸⁶⁴ designed and synthesized an α-d-glucopyranoside-based hydrogelator (380) containing an aldehyde group which forms a hydrogel with a CGC of 0.8 wt %. Due to the existence of aldehyde and acetal groups, the resulting hydrogel not only responds to the pH, but also reacts with cysteine, which may lead to a new approach to design smart delivery systems. Birchall and Edward et al.⁸⁶⁵ reported a class of supramolecular hydrogels (381 and 382) derived from glucosamine and Fmoc. Notably, the authors suggested that CH−π interaction, rather than π–π stacking and H-bonding, drives the self-assembly and subsequently hydrogelation. Pfannemuller and Welte et al. reported that N-octyl-d-gluconamide (383) is able to form a hydrogel which is converted to crystallites over a few hours.⁸⁶⁶ By adding a nonionic surfactant (e.g., polyethylene glycol (PEG)), Rowan et al.⁸⁶⁷ elucidated not only that the resulting hydrogel is stable for more than one year, but also that changing the ratios of the components in the gel allows systematic tuning of the thermomechanical properties of the hydrogels. One notable result is that hydrogelator 383 exhibits potent activity to inhibit ice crystal formation at a concentration of 0.5 mM.⁸⁶⁸

Ikeda and Hamachi et al.^43,869 reported an intriguing example in which a well-designed bolaamphiphile (384) forms a hydrogel via “retro-Diels–Alder” reaction induced by heat. The authors used TEM to show the morphology transition from a twisted ribbon to a helical ribbon, and suggested that this simple and versatile molecular design should produce smart materials for various applications. Altenbach et al.⁸⁷⁰ designed and synthesized a d-glucose-based hydrogelator (385) via two simple steps. The resulting hydrogelator, acting as a surfactant and emulsifier, affords a hydrogel with a CGC of 2.5 wt %. Hamachi et al.⁸⁷¹ prepared a glycolipid-based supramolecular hydrogelator (386) that forms a hydrogel with a CGC of 0.1 wt %. The most interesting feature of this work is that the resulting hydrogel exhibits a color change when heated or upon the addition of relevant glycosidases to induce a gel–sol transition. Employing click chemistry, Mishra and Rao et al.⁸⁷² reported a glucose-based lipid (387) that forms a hydrogel with a CGC of 0.03 wt % in a mixture of water and methanol (50:50). Noto et al.⁴⁴⁰ designed and synthesized a molecule (388) which affords a hydrogel with a CGC of 1.0 wt % in the presence of α-cyclodextrin. Meanwhile, simply changing the ratio of cyclodextrin and 388 can easily tune the gelation properties.

4.5.2. Oligosaccharide-Based Hydrogelators

As shown in Scheme 49, Britt et al. prepared the conjugates 389 and 390 of lactose and fatty amine or fatty acid, which act as gelators and afford gels in a mixture of water and propanol (50:50).⁸⁷³ Thompson et al.⁸⁷⁴ designed and synthesized a series of α-cyclodextrin–aldonamide conjugates. Upon the addition of glucose, the solution of hexaaldonamide-substituted α-cyclodextrin turns into a hydrogel. It was suggested to be useful in developing glucose sensors and glucose-sensitive drug delivery systems.

Scheme 49. Some Oligosaccharide-Based Hydrogelators.

Oriol et al.^875,876 designed and synthesized a class of maltose-based supramolecular hydrogelators (391–393) via click chemistry. 391 and 393 self-assemble in water to form typical ribbons with a left-handed twist and result in hydrogels with a CGC of 1.0 wt %, while 392 forms ribbons with a right-handed twist and affords a hydrogel with a CGG of 0.5 wt %. By incorporating azobenzene into the gelator 391, Oriol et al.⁸⁷⁷ also prepared another hydrogelator (394) that forms a stable hydrogel with a CGC of 5.0 wt %. Interestingly, CD reveals that UV irradiation is unable to induce cis–trans isomerization of azobenzene. The authors suggested that the dense packing of azobenzene in the gel state hinders the photoisomerization, which may serve as a useful caution for designing photomechanical actuators based on gels. Kirimura et al. synthesized a maltoside-based hydrogelator (395) via an enzymatic reaction.⁸⁷⁸ The resulting hydrogelator is able to gel water at a concentration of 3.0 wt % and 12 °C. It is worth noting that its stereoisomers fail to form a hydrogel. Mathiselvam et al. prepared a family of urea–glycolipid-based hydrogelators (396 and 397) and found that 396 forms a hydrogel at a concentration of 0.5 wt %, while its stereoisomer 397 fails to form a hydrogel.⁸⁷⁹ This observation underscores that the orientation of the hydroxyl group in the saccharide has a profound influence on the self-assembly of this class of hydrogelators.

5. Applications

In recent years, on the basis of the increased understanding of protein functions from cell biology and structural biology, considerable efforts have focused on the incorporation of peptide epitopes as the functional motifs on supramolecular hydrogelators for a wider range of biological applications.^880−882 These endevors also stimulated the determination of the protein targets of supramolecular hydrogels.^883,884 In this section, we mainly focus on the recent advances in the design and development of supramolecular hydrogels for biological and biomedical applications. We discuss the different types of hydrogels and highlight some representative applications. Since most of the biomedical applications demand multiple functionalities of the hydrogel network and dynamic interactions between the surrounding matrixes and cells, we first discuss hydrogelators for cell-related applications, followed by fluorescent hydrogelators used for imaging, and then hydrogels for tissue engineering, drug delivery, immunomodulation, and wound healing. We finally describe the unique applications of supramolecular hydrogels and hydrogelators in a cell environment.

5.1. Cell-Related Applications

Because the most obvious features of hydrogels are soft and wet, which resemble the cellular environment, it is not surprising that the most attempted applications of the hydrogels are to mimic the ECM for cell culture (or tissue engineering), and the necessary initial test for a hydrogelator is its cell compatibility.^29,885 Since SPPS allows oligomeric peptides to be made quickly, most of the hydrogelators examined for cell-related applications are self-assembling peptides or peptide derivatives. In the following subsections, we discuss various hydrogelators that have been evaluated for the applications related to cells,^886−888 such as hydrogelators for cell culture,^889,890 cell-compatible hydrogelators,^891,892 cytotoxic hydrogelators,^893,894 and hydrogels for cell adhesion.

5.1.1. Three-Dimensional Cell Culture

Ulijn et al. reported the first case of cell culture using Fmoc-dipeptides. As first reported by Xu et al., the hydrogel of Fmoc-Gly-Gly-OH (398; Scheme 50) consists of nanofibers with average diameters of 33 nm and exhibits a CGC of 0.15 wt % at pH < 4.¹¹ Later, Ulijn used the hydrogel of a mixture of 398 and Fmoc-Phe-Phe-OH (6) for the 2D or 3D cell culture.⁹⁶ In addition, at pH 7, 6 itself forms hydrogels with a concentration between 0.22 and 2.14 wt % which contain polydispersed nanofibers with average diameters of 56 nm. On the basi sof the fact that the Fmoc-dipeptide building blocks are approximately 2 nm in length, the authors suggested that the nanofibers consist of bundles of supramolecular stacks. Although the results of cell viability indicate that 6 shows a relatively high cytotoxicity to bovine chondrocyte cells or Caco-2 and HGF-1 cells⁷⁰⁵ after 7 days, Ulijn et al. demonstrated that 6 can still be applied to 2D and 3D cell culture. According to the authors, the hydrogel is stable under cell culture conditions and consists of nanofibers that have dimensions similar to those of the fibrous components of the ECM.⁹⁶ Besides 6, Ulijn et al. also studied two other diphenylalanine analogues, Nap (naphthalene)-Phe-Phe-OH (3)¹⁴ and Cbz ((benzyloxy)carbonyl)-Phe-Phe-OH (399), and compared their self-assembly properties and cell culture applications with those of 6. After demonstrating that all three hydrogelators form hydrogels consisting of nanofibers with β-sheet arrangements and varying fibril dimensions, the authors used LDH (lactate dehydrogenase) assays to prove that all three structures can support cell proliferation and cell culture of chondrocytes in both two and three dimensions for up to 10 days.⁸⁹⁵ On the basis of the early work of Ulijn et al., Liebmann et al. evaluated the hydrogel of 6 as 3D cultures of COS-7 and MDCK cells for 7 days.⁸⁹⁶ However, 6 still has limitations, especially in terms of long-term gel performance, stability, and cytotoxicity when being used for culturing other cell types (e.g., skin cells such as human dermal fibroblasts and mouse 3T3 cells). Thus, Ulijn et al. mixed 6 with positively charged Fmoc-Lys-OH (202), uncharged/polar Fmoc-Ser-OH (400), and negatively charged Fmoc-Glu-OH (203) in the same hydrogel for examining the proliferation of chondrocytes, 3T3, and human dermal fibroblast (HDF) cells.⁸⁹⁷ Besides confirming that these heterotypic hydrogelators undergo self-assembly to form fibrous scaffolds by mainly adopting an antiparallel β-sheet arrangement, the authors used the LIVE/DEAD staining assay to show that these three types of mixed hydrogels maintain the viability of bovine chondrocytes. The hydrogel of 6 + 400 (Fmoc-FF/Fmoc-S) and the hydrogel of 6 + 203 (Fmoc-FF/Fmoc-E) are compatible with HDF cells, and only the hydrogel of 6 + 400 (Fmoc-FF/Fmoc-S) supports the proliferation of 3T3 fibroblast cells.^898,899

Scheme 50. Representative Molecular Structures of Hydrogelators for 3D Cell Culture.

Besides the above three Fmoc-peptide mixtures, Ulijn et al. designed another hydrogel, a mixture of 6 and Fmoc-Arg-Gly-Asp-OH (Fmoc-RGD, 401), as a 3D scaffold for HDF cells. They found that this mixed hydrogel provides a highly hydrated, stiff nanofiber network with β-sheets interlocked by π–π stacking of the Fmoc groups. The authors suggested that the RGD motif plays a dual role: as a structural component that locates at the surface of the unique, interwoven cylindrical nanofiber structure and as a biological ligand that forms the specific RGD–integrin binding to promote adhesion, spreading, and proliferation of cells.⁹⁰⁰ Using a similar design principle, Hamley et al. found that 401 itself forms a self-supporting hydrogel consisting of well-defined amyloid fibrils with β-sheet features at a concentration of 2 wt %. In addition, the preliminary cell culture experiments showed that 401 can be used to culture bovine fibroblasts.⁹⁰¹

On the basis of the earlier report of a hydrogel made of a hexadecapeptide (RADA16,^181,902248a), Hirose et al. used the 248a self-assembling peptide solution (PuraMatrix)^903−906 to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs) that are derived from rat bone marrow. The authors reported that over 80% of the MSCs in the hydrogel are alive and have spread within the hydrogel of 248a, and suggested that 248a acts as a scaffold for three-dimensional culture of MSCs. The authors observed a significantly higher expression of alkaline phosphatase (ALP) activity and osteocalcin (OC) contents at both the protein and mRNA levels for 3 or 4 weeks, and thus concluded that MSCs in the 248a hydrogel differentiate into mature osteoblasts, followed by the growth of a mineralized extracellular matrix. Although it is suggested that the biodegradable/biocompatible hydrogel 248a may become an attractive option in bone tissue engineering,⁹⁰⁷ the complexity of the bone remolding and growth process likely requires more than one component in the hydrogel.⁹⁰⁸ The commercial availability of 248a allows many research laboratories to evaluate the use of the hydrogel of 248a for 3D cell culture.^909−911 For example, Zhao et al. studied the cellular behavior of human lung cancer cells A549 within a 248a nanofiber scaffold. They found that the cells show morphologies in a 3D scaffold different from those on a 2D Petri dish, an observation that is consistent with RADA being a cell adhesion motif.⁹¹² Xie et al. mixed 248a and RGDA16 (Ac-RADARGDARADARGDA-CONH₂, 402) solutions at a concentration of 10 mg/mL (1%, w/v) and found that the mixture scaffold can significantly promote the cell attachment and proliferation of MC3T3-E1 cells compared with the 248a scaffold.⁹¹³ Semino et al. used the hydrogel of 248a functionalized with biologically active motifs (e.g., GRGDSP, 403, or YIGSR, 404) to replace the use of collagen I in the traditional culture sandwich technique for maintaining functional hepatocytes in vitro.⁹¹⁴ Moreover, Wang et al. used the mixture of the peptide solutions of RLN (405) and 248a to guide rabbit nucleus pulposus cells (NPCs), and demonstrated that NPCs migrate from the surface into the hydrogel in the 3D cell culture experiments and exhibit stimulated synthesis of the ECM.⁹¹⁵ Narmoneva et al. used RAD16 (406) peptide nanofibers for vascular tissue engineering. They reported enhanced angiogenesis in vitro and in vivo, and suggested that the observation results from low-affinity integrin-dependent interactions of microvascular endothelial cells (MVECs) with the RAD motifs.⁹¹⁶ These results imply that the development of multicomponent hydrogels may address several limitations of single-component hydrogels. Using 406 to form a hydrogel, Urtti et al. established 3D hepatic cell cultures in the hydrogel to improve the 3D phenotype of Hep G2 cells, a human liver carcinoma cell. The authors reported that Hep G2 cells formed multicellular spheroids which consist of filamentous actin accumulation and large tubular bile canalicular structures to indicate apicobasal cell polarity.⁹¹⁷

Realizing the merits of multicomponent hydrogels, Collier et al. designed and examined multipeptide coassembling hydrogels based on peptides RGDS-Q11 (408) and IKVAV-Q11 (409) consisting of two segments: a nanofiber-forming peptide, Q11 (407), which self-assembles to form a β-sheet, at the C-terminal and a ligand of integrins, RGDS or IKVAV, at the N-terminal. The authors suggested that such a design allows the ligands to be presented on the surface of the nanofibers. In coassemblies of the ligand-bearing peptides containing 407, the amount of the incorporated ligands is able to influence the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) without significantly altering the materials’ properties, such as fibrillization, β-turn secondary structure, or stiffness. The authors reported that while 408, being coassembled into the gels of 407, specifically increases HUVEC attachment, spreading, and growth, 409 exerts a more subtle influence on the attachment and morphology of the cells. Additionally, they reported that 407 and 408 are minimally immunogenic in mice, making the 407-based biomaterials attractive candidates for applications in vivo.⁹¹⁹ However, the proteolytic stability of these peptides remains to be established. Recently, on the basis of the sensitivity of 407 to the ionic strength, Collier et al. developed a microgel made of RGD-Q11 (410) by triggering peptide self-assembly within the aqueous phase of water-in-oil emulsions. According to the authors, one of the advantages of microgels is that they can be embedded within other self-assembled peptide matrixes for generating composites of different peptide formulations. The authors, indeed, demonstrated an example of microgels that are cytocompatible and encapsulate NIH/3T3 fibroblasts (Figure 8) and C3H10T-1/2 mouse pluripotent stem cells with good viability.⁹¹⁸

Figure 8.

The viability of NIH/3T3 cells encapsulated in 30 mM 407 microgels was quantified with calcein/ethidium homodimer staining. The assay was conducted 2 h after the incubation of (a) 1 day, (b) 2 days, and (c) 3 days. The scale bar in (a) represents 100 mm. The magnification is the same in (a)–(c). Adapted with permission from ref (918). Copyright 2011 Royal Society of Chemistry.

On the basis of their seminal works of the applications of peptide amphiphiles for cell cultures,^{557,920−922} Stupp et al. designed and synthesized a peptide amphiphile molecule, 411 (Scheme 51), containing both the photocleavable 2-nitrobenzyl group as well as the bioactive epitope Arg-Gly-Asp-Ser (RGDS). The 2-nitrobenzyl group of 411 can be photocleaved to afford 412, which self-assembles to form high-aspect-ratio nanofibers in the presence of charge-screening salts. In vitro experiments with NIH/3T3 mouse fibroblasts indicate that 412 and the byproducts of the photoreaction at a concentration of 7.9 × 10^–3 M are not toxic to the cells and that cell proliferation is normal after the irradiation.⁹²³ In another case, Stupp et al. reported a photoresponsive, synthetic ECM mimic through linking peptide amphiphiles 413 to the ECM-derived cell adhesion epitope RGDS by a photocleavable nitrobenzyl ester group. This derivative self-assembles to form cylindrical nanofibers, and light irradiation on the photolabile group in the peptide backbone efficiently removes the RGDS epitopes without affecting the nanofibers. The authors demonstrated that the adhesion of mouse NIH/3T3 fibroblast cells on the surface of peptide amphiphile (PA) hydrogels can be dynamically controlled by rapid photolytic removal of the RGDS peptide from the supramolecular nanofibers.⁹²⁴

Scheme 51. Representative Molecular Structures of Hydrogelators for 3D Cell Culture.

As shown in Scheme 52, Schneider and Pochan et al. reported a hydrogel based on TSS1 (414), a de novo designed three-stranded β-sheet. 414, containing 29 amino acids with 12 lysine residues and 12 valine residues, undergoes thermally triggered folding and self-assembly to afford a network of well-ordered β-sheet-rich fibrils that constitute a mechanically rigid hydrogel. A gelation test indicated that 414 remains unfolded at lower temperatures but folds and self-assembles into rigid hydrogels upon raising the temperature of the aqueous solutions (pH 9.0 or 7.4 (150 mM NaCl)) of 414. TEM images and SANS show that 414 self-assembles into monodispersed fibrils with a width of around 3 nm, which corresponds to the width of the peptide in its folded state. The authors demonstrated the in vitro culture of C3H10t1/2 mesenchymal stem cells on the gel surface for 24 h, and suggested that the surface of the hydrogel supports cell adhesion and allows cell migration.⁹²⁵ Schneider and Pochan et al. developed a class of self-assembling β-hairpin peptides^188,608,926 to create physical hydrogels as injectable therapeutic delivery vehicles. On the basis of their works on peptide hydrogels, the authors studied the behavior of β-hairpin peptide-based hydrogels Max1 (251) and Max8 (257) during and after flow. Importantly, the authors verified that the observed shear-thinning and rehealing, after flow, represent the authentic bulk gel properties. In another experiment, the author utilized the hydrogel of 257 (at a concentration of 0.5 wt %) to encapsulate MG63 cells, a progenitor osteoblast cell line from rat. However, 3 h after injection, some cells were already dead in the 3D gel–cell construct.⁹²⁷ A scaffold with biocompatibity and in vivo stability needs to be designed in the future.

Scheme 52. Representative Molecular Structures of Hydrogelators for 3D Cell Culture.

Conjugating peptide epitope IKVAV from laminin to a peptide amphiphile, C₁₆H₃₁O-A₃G₄D₂ (415), Song et al. generated peptide amphiphile C₁₆H₃₁O-A₃G₄D₂IKVAV, 416,⁹²⁸ which is similar to the IKVAV peptide amphiphiles reported by Stupp et al.⁵⁵⁷ After observing that a 1 wt % concentration of the peptide amphiphile self-assembles to form a hydrogel in cell media, the authors investigated 2D⁹²⁹ and 3D⁹³⁰ culture of neural stem cells (NSCs) using the hydrogel, and found that mice NSCs proliferate and differentiate into neurofilament (NF)-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes on the surface of the hydrogel. Zheng et al. synthesized a peptide with the sequence of KLD-12 (417) and found that 417, at 0.5 wt %, self-assembles to produce a hydrogel consisting of nanofibers (diameters of 30–40 nm). The authors reported that rabbit MSCs, being encapsulated within the hydrogel of 417 for 3D culture for 2 weeks, grow well and proliferate with the culture time.⁹³¹ Kim et al. reported that MSCs encapsulated in the hydrogel of 417 decelerate the progression of cartilage destruction in osteoarthritis in a rat knee model. The authors suggested that the beneficial effect may result from the prevention of chondrocyte apoptosis, the alteration of the subchondral bone mineral density, a reduction of inflammation, and a potential chondrogenic mechanism.⁹³² Gelain et al. developed a co-assembly of peptides, which are analogs of 417, for culturing neuronal cells.⁹³³

Hartgerink et al. designed and synthesized a series of amphiphilic multidomain peptides (MDPs; 418, 419, and 420) with an innovative modular ABA block motif in which the amphiphilic B block drives self-assembly and the flanking A blocks bear charges for controlling the conditions of self-assembly. In their peptide design, the authors created four different variants with a matrix metalloprotease 2 (MMP-2)-specific cleavage motif, an RGDS adhesion sequence, and either one or two lysine residues in the flanking regions. The lyophilized peptides self-assemble to form hydrogels after being dissolved in a sucrose solution. With a final peptide concentration of 1.0 wt %, the hydrogels consist of β-sheet fibrils formed by the cross-linking of lysine-containing peptides due to the presence of negatively charged phosphate ions in the buffer. The authors illustrated that the structures of the peptides control the lengths and diameters of self-assembled nanofibers, the gelation conditions, and the viscoelastic properties of the formed hydrogels, which highlights the promises of this approach for materials and biological applications. More interestingly, in an in vitro experiment with mesenchymal stem cells from human exfoliated deciduous teeth (SHED), the authors demonstrated that the incorporation of an MMP-2-specific cleavage site and a cell adhesion motif increases the cell viability, cell spreading, and cell migration into the hydrogel matrix.⁹³⁵ Later, the authors designed another MDP, E₂(SL)₆E₂GRGDS (421), which self-assembles to form β-sheet nanofibers approximately 8 nm wide, 2 nm high, and micrometers in length in the presence of Mg²⁺. The corresponding hydrogel undergoes shear thinning and recovers nearly 100% of its elastic modulus after shearing, making it ideal for being used as an injectable material. Interestingly, in the in vitro experiments with human embryonic stem cells (ESCs), the hydrogel acts like a sponge, soaking up most of the growth factors and cytokines released by the ESCs. Using in vivo experiments, the authors demonstrated a promising application of the hydrogel—as a depot to release stem cell secretome gradually over time.⁹³⁶ By changing the serine residues in the amphiphilic region to threonine, Hartgerink et al. also designed another MDP, K(TL)₂SLRG(TL)₃KGRGDS (422), which forms porous hydrogels with antiparallel β-sheet nanofibers. The authors also used this hydrogel to encapsulate the SHED cells (Figure 9), and observed more fibroblast-shaped cells after 7 days in culture.⁹³⁴ Recently, they also demonstrated the angiogenic properties of the MDP hydrogels.⁹³⁷

Figure 9.

Confocal microscopy of SHED cells 1, 3, or 11 days after 3D encapsulation in 422 hydrogels. Adapted from ref (934). Copyright 2014 American Chemical Society.

Aggeli et al. reported a class of positively charged tape-forming and gel-forming amphiphilic peptides in physiological solutions, all of which bear 2 positive charges and 11 amino acid residues. By changing the peptides to be amphiphilic or completely polar, they systematically synthesized several derived peptides. Each of them has a different polar uncharged group: P₁₁-8 (423, based on glutamine Q, sequence Ac-QQRFOWOFEQQ-NH₂; O represents ornithine), P₁₁-12 (424, based on serine S, sequence Ac-SSRFOWOFESS- NH₂), P₁₁-16 (427, based on asparagine N, sequence Ac-NNRFOWOFENN- NH₂), and P₁₁-18 (428, based on threonine T, sequence Ac-TTRFOWOFETT- NH₂). They found that all of these amphiphilic peptides carrying a +2 charge at neutral pH form self-supporting gels at concentrations above 25 mg/mL (ca. 1.8%, w/v) in physiological solutions. All these hydrogels contain a network of semiflexible, micrometer long nanofibers. In addition, all of these self-assembling peptide hydrogels show biocompatibility with L929 murine fibroblast cells, on the basis of the contact cytotoxicity test. However, only 423 supports L929 cell growth in 3D cell cultures inside 2% (w/v) gels for 14 days without observation of macroscopic degradation of the peptide gel matrix during the experiment, while the other peptides are unable to support cell growth.⁹³⁸ Later, Aggeli and Ingham et al. designed complementary self-assembling peptides comprising the unimers P₁₁-13 (425) and P₁₁-14 (426), which exhibit negative and positive charges, respectively, under physiological conditions. Being mixed in equal quantities, they instantaneously form a self-supporting hydrogel that consists of long fibrils with widths ranging from 10 to 20 nm. Although the hydrogels of 425 + 426 appear to be cytocompatible with primary human dermal fibroblasts, they fail to support the proliferation of this cell type, and the cell numbers began to decline after 7 days.⁹³⁹

As shown in Scheme 53, Yang et al. reported several supramolecular hydrogels based on adamantane-derivatized peptides that respond to the presence of β-cyclodextrin (β-CD, 155) motifs. The authors used dithiothreitol (DTT) or GSH to reduce the disulfide bond in the precursors (429) to convert the solutions to hydrogels for encapsulations of cells and drugs. Since the hydrogel is transparent and stable over months and undergoes a gel–sol transition upon the addition of 155-containing molecules, the authors applied them for cell culture and postculture cell recovery in an in vitro experiment of mouse fibroblast 3T3 cells.⁹⁴⁰ Later, Yang et al. designed several other hydrogels formed via GSH reduction, and demonstrated the use of the concentrations and structures of the hydrogelators to regulate the mechanical properties and ζ potential of the hydrogels. Among these hydrogels, 430, having a storage modulus (G′) of hundreds of pascals, is suitable for 3T3 cell spreading and proliferation.⁹⁴¹ In addition, the authors also reported the formation of 431, a curcumin-based hydrogelator, after disulfide bond reduction, to inhibit cancer cells and tumor growth in vitro and in vivo.⁹⁴² Taking advantage of the self-assembling ability of Phe-Phe, Yang et al. have developed useful hydrogels made of D-peptides as potential adjuvants for HIV vaccine⁹⁴³ or for self-assembling on cell surface.⁹⁴⁴

Scheme 53. Representative Molecular Structures of Hydrogelators for 3D Cell Culture.

As shown in Scheme 54, Zhang et al. reported an intriguing example tin which a hydrogel of dipeptide Tet-GA⁹⁴⁵ (432) serves two functions: as the medium for 3D cell culture and as the carrier for the delivery of miRNA into live cells. The authors found that 432 is able to form a transparent and stable hydrogel at concentrations higher than 0.15 wt % in PBS buffer. After confirming the cell compatibility of the hydrogelator, the authors used repressed target gene expression in an in vitro experiment to indicate the delivery of the miRNA, encapsulated together with cells in the hydrogel matrix, into the encapsulated cells. It would be important to elucidate the mechanism of the observed delivery. Luo et al. reported a hydrogel made of a d-form peptide, d-EAK16 (433), for 3D cell cultures. After confirming the proteolytic resistance of the d-peptide, the authors used the hydrogel of 433 for 3D cell culture and reported the human hepatoma cell SMMC-7721 to show a high cell viability and low-level cell apoptosis for weeks in the hydrogel.⁹⁴⁶ Later, Li and Ding et al. reported an EFK8-based small peptide, Ac-FEFKFEFK-CS-EEE (434), that self-assembles to form a hydrogel via disulfide bond reduction with a concentration of 0.5 wt % at physiological pH. Using the LIVE/DEAD assay, the authors demonstrated that the hydrogel of 434 is suitable for the 3D cell culture of NIH/3T3 cells.⁹⁴⁷ Furthermore, Saiani et al. reported the use of thermolysin, a protease, to trigger the gelation of FEFEFKFK octapeptide (228), which is able to encapsulate human dermal fibroblast cells for 3D cell culture for 5 days.⁹⁴⁸ By replacing the alanine residue in EAK16 (435) with a more hydrophobic residue, leucine, Lu et al. designed a new peptide, ELK8 (436), and directly attached three kinds of functional motifs (e.g., an osteogenic growth peptide, an osteopontin cell adhesion motif, and a two-unit RGD binding sequence) to the C-terminal of 436. Using in vitro experiments, several labs illustrated that the mixtures of these peptides (228, 435, and 436) are suitable for the 3D cell culture of mouse preosteoblast MC3T3-E1 cells and promote the attachment, proliferation, and osteogenic differentiation of those cells.^949,950

Scheme 54. Representative Molecular Structures of Hydrogelators for 3D Cell Culture.

Hamachi et al. designed a series of glycolipid mimics with muconic amide as the spacer and found that 437, consisting of N-acetylglucosamine as its hydrophilic head and methylcyclohexyl groups as hydrophobic tails, forms a stable hydrogel with a CGC of 0.05 wt %. Besides using TEM to elucidate that hydrogelator 437 self-assembles into a helical, bilayer-type nanofiber with a well-defined network of nanofibers of a high aspect ratio, the authors unexpectedly found that polystyrene nanobeads (100–500 nm in diameter) greatly facilitate the homogeneous 3D dispersion of the supramolecular nanofiber networks. Since 437 also forms a stable hydrogel in cell culture media, such as RPMI1641 or Dulbecco’s modified Eagle’s medium (DMEM), the resultant hybrid supramolecular matrix efficiently encapsulates and distributes live Jurkat cells (a human T cell lymphoblast-like cell line) in 3D cell culture under physiological conditions.⁹⁵¹ Recently, Hamachi et al. also reported photoresponsive hydrogels for controlling cell motions⁹⁵² and chemically responsive hydrogels for enhancing analyte sensitivity.⁹⁵³

5.1.2. Cell-Compatible Hydrogelators

Chou et al. proposed a simple and economical methodology to synthesize dimeric cholesterol derivatives (DCDs) with high yields. The dynamic light scattering analysis and TEM images showed that in aqueous solution most DCD dispersions are irregularly angular shaped with two peaks in the size distribution centered at 204 and 837 nm. In addition, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based cell viability assay indicated that 438 (Scheme 55) is innocuous to human keratinocyte (HaCaT) and squamous cell carcinoma (SCC25) cells at a concentration of 0.05 mM after 24 h incubation with 438.⁹⁵⁴ As a representative example of the de novo β-hairpin peptides, 251 folds as a β-hairpin peptide upon the addition of saline solution, and the β-hairpin peptide self-assembles to form a hydrogel at 2 wt %. Thus, DMEM cell culture media initiate the folding and consequent self-assembly of 251 to afford a hydrogel which is cytocompatible with NIH/3T3 cells.^955,956

Scheme 55. Representative Molecular Structures of Cell-Compatible Hydrogelators.

Woolfson et al. reported supramolecular hydrogels formed by rationally designed standard linear peptides (439–444). Consisting of two 28-residue peptides designed to coassemble, the pair of peptides results in an offset α-helical dimer with complementary sticky ends which promote longitudinal assembly into α-helical coiled-coil fibrils bundling to form nanofibers. On the basis of the coiled-coil heptad repeat that is rich in alanine (for hydrophobic interaction) and glutamine (for hydrogen bonding) residues, the authors demonstrated that the glutamine-rich peptide (441 + 442) forms a gel at low temperature which melts on warming, whereas the alanine-rich peptide (439 + 440) forms a weak gel at low temperature that strengthens on warming. By replacing one of the surface-exposed alanine residues with the more hydrophobic tryptophan, the authors obtained 443 and 444, which form hydrogels that support the proliferation and differentiation of rat adrenal pheochromocytoma (PC12) cells.^632,957 Gazit et al. also extended the family of the aromatic Fmoc-dipeptides with a series of new Fmoc-peptides which consist of natural and synthetic amino acids with an aromatic nature for making supramolecular hydrogels. With the assistance of DMSO as the cosolvent, the authors produced the hydrogels at a final peptide concentration of 0.5 wt %. TEM and SEM analysis indicated that the self-assembly of these Fmoc-peptides results in various structures and distinctive molecular and physical properties. A pair of notable peptides in their work are Fmoc-FRGD (445) and Fmoc-RGDF (446), which self-assemble to form β-sheet-based nanofibers. Using the MTT assay, the authors demonstrated that Chinese hamster ovary (CHO) cells on the hydrogels of 445 and 446 show a high viability after 24 h. However, the cell viability decreases significantly at 72 h.⁹⁵⁸

Palocci et al. used a lipase to trigger the self-assembly of peptide hydrogels of Fmoc-FFF (447) via reverse hydrolysis to control or modulate the functions and responses of the hydrogels according to their preparation conditions. Under physiological conditions, the authors obtained amphiphilic building blocks consisting of tripeptides (Phe-Phe-Phe) linked to Fmoc. SEM and AFM images indicated that the hydrogels of 447 consist of a nanofiber network at 0.14 wt %.¹⁶⁹ On the basis of the cell viability of rat microglial cells incubated with 447 at concentrations up to 300 μg/mL, the authors suggested that the hydrogelator is biocompatible. Later, the authors reported that the hydrogelator also stimulates the production of neurotrophic factor NGF (nerve growth factor) from the microglial cells.⁹⁵⁹ Liu et al. reported a hydrolase model based on the nanotubes formed by the self-assembly of a synthetic Fmoc amphiphilic short peptide (Fmoc-FFH, 448). According to the authors, the imdazolyl groups on the surface of the nanotubes act as the catalytic centers for the hydrolysis of p-nitrophenyl acetate (PNPA). Replacing the histidine of 448 with arginine, the authors produced a structurally similar peptide, Fmoc-FFR (449), the guanidyl groups of which reside in the nanotubes through the coassembly of these two molecules to stabilize the transition state of the hydrolysis. The authors also reported that this model of peptide hydrolase is compatible with HeLa cells and suggested the applications of these peptides as a substitute for natural hydrolases.⁹⁶⁰ Liu et al. synthesized an FGL pepitide amphiphile (450) that self-assembles to form nanofibers (10–20 nm) as the scaffold for NSCs. Besides self-assembling to form a hydrogel at a CGC of 1 wt %, 450 at a concentration of 50, 100, or 200 mg/L promotes the proliferation of NSCs, which agrees with the NCAM mimietic properties of the FGL peptide.^961,962 Meanwhile, the authors found that the nanofibers of 450 increase the rate of neuron differentiation from NSCs and concluded that the self-assembled nanofibers of 450 have good biocompatibility with NSCs.⁹⁶³

As shown in Scheme 56, Xu et al. designed a series of hydrogelators based on the conjugates of a dipeptide and (naphthalen-2-yloxy)acetic acid. Among these hydrogelators, Nap-Gly-d-Ala (451) and Nap-Gly-Ala (452) form hydrogels efficiently with CGCs of 0.07 wt %. The hydrogen bonding between dipeptides and aromatic–aromatic interactions of the naphthyl groups cooperatively result in the excellent hydrogelation ability of these hydrogelators. Besides demonstrating that the handedness of the helical fibril structures in the hydrogels correlates with the chirality of the hydrogelators, the authors also found that these molecular hydrogelators are compatible with HeLa cells when the concentration of the hydrogelators is 200 μM and the incubation time is 24 h.⁹⁶⁴ Recently, Xu et al. reported a new class of hydrogelator (453 and 454) based on conjugates of nucleobases (e.g., thymine, adenine, cytosine, and guanine) and ultrashort peptides which self-assemble in water upon application of a pH or enzymatic stimulus to afford a new class of supramolecular hydrogels that are biocompatible (Figure 10). The studies on the gelation properties indicate that all these nucleopeptides self-assemble to generate β-sheet nanostructures at a concentration of 2 wt %. In addition, the hydrogelators also exhibit significant resistance to proteinase K, which makes them attractive materials for applications in vivo. Besides illustrating the first example of nucleopeptides as hydrogelators made by an enzymatic reaction, the approaches illustrated by the authors provide a facile way to explore the potential applications of nucleopeptides as soft biomaterials.⁹⁶⁵ One possible application may be kinase detection, as shown by Yang et al.⁹⁶⁶ Meanwhile, Xu et al. also developed simple conjugates of a nucleobase, amino acids, and a glycoside as a new class of supramolecular hydrogelators (455 and 456). Consisting of the three unified building blocks of life,⁹⁶⁷ these hydrogelators self-assemble in water to yield ordered nanostructures and supramolecular hydrogels at a concentration of 3 wt %. The conjugates not only exhibit exceptional biocompatibility and biostability, but also facilitate the entry of nucleic acids into the cytosol and nuclei of cells through interbase interactions with nucleic acids. In addition, the integration of a saccharide at the C-terminal into the hydrogelators significantly enhances their resistance to proteinase K,⁹⁶⁸ which greatly expands the use of this kind of hydrogel in vivo. As a facile way to generate a fundamentally new molecular architecture from the unified building blocks of life,⁹⁶⁷ this approach promises the development of sophisticated soft biomaterials from a rather simple pool of building blocks.

Scheme 56. Representative Molecular Structures of Cell-Compatible Hydrogelators.

Figure 10.

(A) Dephosphorylation process catalyzed by ALP with 454A to result in nanofibers and a hydrogel. (B) Cell viability test for 72 h of 454. (C) Optical images of HeLa cells on the surface 0 and 20 h after creation of scratchs in the presence of hydrogel 454T (by adding 27.7 mM 454T to the media). Adapted with permission from ref (965). Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

Encouraged by the self-assembly of the conjugates of a nucleobase, amino acids, and a glycoside, Xu et al. explored the biological functions of the self-assembly of this kind of conjugate. They engineered a multifunctional small molecule that consists of adenine (as an assembly domain), an Arg-Gly-Asp sequence (RGD, as a binding domain), and glycosamine (as a glycogen), and found that the assemblies of the conjugates (457) promote the proliferation of mES cells and the development of zygotes into blastocysts of mouse.⁹⁶⁹ In addition, they found that each module (i.e., nucleobase, RGD, and glycosamine) in the conjugate is indispensable for the observed functions according to the cell proliferation test of the “structural mutants” of 457. On the basis of this work, the authors suggested that the self-assembly of this kind of de novo glycoconjugate (457) promises a potential approach to use supramolecular assemblies as multifunctional mimics of glycoconjugates,⁹⁶⁹ including glycoproteins. Furthermore, Xu et al.^970,971 replaced the adenine of 457 with thymine, generating an analogue of 457 which belongs to an unprecedented type of small molecules that consist of unified building blocks of life.⁹⁶⁷ This analogue self-assembles in water to form nanofibrils and results in a hydrogel at a concentration of 3.0 wt %. One important observation of this work is that the glycoside at the C-terminal of the peptide greatly enhances the proteolytic resistance of RGD in the hydrogelator (457).

Later, Xu et al. synthesized another glycoconjugate, 458, and its analogues based on the three fundamental biological building blocks (i.e., saccharides, amino acids, and nucleobases) by SPPS. They found that all these conjugates were compatible with HeLa cells even at a concentration of 415 μg/mL.⁹⁷² Xu et al. reported the first hydrogelator (459) consisting of both proteinogenic amino acids (e.g., phenylalanine) and a nonproteinogenic amino acid (e.g., taurine) by attaching taurine at the C-terminal of a well-established self-assembly motif (3). The authors found that, besides the pH, the temperature and ultrasound affect the gelation behavior of 459 to result in different morphologies of the nanostructures. In addition, the MTT-based cell viability assay indicated that 459 is biocompatible with HeLa cells for 3 days at a concentration of 500 μM.⁸⁷ On the basis of the study of the nucleobase–amino acid–saccharide conjugates, Xu et al. also designed another kind of hydrogelator which is a nucleobase–saccharide–amino acid conjugate.⁹⁷³ Among all the hydrogelators, 460 forms a typical hydrogel (with a CGC of 0.8 wt % at pH 7.0) which turns into a solution at 59 °C or at pH above 9.0. This study illustrates the incorporation of l-3-(2-naphthyl)alanine as an effective strategy to promote molecular self-assembly in water. Furthermore, the addition of T₁₀ appears to result in a mechanically stronger hydrogel which consists of nanofibers with widths increasing from 7 ± 2 to 17 ± 2 nm. The in vitro experiments indicate that 460 is compatible with HeLa cells at concentrations up to 500 μM for 3 days.⁹⁷⁴

As shown in Scheme 57, Stupp et al. reported a peptide amphiphile (461) with its sequence (KRRASVAGK[C₁₂]-NH₂) containing the specific consensus substrate (RRXSO; X = any residue; O = hydrophobic) for protein kinase A (PKA), a ubiquitous kinase in intracellular signaling and metabolism that has also been demonstrated to be an extracellular cancer biomarker. 461 is able to form a hydrogel with a β-sheet secondary structure in the nanofibers, and its assembly and disassembly can be reversibly controlled by PKA. In addition, the authors suggested that the disassembly of the nanofibers of 461 by using PKA might contribute to an enzyme-triggered release of an encapsulated cancer drug. The authors also reported an in vitro experiment to show the peptides themselves to be compatible with cells while the drug-loaded nanofibers of 461 induce preferential cytotoxicity in a cancer cell line that is known to secrete high levels of PKA, such as the MDA-MB-231 human breast cancer cell line.⁹⁷⁵ Ryadnov et al. designed a self-assembling peptide (462) which contains two domains that oligomerize by forming a parallel coiled-coil heterodimer. In this arrangement, each domain pairs with its complementary partner from another copy of the same peptide, connected through two short linkers and cyclized antiparallel to each other such that interactions occur between different peptides. One unusual feature of 462 is that it forms hyperbranched fibrillar networks spanning from nano- to micrometer dimensions. Although this elaborately designed peptide is less effective than collagen for promoting the proliferation of human dermal fibroblast cells, the decoration of a cell attachment motif (e.g, a mimic of YIGSR) results in a 20% increase of cell proliferation compared with the bare scaffold.⁹⁷⁶

Scheme 57. Representative Molecular Structures of Cell-Compatible Hydrogelators.

Yu et al. designed four oligopeptides; the two positive sequences (l⁺ (463) and d⁺ (463)) contain alternating neutral (W and A) and positively charged (K) residues, while the two negative sequences (l^– (464) and d^– (464)) replace lysine by negatively charged glutamic acid (E). The oppositely charged oligopeptide modules can interact with each other electrostatically, coassemble, and form a hydrogel. According to the in vitro experiments reported by the authors, the l-homochiral hydrogels of 463 are the most cell compatible, leading to the highest human mesenchymal stem cell (hMSC) viability and proliferation, but the peptides are susceptible to proteases. However, the d-oligopeptide hydrogels of 463, which resist proteases, are unable to support cell hMSC proliferation. The authors found that negative charges significantly improve hMSC growth in the d-oligopeptide hydrogels of 464 but have little effect on their interactions with the l-oligopeptide hydrogels of 464. This interesting observation indicates that negative charges can compensate for the disadvantage of the d-homochiral hydrogels.⁹⁷⁷ Gu et al. reported a hydrogelator connecting 7-(carboxylmethoxy)coumarin molecules and hydrazine linked by l-lysine (DCOU-Lys-CONH-NH₂, 465) to act as lipophilic and water-soluble moieties. 465 self-assembles to form hydrogels in distilled water with a CGC of 1 wt %. 465 starts to self-assemble into short fibrils even at concentrations lower than the CGC. In addition, ultrasound accelerates the gelation and induces homogeneous self-assembly to form nanofibers with average diameters between 30 and 40 nm (Figure 11). On the basis of a LIVE/DEAD assay, the authors reported that the hydrogel is preferable for the migration and proliferation of NIH/3T3 fibroblast cells.⁸⁵

Figure 11.

Formation mechanism of hydrogel 465: (A) hydrogen-bond-driven self-assembly, (B) self-assembled fibrils, (C) fibrils with a hydrogelator concentration lower than the minimum gelation concentration (MGC), (D) entangled fibrils with a hydrogelator concentration higher than the MGC, (D) well-organized 3D hierarchical nanoarchitectures with ultrasound treatment, (F) cells seeded in hydrogels, (G) optical image of the hydrogel (the transition from solution to hydrogel was reversible). Adapted with permission from ref (85). Copyright 2013 Royal Society of Chemistry.

Lin et al. have reported the detailed study of a new series of small molecular hydrogelators, among which the intramolecular alternative packing of the phenyl/perfluorophenyl pair promotes the formation of supramolecular nanofibers and hydrogels at pH 5 with a CGC of 1.0 wt %. The authors also reported that 466 is compatible with the CTX TNA2 cells in a concentration range of 10–500 μM for 48 h.⁹⁷⁸ The same lab recently reported a co-assembled hydrogel based on naphthalene diimide for treating MCF-7 cells.⁹⁷⁹ Marchesan et al. designed a series of uncapped hydrophobic heterochiral tripeptides with all combinations of d- and l-amino acids to minimize the disadvantages of l- or d-peptides. Rheology and XRD results indicated that, among all the heterochiral tripeptides, 467 forms a hydrogel with a β-sheet amyloid structure. According to the LIVE/DEAD assay, the authors showed that 467 maintains the viability and proliferation of L929 mouse fibroblast cells in vitro for 3 days.⁹⁸⁰ Taking advantage of click chemistry, Barthélémy et al. designed and synthesized two glycosyl-nucleoside fluorinated amphiphiles (GNFs, 468) which feature either β-d-glucopyranosyl or β-d-lactopyranosyl moieties linked to a thymine nucleobase. On the basis of the air–solution surface tension (γ) measurements, the authors reported that the critical aggregation concentrations (CACs) are 5.9 and 3.7 μM, respectively. Gelation tests indicate that both of the GNFs self-assemble to form entangled nanofibers roughly 10–20 nm in diameter and the β-d-glucopyranosyl-based GNF shows a CGC of 0.1% (w/w). The authors also reported that β-d-glucopyranosyl is compatible with a human cell line (Huh-7, human hepatocarcinoma cell line).⁹⁸¹ The same lab also reported the hydrogels made of glycosyl-nucleoside bola-amphiphiles (GNBAs) for culturing human mesenchymal stem cells isolated from adipose tissues.⁹⁸² Wang et al. designed and synthesized three amino acid derivative–saccharide conjugates, among which 469 self-assembles to form stable hydrogels containing nanofibers with diameters of 80–300 nm at a concentration of 0.2 wt %. The authors found that the extensive hydrogen bonds between sugar rings contributed to the formation of π–π stacking between aromatic naphthalene groups, which results in the formation of stable hydrogels in aqueous solutions. Using an MTT-based cell viability assay, the authors verified that these kinds of saccharide-based hydrogels are compatible with NIH3T3, HepG2, AD293, and HeLa cells. In addition, these cells show a good adhesion and proliferation rate on the surface of hydrogels in a 2D environment.⁹⁸³ It would be interesting to know how the hydrogelators affect the morphological properties of these cells.

5.1.3. Cytotoxic Hydrogelators

While most of the research activities are centered on the use of supramolecular hydrogels for promoting cell proliferation, Xu et al. have been working on the design of supramolecular hydrogelators to inhibit cell selectively. For example, as shown in Scheme 58, Xu et al. designed and synthesized a new class of supramolecular hydrogelators (304, 470, and 471) consisting of N-terminated diphenylalanine and naphthalene motifs. They found that the hydrogelators self-assemble to result in nanofibers and hydrogels at a concentration of less than 0.8 wt %, but within a relatively narrow pH range (5.0–6.0). Interestingly, the authors found that these hydrogelators exhibited significantly higher cytotoxicity to HeLa cells than to Ect1/E6E7 cells, which proves that hydrogelators selectively inhibit cancer cells.⁷⁶³ Thordarson et al. reported the synthesis of a new hydrogelator (472) with an indole capping group which forms exceptionally strong hydrogels in a variety of environments with a CGC of 0.4 wt %. Cell viability studies of HeLa cells indicate that 472 exhibits compatibility with cells at lower concentrations while being cytotoxic at concentrations up to 0.1 wt %.⁹⁸⁴ Liang et al. reported heptapeptide hydrogelators 473 based on the DEVD peptide sequence, which is a specific substrate for caspase-3. The cryo-TEM photograph indicates that 473 self-assembles to form a hydrogel containing flexible and long nanofibers with an average width of 6.1 ± 1.2 nm. The MTT cell viability assay shows that 473 is slightly more compatible with Hep G2 cells than its isomeric control hydrogelator (474) at 400 μM for 3 days. Western blot analysis indicated that the isomer 474, which is not a substrate of caspase-3, at 400 μM, obviously is able to activate caspase-3 to induce cell death via apoptosis.⁹⁸⁵

Scheme 58. Representative Molecular Structures of Cytotoxic Hydrogelators.

Numata et al. reported the high-yield chemoenzymatic synthesis of linear oligo(l-phenylalanine) by proteinase K from Tritirachium album. By connecting the synthesized linear oligo(l-phenylalanine) with tris(2-aminoethyl)amine, they obtained a star oligo(l-phenylalanine) (475) that self-assembles into fluorescent fibers with various branching ratios. The authors reported that the oligo(l-phenylalanine) analogues exhibit slight cytotoxicity to human embryonic kidney 293 cells (HEK293) at a concentration above 25 mM after 8 h.⁹⁸⁶ Zhang et al. designed and synthesized a glycopeptide (476) consisting of the Fmoc-Phe-Phe-Asp sequence and a therapeutic glucosamine moiety. 476 dissolves well to form a homogeneous solution at an elevated temperature and a concentration of 1 wt % and forms a stable hydrogel at pH 7.4 upon being cooled to room temperature. An MTT-based cell viability assay indicated that the glycopeptide slightly inhibits the NIH/3T3 fibroblast cells on the surface of the hydrogel. Most importantly, the authors reported that the hydrogel of 476 is able to inhibit postoperative fibrosis in eye surgery (Figure 12), as evidenced by the fact that the filtration fistula is constantly smooth and the mean intraocular pressure is significantly lower within 21 days postsurgery compared with the results from conventional antiproliferative drug injections.⁹²

Figure 12.

Histological section of rabbit eyes which underwent filtration surgery (a) alone at 14 days postsurgery, received the Fmoc-FF (6) hydrogel at (b) 7 and (c) 14 days postsurgery, and received the glycopeptide hydrogel (476) at (d) 7, (e) 14, and (f) 21 days postsurgery. Hematoxylin–eosin; magnification 100×. Adapted with permission from ref (92). Copyright 2012 Royal Society of Chemistry.

5.1.4. Cell Adhesion

As shown in Scheme 59, Stupp et al. linked a cyclic RGD motif at the side chain of a peptide amphiphile to construct a branched architecture in the monomer 477, which self-assembles to form cylindrical nanofibers having a very high aspect ratio and therefore mimicks the soft fibrous environment in the ECM. By changing the local dynamics either through the architecture of the molecules or dilution of the epitopes, the authors were able to tailor the density of RGD epitopes on the nanofibers to an extremely high level. In addition, the authors found that branched architectures of the monomers and additional space for epitope motion improve signaling for cell adhesion, spreading, and migration of NIH/3T3 fibroblast cells in 2D and MDA 231 cells in 3D cell migration.⁹⁸⁷ In a related study, Zhou and Zhang et al. reported cyclic RGD exhibiting synergistic effect with a BMP-7 derived peptide in the differentiation of mesenchymal stem cells.⁹⁸⁸ Besides the branched architecture with the cyclic RGD epitope reported by Stupp et al., Hamley et al. investigated the Fmoc-tetrapeptide Fmoc-RGDS (478) consisting of the RGDS cell adhesion motif from fibronectin. Circular dichroism and fiber X-ray diffraction indicated that the self-supporting hydrogel formed by sonication and heating/cooling at a concentration of 1 wt % is comprised of parallel β-sheet nanofibers with a diameter of approximately 10 nm. The authors suggested that 478 may be used to produce collagen-based gels for growing corneal fibroblasts.⁹⁸⁹

Scheme 59. Representative Molecular Structures of Hydrogelators for Cell Adhesion.

Yang et al. reported an intriguing example in which the peptide 479 self-assembles to form nanofibers and results in a hydrogel with a CGC of 0.3 wt %. The resulting hydrogel is able to selectively form a thin layer of hydrogel at the surface of platelets, thus preventing human platelet aggregation induced by various agonists such as collagen.⁹⁹⁰ Yang et al. also designed and synthesized a class of supramolecular hydrogelators consisting of the tripeptide sequence glycine-Xaa-4(R)-hydroxyproline (GXO; Xaa is any one of the natural amino acids) from collagen. Among all these hydrogelators, 480 self-assembles in aqueous solution to form nanofibers with a diameter of 20–30 nm and with a CGC of 0.06 wt %. Furthermore, 480 promotes the cell adhesion of NIH/3T3 fibroblasts, a property similar to that of collagen, which makes it suitable for 2D cell culture.⁹⁹¹ They reported another collagen mimic hydrogelator, 481, that self-assembles to form a thixotropic hydrogel, consisting of flexible nanofibers of about 9 nm, at a concentration of 2 wt %. Importantly, the authors found that the hydrogel of 481 selectively enhances Flk1 expression in differentiated murine embryonic stem (mES) cells.⁹⁹² Mihara et al. designed a glutamic acid residue-conjugated β-sheet peptide, E1Y9 (482), which, at a concentration of 2 wt %, undergoes hydrogelation in the presence of Ca²⁺. The hydrogel contains disentangled and wider nanofibers than the original Y9 nanofibers. The hydrogel maintains its shape well to allow it to be molded to a short string. The authors conjugated the RGDS sequence to the C-terminals of 482 peptides and obtained a new peptide, E1Y9- RGDS (483), which can be mixed with 482 to form hydrogel strings. One impressive result is that PC12 adheres to the hydrogel string and differentiates in 6 days (Figure 13), suggesting that the surface of the hydrogels resembles that of fibronectin surfaces.⁹⁹³

Figure 13.

(A) Percentages of strongly attached 3T3-L1 cells. 3T3-L1 cells were incubated on the flat hydrogels composed of 482 containing 0%, 10%, or 20% 483, fibronectin (FN), tissue-culture-treated plates (TCTPs), or nonadhesive plate surfaces in Dulbecco’s modified Eagle’s medium containing 5 mM Ca²⁺. (B) Fluorescence microscopic images of cell-adhered peptide gel strings. PC12 cells were cultured in Dulbecco’s modified Eagle’s medium containing 5 mM Ca²⁺ for 6 days. The scale bar represents 100 μm. Adapted with permission from ref (993). Copyright 2012 The Society of Polymer Science, Japan (SPSJ).

As shown in Scheme 60, Feng et al. reported a series of PEG-containing hydrogelators by coupling ethylene glycol (EG) monomers and the RGD motif onto C₂-benzene cores to resist protein adsorption and promote cell adhesion. TEM images indicated that 484 self-assembles to form entangled fibrous gel networks with fiber diameters of 68.9 ± 4.3 nm at a CGC of 0.07 wt %. The incorporation of the RGD sequence into 484 not only influenced the supramolecular structure and viscoelasticity of the fibers, but also contributed to overcoming nonspecific protein adsorption and promoting adhesion of encapsulated cells, which makes 484 suitable for 2D and 3D culture of human hepatoma cells and normal human skin fibroblasts.⁹⁹⁴ Furthermore, it is feasible to vary the supramolecular self-assembly of 484 for controlling the cell adhesion and proliferation in 2D and 3D microenvironments.^995,996 Meanwhile, Feng et al. also reported a new kind of hydrogel derived from the combination of a C₂-phenyl-derived gelator and a polysaccharide (alginate). After addition of Ca²⁺, the conjugate self-assembles to form flexible nanofibers with branches and twists. The LIVE/DEAD cell viability assay indicates that the hydrogel exhibits no cytotoxicity to normal human skin flbroblasts (NHSFs) and promotes cell adhesion and spreading in vitro.⁹⁹⁷ The hydrogels formed by the coassembly of C₂-phenyl-based hydrogelators and sodium hyaluronate showed a high swelling property to ensure cell migration and proliferation inside the bulk of the hydrogels.⁹⁹⁸ The authors also investigated the influence of the chirality of the nanofibers on cell adhesion and proliferation by using two enantiomers of C₂-phenyl-derived hydrogelators. They found that left-handed helical nanofibers (containing an l-phenylalanine derivative) can increase cell adhesion and proliferation, whereas right-handed nanofibers (containing a d-phenylalanine derivative) have the opposite effect.⁹⁹⁹ It would be more informative if the authors had examined the biostability of the hydrogelators.

Scheme 60. Representative Molecular Structures of Hydrogelators for Cell Adhesion.

Zhang et al. reported a class of photoresponsive small molecular hydrogels (Tet(I)-GFF (485) and Tet(II)-GFRGD (487)) formed by the self-assembly of short peptides linked with a biaryl-substituted tetrazole-moiety-based phototrigger. At pH 7, 485 forms a clear and stable hydrogel with a CGC of 0.08 wt %. Upon mild light irradiation, 485 undergoes fast intramolecular photo click ligation, and the complete transformation from 485 to 486 takes <2 min. This photoreaction disturbs the self-assembled hydrogel matrixes and induces the photodegradation of the hydrogel, which modulates the cellular microenvironments when the hydrogel of 485 serves as the scaffolds for cell cultures. The authors demonstrated that the irradiation of the hydrogel causes the cell to express much higher levels of differentiation markers. The authors also demonstrated that the hydrogel of 487 at a concentration of 0.9 mg/mL can mimic the 3D microenvironment for the hMSCs.¹⁰⁰⁰ On the basis of the Fmoc-FF (6) hydrogelator, Gazit et al. designed the DOPA-containing DOPA-DOPA and Fmoc-DOPA-DOPA peptides 301 that self-assemble to form a hydrogel at 0.25 wt %. The authors reported that the hydrogels of these DOPA-containing peptides reduce ionic silver into silver nanoparticles. In addition, the conjugation of lysine (Lys) with 301 generates 488, which self-assembles to form ordered nanostructures in the presence of dimethyl sulfoxide (DMSO) and water. The authors envision that it may serve as a multifunctional platform for various biotechnological applications.⁷³² He et al. reported an Fmoc-protected tetrapeptide amphiphile for fabricating a bioadhesive hydrogel with DOPA groups as affinity fusion tags (Fmoc-LFF-DOPA, 489). A 2000 U/mL concentration of of metalloprotease can trigger 489 to form a transparent yellow molecular hydrogel. On the basis of a LIVE/DEAD assay, the authors inferred that the hydrogel of 489, containing the catechol groups, could successfully promote the adhesion and proliferation of adult human dermal fibroblast cells in vitro.¹⁰⁰¹ It would be useful to know the mechanism behind this adhesion.

5.1.5. Others

As shown in Scheme 61, Liang et al. designed a radioactive probe (490) that intracellularly forms radioactive nanoparticles under the action of furin in living tumor cells. They found that, upon 160 min of cellular efflux, the radioactivity retained in MDA-MB-468 cells incubated with 490 remains at a high level.¹⁰⁰² Wang and Long et al. intended to design the peptide sequence FEFKFEFKGRGD (491) by adding a hydrophilic peptide (RGD) to the EFK8 peptide to decrease the strong aggregation properties of EFK8, therefore allowing hydrogels to form in neutral pH conditions. However, the results proved that this strategy was unsuccessful. Interestingly, they found that the hydrogel of 491 specfically supports and stimulates the growth of Delftia XD, a bacterium.¹⁰⁰³ Chen and Yang et al. reported another strategy to form hydrogels of EFK8 peptide derivatives at neutral conditions by the replacement of F with ^DF and the introduction of a hydrophilic RGD tripeptide (^DFEFK^DFEFKYRGD, 492). They found that 492 self-assembles to form hydrogels in PBS buffer with a CGC of 0.15 wt %. In addition, the LIVE/DEAD assay showed that the hydrogel of 492, at a concentration of 2 wt %, is suitable for cell proliferation and produces a colony of HeLa cells in vitro.¹⁰⁰⁴ This ingenious doping of d-amino acid residues for controlling the self-assembly behaviors of the hydrogelator may be general and applicable for other peptide sequences.

Scheme 61. Representative Molecular Structures of Hydrogelators.

5.2. Chemo/Biosensors

Chemo/biosensors for visual detection are a class of increasingly attractive tools for the analysis of many targets (e.g., biological markers, enzymes, ions, gases, etc.).⁵⁶² They are extremely useful for rapid and high-throughput diagnostics or detection in situations where low cost, speed, and ease are required. “Stimulus-responsive” or “smart” supramolecular hydrogels, thus, attract tremendous attention as a platform¹⁰⁰⁵ for chemosensors because they have the following properties/advantages: (i) A variety of biological, chemical, or physical triggers (e.g., temperature, pH, ionic strength, electric field, enzyme, etc.) instruct the formation of supramolecular hydrogels which report the presence of the targets.^1006,1007 For example, hydrogel formation triggered by enzymes can serve as an indicator of certain enzymes.^1008,1009 (ii) Supramolecular hydrogels are able to incorporate/immobilize a variety of colorimetric reagents, such as visible dyes, both covalently and noncovalently.¹⁰¹⁰ A range of diverse yet selective molecular interactions can lead to a color change of the hydrogel, for example, stimulus-induced release or absorbance of dye molecules and color changes of the hydrogels initiated by target binding. (iii) Supramolecular-hydrogel-based chemosensors can work in aqueous conditions, which is of extreme importance because most biological substances (e.g., enzymes, biomarkers, etc.) remain active only in physiological conditions (i.e., in aqueous solution).¹⁰¹¹ In the following subsections, we briefly describe these applications.

5.2.1. Dye/Molecule Absorption

Supramolecular hydrogels show solidlike, yet soft, properties and contain three-dimensional networks, formed by hydrogelators, to not only imbibe water, but also immobilize other components, such as small molecules, enzymes, and ions, especially when the hydrogels serve as chemo/biosensors. Before discussing supramolecular-hydrogel-based chemo/biosensors, we first highlight some recent works on hydrogels used as efficient absorbents of dyes, metal ions, and other molecules.^1012−1015 Due to the use of a wide range of dyes in several industries (e.g., paper, plastics, textiles, and cosmetics), it is necessary to remove the dyes from industrial discharge to prevent pollution. Among all kinds of methods, absorption is more preferred due to its low cost, high efficiency, and easy handling. Supramolecular hydrogels which contain both hydrophilic and hydrophobic groups can absorb a variety of dyes^1016−1018 and may have superiority in the recycle and adsorption rate compared with some traditional methods. As shown in Scheme 62, Banerjee and co-workers report a phenylalanine-based bolaamphiphile, 493, containing a centrally located oligomethylene group, which affords a hydrogel at pH 6.5–7.2 in the presence of divalent metal salts (e.g., MnCl₂, CoCl₂, CuSO₄, and NiCl₂). By studying the hydrogelation behaviors of these molecules, Banerjee et al. concluded that these hydrogels not only can entrap and release a biological substance, but also can efficiently adsorb various toxic dyes, such as crystal violet and naphthol blue black from water.¹⁰¹⁹ The same group reported several tripeptide-based hydrogels and their use in removal of dyes from wastewater.¹⁰²⁰ Sanchez et al. used a supramolecular hydrogel formed by triangular-shaped dendronized oligo(phenyleneethynylene) amiphiphiles 494 and 495 for dye encapsulation, such as disperse orange 3, a hydrophobic dye (Figure 14).¹⁰²¹

Scheme 62. Representative Molecular Structures of Hydrogelators.

Figure 14.

Temperature-dependent UV/vis spectra of aqueous solutions (∼10^–5 M) of (A) 494 and (B) 495 containing 1 equiv of disperse orange 3. Arrows indicate the spectroscopic changes with increasing temperature. The insets depict the changes in the absorbance at 400 nm as a function of temperature. Adapted with permission from ref (1021). Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Feng et al. recently developed two C₂-symmetric benzene-based hydrogelators, 496 and 497. They easily obtained hydrogels by changing the pH of the solution of 496, or by heating and then cooling the solution of 497. Both of the hydrogels exhibit a unique layered structure of activated carbon and are capable of the controllable adsorption of 97–99% of certain organic dyes, such as methylene blue and methyl violet 2B, within 2 min.⁴⁰⁴ Srivastava et al. used Nap-F (498), which gels water even at a concentration 0.025 wt %, as a network for dye entrapment. Besides, they demonstrated that the addition of chaotropic reagents, as well as increasing the pH value, disassembles the gel and promotes the release of the entrapped molecules.¹⁰²² Song et al. studied the hydrogenation behavior of lithocholate (87) by introducing alkali-metal ions and NH₄⁺ into the aqueous solution of 87. This hydrogelator shows a CGC varying from 75 to 130 mM (from 2.8 to 4.9 wt %), depending on the ions added. The authors demonstrated that these hydrogels show high efficiency and the capability of absorption of cationic dyes, and thus may be a promising candidate for the removal of toxic substances.^1023,1024

5.2.2. Chemosensors

The networks of supramolecular hydrogels can reversibly entrap a variety of probe molecules, which allows the development of various readout systems, such as fluorescence enhancement or quenching, color changes, or fluorescence resonance transfer (FRET), based on the hydrogels. Acting as a class of chemosensor, hydrogels help monitor the signal changes associated with molecular recognition.^1025,1026 As a pioneer in the applications of hydrogels for chemosensing, Hamachi et al. developed a supramolecular hydrogel formed by 499 molecules (Scheme 63) as a platform for a semiwet sensor chip.⁸⁴⁵ They created a hydrogel-based array on a glass plate by incorporating artificial receptors into the heated solution of the hydrogelator 499 and spotting them on a glass plate. They found that this semiwet sensor chip not only recognizes a variety of cations by simply changing the incorporated artificial receptors/probes, but also can work as a pH probe. They also demonstrated that the integrated supramolecular sensor chip can accept mixtures without tedious isolation steps.¹⁰²⁷ Hamachi et al. also used the same supramolecular hydrogel to construct a fluorescent lectin array for detecting saccharides. By noncovalently fixing the fluorescent lectins into the hydrogel matrix to act as a molecular probe for various glycoconjugates, they demonstrated that one can read a series of saccharides on the basis of the selectivity and affinity of the immobilized lectins.¹⁰²⁸ Using similar molecules, 367 and 500, Hamachi et al. designed a novel polyanion-selective fluorescence sensing system composed of a hybrid material of supramolecular hydrogels, enzymes, and aminoethyl-modified MCM41-type mesoporous silica particles with cationic nanopores encapsulating anionic fluorescent dyes (e.g., 501). This system efficiently coordinates (i) the release of an anion-selective probe (e.g., 501) from MCM41 and (ii) the translocation of the probe facilitated by enzymatic reaction (e.g., dephosphorylation catalyzed by phosphatases) with (iii) FRET sensing in the hydrogel form by 367 and 500.¹⁰²⁹ On the basis of similar strategies, Hamachi et al. also developed several fluorescent sensors for rapid and convenient detection of chemicals, such as phosphate derivatives,¹⁰³⁰ polyamines,¹⁰³¹ and polyols.¹⁰³² Kim et al. prepared a library of amphiphiles, each comprising a pyrene group and a polar carbohydrate head group. They found that all of the amphiphiles form robust hydrogels with CGC values ranging from 0.07 to 0.30 wt %, but only amphiphile 502 (a derivative of d-gluconolactone) affords a fluorescent hydrogel which is sensitive to the presence of insulin in aqueous media. They suggested that this supramolecular hydrogel can serve as an efficient probe for insulin.¹⁰³³ Rybtchinski et al. demonstrated that molecule 503, based on a perylenediimide chromophore decorated with polyethylene glycol, self-assembles in aqueous media to form extended supramolecular fibers which afford a stable hydrogel. As reported by the authors, the hydrogel of 503 can respond to multiple stimuli, such as temperature changes and chemical reductions. The authors suggested that the dual sensitivity toward chemical reduction and temperature with a distinct and interrelated response to each of these stimuli is especially useful to applications in the area of adaptive functional materials, such as chemosensors.⁷⁷

Scheme 63. Representative Molecular Structures of Hydrogelators for Chemosensing.

As shown in Scheme 64, Kim et al. reported the design of an anisotropic supramolecular hydrogel of γ-CD (504) and an azo dye (505) in which the host–guest interaction between the two molecules leads to hydrogelation. They tested the obtained hydrogel for identifying different classes of metal ions and demonstrated visual detection of lead ions by the naked eye.¹⁰³⁴ In addition to sensing ions in aqueous solution, hydrogel-based chemosensors can serve as probes for gases. For example, Jung et al. designed tetracarboxylic acid-appended thiacalix[4]arene (506), which is able to self-assemble in the presence of Co²⁺. They unexpectedly found that the red color of the filter paper coated with the resulting hydrogel selectively changed to a blue color by exposure to a toxic VGCl (volatile gas containing a chlorine atom), such as HCl, SOCl₂, (COCl)₂, and COCl₂, which hydrolyzed to yield HCl. They concluded that the strategy may lead to useful applications in sensing other chemical vapors.¹⁰³⁵ Singh et al. developed a Hg²⁺ coordinate complex of a 1,4-dioxa-7,13-dithia-10-azacyclopentadecane–BODIPY dyad (507) which selectively recognizes l-cysteine over other amino acids via a reversible complexation/decomplexation. As reported by them, the detection relies on the switch of fluorescence upon sequential addition of Hg²⁺ and a cysteine solution.¹³³⁰

Scheme 64. Representative Molecular Structures of Hydrogelators for Chemosensors.

5.2.3. Biosensors

The most attractive feature of supramolecular hydrogels is that the 3D, semiwet nanofiber network can entrap biological substances without a detrimental effect on the activities or functions of the entrapped substances.^1037−1039 For certain hydrogels formed by enzyme instruction, the state changing from a solution to a hydrogel itself can serve as a signal for reporting the existence of certain enzymes.^{149,966,1040,1041} For example, Xu et al. developed a simple assay based on the hydrogelation of small molecules Fmoc-Y_P (14) for the rapid detection of the inhibitors of enzymes (i.e., acid phosphatase). On the basis of the fact that phosphatase catalytically transforms the solution of 14 into a solid hydrogel within 30 min, Xu et al. demonstrated that the sol–gel transition can serve as a visual assay for screening the inhibitor of the enzymes.¹⁵⁸ Similarly, Wang et al. developed a strategy that utilized an aptamer-functionalized hydrogel to detect human thrombin through a diffraction measurement (508; Figure 15). Being a serine protease, thrombin acts as a model protein to test its binding with an aptamer. Wang et al. constructed the thrombin-responsive hydrogel by functionalizing the hydrogelator with both the aptamer and its complementary sequence as the physical cross-linking points. When exposed to human thrombin solution, the aptamer tends to bind with thrombin rather than its complementary sequence, which causes the hydrogel to swell due to the decrease of cross-linking and the change in the diffraction efficiency.¹⁰³⁶ Park et al. employed a self-assembled peptide hydrogel consisting of Fmoc-FF (6) as a biosensing platform. By encapsulating enzymes (e.g., glucose oxidase or horseradish peroxidase) and fluorescent reporters (e.g., CdTe, and CdSe quantum dots) physically within the hydrogel matrix via simply mixing them in a peptide solution, they successfully applied the system to detect analytes (e.g., glucose or phenolic compounds) on the basis of a photoluminescence quenching of the hybridized quantum dots.¹⁰⁴² Shimizu reported that an unsymmetrical bolaamphiphile, 509 (Scheme 65), with glucose and triglycine groups at both ends, exclusively self-assemblies into nanotubes. The nanotubes allow the doping of a compound (510) containing a chromophore. They demonstrated that the self-assembled nanotubes with an interior recognition probe on the inner surface not only detect the encapsulation, transportation, and release behavior of GFP in real time, but also report the stability of GFP in the hollow cylinder.¹⁰⁴³

Figure 15.

Schematic illustration of the sensing strategy of diffraction grating for human thrombin detection. (A) The hydrogel 508 contains an aptamer and its complementary sequence as the supermolecular cross-linking points and swells when exposed to the human thrombin. (B) Response of the hydrogel grating to human thrombin in the solution. Adapted with permission from ref (1036). Copyright 2013 Royal Society of Chemistry.

Scheme 65. Representative Molecular Structures of Hydrogelators for Biosensors.

Recently, Yang et al. reported a self-assembling vacomycin derivative (511) for bacterial detection and inhibition. They demonstrated that the conjugation of vancomycin to the side chain of the peptide derivatives increases its antimicrobial activity by 7-fold. By monitoring the fluorescence response of the solution of 511 to the bacteria, they found that the fluorescence of 511 increases gradually with increasing concentration of the bacteria. Similar to the bacterial surface-induced self-assembly of 23, this specific peptide–antibiotic interaction initiates the self-assembly of an environment-sensitive conjugate (511), which may find applications for simultaneous detection and inhibition of bacteria.¹⁰⁴⁴ Vemula et al. recently developed a self-assembled nanofibrous hydrogel using a biologically inert amphiphile (512) which possesses unique physical/mechanical properties and easily carries a diverse range of payloads. The authors found that 512 exhibits excellent self-assembly ability in multiple solvents, including aqueous and organic solvents, typically at a concentration of 1–4 wt %. By noncovalently encapsulating a pH dye (pHrodo) into the self-assembled hydrogel/fibers of 512, they obtained a pH sensor which can be internalized into macrophages at both physiological and subphysiological temperatures through an energy-dependent, passive process and report the pH in both the cytoplasm and phagosomes as well as the nucleus.¹⁰⁴⁵ Yang et al. synthesized compounds of dabcyl-GF_nG_3–nDEVDGK(FITC/rhodamine) (513) (n = 0–3) with and without F-substitution on the 4-position of the benzyl ring of phenylalanine as the self-assembling probes for caspase-3. They demonstrated that the incorporation of one or two amino acids of phenylalanine (F), especially 4-fluorophenylalanine (^FF), would greatly lower the background fluorescence intensities of conventional quenched probes with quenchers (dabcyl) by the synergistic effect of FRET and aggregation-caused quenching (ACQ). By varying the amount of ^FF, they optimized the properties of the resulting probes, such as self-assembly ability, fluorescence recovery, and kinetics of enzyme cleavage. They found that these probes can detect caspase-3 in complex environments such as that in apoptotic cells, which offers a simple strategy to design fluorescent molecular probes with better signal-to-noise ratios.¹⁰⁴⁶

5.3. Fluorescence/Imaging

Due to the promising applications of hydrogels in drug delivery, biosensors, tissue engineering, immunology, and other biomedicine, it is necessary to gain a comprehensive understanding of the self-assembly behavior of small molecules in the biological environment, aiming for optimal molecular design. Such being the case, imaging would be one of the most direct and revealing methods to distinguish, depict, and record the supramolecular self-assembly during the biological events or cellular processes. Compared to other imaging modalities (e.g., positron emission tomography (PET), MRI, etc.), the use of fluorescence has many advantages, such as easy access, low damage, and ready adaptability to specific molecular events despite poor depth penetration. Most importantly, fluorescent imaging provides the highest spatial resolution for imaging the molecular process at the cellular level. Various successful examples have been established to use specific fluorophores noncovalently staining supramolecular self-assemblies to reveal their existence, formation, and degradation, such as using Congo red to stain amyloids. In contrast, the covalent incorporation of a suitable fluorophore into a self-assembling small molecule (e.g., a hydrogelator) not only allows the self-assembly process to align the hydrogelators into nanofibers or other ordered structures, but also forces the appended fluorophore to comply with the ordered organization. In this subsection, we focus on the applications of fluorescent hydrogelators for imaging in a cellular environment, and introduce it as a powerful and facile method to reveal the emergent properties of supramolecular self-assemblies because it couples fluorescence with the self-assembly process.

5.3.1. Fluorescent Hydrogels

Before describing imaging of molecular self-assembly in a cellular environment, we present several typical examples of fluorescent hydrogels formed by small molecules. There are two kinds of fluorescent hydrogels: one consists of fluorescent hydrogelators,^{117,1047−1055} and the other forms by fluorescent dyes diffusing into the matrix of the hydrogel.^{197,1056−1062} For example, Shinkai et al. developed a supramolecular hydrogelator (β-d-glucopyranoside–azonaphthol conjugate, 514 or 515; Scheme 66) which affords a fluorescent hydrogel in a mixture of water and ethanol (80:20, v/v). The azonaphthol moiety serves not only as an aggregative functional group but also as a probe for microscopic solvent polarity. On the basis of a UV–vis spectral change induced by the azo–hydrazone tautomerism, it is possible to estimate the microenvironmental polarity in the fibrous aggregates of the hydrogelators.¹⁰⁴⁷ Jung et al. reported two fluorescent hydrogels formed by amide-linked tripyridine derivatives 516 and 517, with para or meta substituents. They demonstrated that both molecules gel water or water–DMSO and the hydrogelation ability depends mainly on CH−π and π–π stacking or strong intermolecular hydrogen bonding between the amide groups.¹⁰⁴⁹ Another interesting example is the ruthenium(II) tris(bipyridine) complex (10), developed by Xu et al. The integration of a tripeptide derivative, a versatile self-assembly motif, with a ruthenium complex affords the first supramolecular metallohydrogelator that not only self-assembles in water to form a hydrogel but also exhibits a gel–sol transition upon oxidation of the metal center (Figure 16). They found that this hydrogel formed by 10 exhibits strong fluorescence upon the irradiation of UV light.¹¹⁷ It is also noteworthy that the long lifetime and photostability of [Ru(bipy)₃]²⁺ will likely find applications in molecular imaging in cells.¹¹⁷

Scheme 66. Representative Molecular Structures of Fluorescent Hydrogelators.

Figure 16.

(A) Optical images of the oxidation-induced gel–sol transition and the TEM images corresponding to the samples at different states of transition. The hydrogel (reduced state) is formed by 0.8% (w/v) 10 in water at pH 1. The scale bar represents 10 nm. (B) Fluorescent images of a HeLa cell incubated with 3 (200 μM, 24 h). Adapted from ref (117). Copyright 2013 American Chemical Society.

5.3.2. Imaging Self-Assembly in a Biological Environment

Supramolecular hydrogelators serve as an excellent system for exploring the properties of molecular nanofibrils in a cellular environment. One of the successful cases is imaging of phosphatases inside living cells. Xu et al. developed a method to image enzyme-instructed self-assembly of small molecules inside live cells (see section 5.9.2).^156,1063 In a different study, Tomasini et al. found that a physical hydrogel prepared with small molecules of CH₂(C₃H₆CO-l-Phe-d-Oxd-OH)₂ (518; Scheme 67) is a potential “Trojan horse” carrier into cells. To check the internalization process by confocal microscopy, they prepared a fluorescent hydrogelator, introducing the fluorescent dansyl moiety into the molecules (519).¹⁰⁶⁴ Yang and co-workers conjugated the environment-sensitive fluorophore NBD to the peptide FFYEEGGH at its N-terminal and found that the resulting peptide derivatives 520 yield supramolecular nanofibers with enhanced cellular uptake, brighter fluorescence, and a significant fluorescence response to external stimuli (Figure 17).¹⁰⁶⁵

Scheme 67. Representative Molecular Structures of Hydrogelators.

Figure 17.

(A) The nanofibers of 520 could specifically bind to Cu²⁺, leading to the formation of fluorescence-quenched elongated nanofibers. Confocal images (bright field + fluorescence) of (B) HeLa cells treated with 520 (0.05 wt %) at a 2 h time point and (C) HeLa cells pretreated with 100 μM Cu²⁺ and then treated with 520 (0.05 wt %) at a 6 h time point. Adapted from ref (1065). Copyright 2014 American Chemical Society.

5.4. Antibacterial Hydrogelators/Hydrogels

Infectious disease remains a major threat to public health, and there is an urgent need for novel antimicrobial agents with activities against multi-drug-resistant bacteria. The discovery of antimicrobial peptides has stimulated the use of self-assembly of peptide amphiphiles to develop antibacterial hydrogels. Particularly, the pioneering work by Schneider et al. on antibacterial hydrogels has provided useful insights into the development of hydrogelators for antibacterial applications. Several comprehensive reviews have focused on this subject.^1066,1067 In the following section, we only give a brief discussion of the works on antibacterial hydrogelators reported over the past decade.

Schneider and Pochan et al. reported a series of β-sheet peptide-based hydrogels,¹⁰⁶⁹ among which the surface of 251 is inherently antibacterial and exhibits broad-spectrum activity against both Gram-negative (Klebsiella pneumoniae and E. coli) and Gram-positive (Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus pyogenes) bacteria without incorporating exogenous antimicrobial agents. Using the LIVE/DEAD assays by laser scanning confocal microscopy (LSCM), they found that the surface of the hydrogel of 2 wt % 251 displays broad-spectrum antibacterial activity when incubated with bacterial solutions ranging in concentration from 2 × 10³ to 2 × 10⁹ colony-forming units (CFUs)/dm² (Figure 18). On the basis of the β-galactosidase leakage experiments, they suggested that the surface of the 251 hydrogel likely causes inner and outer membrane disruption and controls the release of β-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35, resulting in cell death upon cellular contact with the surface of the hydrogel. Furthermore, coculture experiments showed that, when NIH3T3 fibroblasts and a mixture of Achromobacter xylosoxidans and Stenotrophomonas maltophilia are introduced onto the hydrogel, the surface of the hydrogel inhibits bacterial proliferation yet allows mammalian cell adhesion and proliferation, indicating that the surfaces are selective against bacteria.¹⁰⁶⁸ Later, they switched two lysine residues of 251 to two arginines, generating another β-hairpin peptide, MARG1 (521; Scheme 68). They found that the surface of the hydrogel of 2 wt % 521 imparts potent antibacterial activity against methicillin-resistant S. aureus (MRSA) while it is noncytotoxic toward mammalian cells (murine C3H10t1/2 mesenchymal stem cells).¹⁰⁷⁰ On the basis of this result, Schneider et al. designed another arginine-rich β-hairpin peptide, PEP6R (522),¹⁰⁷¹ which self-assembles to form hydrogels at 1.5 wt % or higher concentration containing NaCl. They found that the hydrogel surfaces of 522 exhibit potent activity for killing both Gram-positive and Gram-negative bacteria, including multi-drug-resistant Pseudomonas aeruginosa, while they exhibit cytocompatibility toward human erythrocytes as well as mammalian mesenchymal stem cells.¹⁰⁷²

Figure 18.

LSCM xy projections taken of 2.5 × 10³ CFUs/dm²E. coli incubated on (A) a borosilicate control surface and (B) the hydrogel of 2 wt % 251 after 24 h. The gel is viewed parallel to the z-axis. Green fluorescence denotes live cells, and red fluorescence denotes dead cells with compromised membranes. (C) LSCM xy projections taken of 2.5 × 10⁹ CFUs/dm²E. coli incubated on the surface of the hydrogel of 2 wt % 251 viewed perpendicular to the z-axis. Arrows denote the gel–bacterial interface. Adapted from ref (1068). Copyright 2007 American Chemical Society.

Scheme 68. Representative Molecular Structures of Antibacterial Hydrogelators.

Recently, Laverty et al. reported a series of cationic, naphthalene-derivatized self-assembling ultrashort peptides, among which 523 self-assembles to form hydrogels with a β-sheet structure at a concentration of 1 wt % and pH of 7.4 in water. The authors found that the hydrogel of 2 wt % 523 significantly reduces the viable S. epidermidis biofilm by 94% while exhibiting little hemolytic side effect toward human red blood cells (hRBCs). On the basis of the cytotoxicity assays against murine fibroblast (NCTC 929) cell lines and hemolysis assays using equine erythrocytes, the authors concluded that the hydrogels are compatible with mammalian cells.¹⁰⁷³ Yang and Wang et al. reported self-assembled vancomycin derivatives 511 based on FF or FFY with aromatic capping groups that showed great self-assembly ability in PBS buffer with a critical micelle concentration (CMC) of 75 μg/mL. Using the standard broth microdilution assay, the authors studied the bacterial inhibition capacity of 511 and found that the minimum inhibitory concentration (MIC) of 511 is about 4.5 μM, which was similar to that of the parent Van molecule (1.3 μM).¹⁰⁴⁴ Meanwhile, Wang and Chen et al. reported a selenium-containing vancomycin derivative with a redox-controllable self-assembly property and antibacterial activity.¹⁰⁷⁴

Das et al. designed and synthesized several dipeptide-based cationic amphiphiles with different head group structures by varying the combinations of l-amino acid residues. Among all the dipeptide derivatives, although 524 requires a relatively high concentration (MGC), 22 wt %, to form a hydrogel, the authors reported that 524 inhibits the growth of several Gram-positive (MIC = 0.1–0.5 μg/mL) and Gram-negative (MIC = 5–10 μg/mL) bacteria as well as fungi (MIC = 1–5 μg/mL). Moreover, the authors reported that 524 is compatible with different mammalian cell lines such as Hep G2, HeLa, and SiHa.¹⁰⁷⁵ Later, the authors reported a new class of antibacterial hydrogelators based on anti-inflammatory Fmoc-amino acid/peptide-functionalized cationic amphiphiles (525). By the incorporation of a pyridinium moiety at the C-terminal of Fmoc-amino acid/peptides, the positively charged hydrogelators self-assemble to form an antiparallel β-sheet arrangement of the peptide backbone and exhibit efficient antibacterial activity against both Gram-positive and Gram-negative bacteria.¹⁰⁷⁶ Das et al. also designed and synthesized several cholesterol-based amino acid-containing hydrogelators (526) that exhibit a high gelation efficiency (MGC of 0.9–3.1 wt %) and biocompatibility with human hepatic cancer-derived Hep G2 cells. After the incorporation of silver nanoparticles (AgNPs), the soft nanocomposite of the amphiphile and AgNPs exhibits a notable bactericidal property against both Gram-positive and Gram-negative bacteria.¹⁰⁷⁷ Sharma et al. designed and synthesized two self-assembled amphiphilic α,β-dehydrophenylalanine-containing small glyco-dehydropeptides, 527 and 528, with glucosamine attached at the C-terminal through a 6-aminocaproic acid linker. The authors found that 527 and 528 self-assemble to form gels in a mixture of methanol and water at a concentration of 0.1 wt %, with the sizes of the nanostructures being ∼197 and ∼235 nm, respectively. In addition, the authors used a disk diffusion assay to test the antimicrobial activity of the peptides 527 and 528, and they found that the peptides display antimicrobial activity against Micrococcus flavus, Bacillus subtilis, and P. aeruginosa.¹⁰⁷⁸

Das and Ramesh et al. reported several structurally diverse quinoline-based amphiphiles containing a fluorescent head group and hydrophobic chain of different lengths. Among these amphiphiles, 529 (Scheme 69) is the most potent antibacterial amphiphile to exhibit a dose-dependent bactericidal activity on target pathogens and even inhibits the growth of a presumptive MRSA strain. The authors found that this bactericidal activity may result from the electrostatic binding of 529 to bacteria. Most importantly, 529 has high antimicrobial selectivity, but hardly decreases the viability of human HT-29 cells.¹⁰⁷⁹ Zhao et al. designed a peptide (530) by connecting two Gram-positive antibacterial peptide sequences (KIGAKI)₃-NH₂ with a central tetrapeptide linker. They found that the electrostatic repulsion of the charged lysine residues balances the hydrophobic collapse of the isoleucine and alanine residues and backbone β-sheet hydrogen bonding to favor the self-assembly of 530, which forms individually dispersed nanofibers with a β-hairpin conformation. Furthermore, after 36 h of incubation, the hydrogel of 530 effectively inhibits E. coli proliferation when the concentrations of the initially introduced E. coli resuspensions are in the range of 10³–10⁶ CFUs/mL. However, when the bacteria reach a density of 10⁷ CFUs/mL, the hydrogel starts to lose its inhibitory capacity.⁹⁵ Yang and Yi et al. designed and synthesized a unique hydrogelator (531) based on (−)-menthol and a lysine. 531 self-assembles to form an opaque hydrogel at a concentration of 0.83 wt %. Interestingly, the hydrogelators form the 3D multiporous networks through acid–base interactions and strong double hydrogen bonding between amino acids for encapsulating some known antibacterial agents such as Zn²⁺ and a series of water-soluble organic antibiotic medicines such as lincomycin, amoxicillin, etc. Using the Oxford cup method, the authors found that the antimicrobial susceptibility of the hydrogels loaded with Zn²⁺ or lincomycin was much more effective than that of the corresponding aqueous solution when they were incubated with E. coli and S. epidermidis. In addition, the hydrogel of 531 is innocuous to mammalian cells such as HeLa cells.¹⁰⁸⁰ Yang and Hedrick et al. reported the synthesis, self-assembly, and antimicrobial activity of a series of oligomeric cationic compounds (532). Containing a rigid hydrophobic terephthalamide–bisurea core flanked by hydrophilic imidazolium groups with short alkyl (C_nH_2n+1, n < 6) or simple aryl tails, all the hydrogelators self-assemble to form nanostructures in aqueous solutions. These cationic hydrogelators exhibit potent, broad-spectrum antimicrobial activity and high selectivity toward Gram-positive bacteria (including clinically isolated MRSA), killing the microbes via the membrane-lytic mechanism. Notably, the bacteria tested fail to develop resistance even after multiple exposures to sublethal doses of the compounds, which is remarkably encouraging.¹⁰⁸¹

Scheme 69. Representative Molecular Structures of Antibacterial Hydrogelators.

Chen and Li et al. reported the preparation of biocompatible hydrogels with antimicrobial activity against Gram-positive bacteria by taking advantage of the intermolecular aromatic–aromatic interactions of Fmoc and the phenyl group. They generated a hydrogel based on the coassembly of Fmoc-Phe (200) and Fmoc-Leu (533), and found that the coassembled (200 + 533) supramolecular hydrogel is bactericidal against Gram-positive bacteria via a mechanism involving cell wall and membrane disruption (Figure 19). Being biocompatible with normal mammalian cells, this type of antibacterial hydrogel may potentially serve as an antimicrobial coating in clinical devices and wound dressings or a topical agent for the treatment of clinical skin and wound infections mainly caused by Gram-positive bacteria such as S. aureus, as suggested by Li and Chen.¹⁰⁸² On the basis of the concept of multidomain peptides (MDPs),^602,661,662 Dong et al. synthesized three hydrogelators, 534, 535, and 536, that self-assemble above critical assembly concentrations (CACs) of 0.87, 1.24, and 1.37 μM, respectively. In addition, they found that the position of tryptophan (W) determines the molecular secondary structure, supramolecular nanostructure, stability, and antimicrobial activity. After incubation with Gram-negative bacteria (E. coli and P. aeruginosa) or Gram-positive bacteria (S. epidermidis and S. aureus), a bacterial killing efficiency study shows that 99% of Gram-negative bacteria are killed by 535 and 536, while less than 40% are killed by 534. A surprising observation is the reverse dose-dependent relationships between the concentration of the peptides and their cytotoxicity toward primary mouse bone-marrow-derived monocytes (BMDMs),¹⁰⁸³ a result that is consistent with the formation of aggregates.^{884,1084−1088}

Figure 19.

(A) Representative SEM images and (B) overlapping fluorescence images for the LIVE/DEAD bacterial staining assay of S. aureus before and after contact with the coassembled (200 + 533) hydrogel for 2 h. Two fluorescent dyes were used in LIVE/DEAD staining in which SYTO 9 with green color labeled both live and dead bacteria while propidium iodide with red color stained only dead bacteria. Adapted with permission from ref (1082). Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.

5.5. Tissue Engineering

If one looks into a mirror, it is not difficult to realize that we are largely made of soft tissues. Because of the striking resemblance between hydrogels and soft tissues, the most attractive and sought-after biomedical application of supramolecular hydrogels is tissue engineering.^1089−1094 However, most of the demonstrations, so far, center on the culture of certain cells in vitro, which is still far away from the repair processes needed for regenerating damaged tissues or diseased organs.^1095−1097 There are several reasons for such a slow progress. First, the complexity and dynamics of biological processes at the tissue level are just beginning to be understood,¹⁰⁹⁸ and the understanding is far from complete. Thus, it remains difficult to devise working engineering principles for tissue engineering without adequate insights into the process. Second, most of supramolecular hydrogels consist of only one or two molecular species, which limits their roles to be only complementary or supplementary to the inherent or endogenous processes. Third, currently, supramolecular hydrogels still lack the sophistication or context-dependent features required for the regeneration of tissues, which usually consist of a myriad of transient biological processes.^1099,1100 Despite these enormous challenges, it is still worthwhile to review the progress made to date in “tissue engineering” by supramolecular hydrogels so that further development can be made to meet the challenges ahead.

In the hope of developing an approach for repairing the degenerated nucleus pulposus (NP) of intervertebral disks, Ulijn et al. tested the growth of bovine nucleus pulposus cells on the hydrogel made of [(fluorenylmethoxy)carbonyl]diphenylalanine (Fmoc-FF, 6)/Fmoc-diglycine (Fmoc-GG, 398) in 1:0 and 1:1 ratios.¹¹⁰¹ Using cryo-SEM, the authors verified that the hydrogels of 6 (1.0 wt %) consist of a dense network of nanofibers, whereas the hydrogels of 6/398 (1:1, 0.7 wt %) contain an overlapping mesh of flat ribbons.⁸⁹⁵ In addition, the authors found that the majority of the NP cells remain in a rounded morphology within both the hydrogels of 6 and the hydrogels of 6/398 after 5 days of culture. On the basis of that observation, the authors suggested that the morphology of the network has a limited effect on the NP cells. Although the authors reported the deposition of collagen and sulfate–glycosaminoglycan by the NP cells cultured within both the hydrogels of 6 and the hydrogels of 6/398 over 3 weeks, it is still too preliminary to establish the application of the hydrogel of 6 for intervertebral disk tissue repair. Recently, Thordarson et al. examined the degradation of the hydrogels of 6 and observed that 6 or its degraded products result in the necrosis of cells in vitro.⁷⁰⁵ This result, indeed, suggests that the fate of 6 in vivo remains to be firmly established.

As shown in Scheme 70, Rowan et al. reported the studies of the hydrogel of a guanosine-based hydrogelator, 8-methoxy-2′,3′,5′-tri-O-acetylguanosine (537), for the cell culture of a murine endothelial cell line (C166). 537 forms hydrogels at as low as 0.5 wt % in 100 mM NaCl. This hydrogelator forms helical assemblies, rather than the macrocyclic quartet assemblies commonly found in guanosine hydrogels. Contrary to the claim of the authors that there is little-to-no cytotoxicity of the hydrogels, the cell viability of the C166 cells, in the presence of hydrogels containing 2 wt % 537, is only about 50% of the control.¹¹⁰² It would be more interesting to elucidate the cell mechanism of cell death caused by the self-assembly of this hydrogelator. Li et al. developed a short peptide derivative containing halogenated phenylalanine and reported that the partially halogenated peptide exhibits better gelation properties than Fmoc-Phe (200) in aqueous solutions.¹¹⁰³ They found that Fmoc-4-fluorophenylalanine is the most efficient gelator (among the molecules derived by them) that gels PBS buffer solution at a minimum gelation concentration of 0.15 wt %. On the basis of this observation, the authors designed and synthesized an Fmoc-peptide (Fmoc-^FF^FFGRGD, 538) and used the peptidic hydrogel to culture NIH/3T3 cells. Although 538 only formed a clear hydrogel in PBS buffer containing 20% DMSO, the authors reported that the hydrogel could efficiently promote the adhesion and proliferation of NIH/3T3 cells.¹¹⁰³ In a related study, Parish and Nisbet et al. reported that Fmoc-self-assembling peptides (i.e., Fmoc-DIKVAV, Fmoc-FRGDF, Fmoc-DYIGSRF) have been used as a vehicle for the delivery and support of cell transplants in vivo.¹¹⁰⁴

Scheme 70. Representative Molecular Structures of Hydrogelators for Tissue Engineering.

On the basis of self-assembling P11-family of peptides reported by Boden,¹¹⁰⁵ McPherson et al. reported the production of self-assembling peptides (QQRFEWEFEQQ, 233)⁵⁹⁷ in a relatively high yield using an E. coli expression system. Being triggered by various physicochemical cues, 233 self-assembles to generate self-supporting isotropic or liquid crystalline hydrogels at peptide concentrations of 10–30 mg/mL (1–3 wt %). Using human dermal fibroblasts, the authors demonstrated that the hydrogels formed by the recombinant peptides display excellent cytocompatibility.¹¹⁰⁶ Zheng et al. synthesized KLD-12 peptide (417) and studied its biocompatibility with the host rabbit and MSCs, also, in the hope for repairing the degenerated nucleus pulposus of intervertebral disks. On the basis of the histological examination, the authors concluded that the 417 peptide hydrogel has a good biocompatibility with the host rabbit and MSCs so that the 417 peptide hydrogel could serve as a good scaffold material for tissue engineering of intervertebral disks.¹¹⁰⁷

Gelain et al. used phage display to identify the peptide sequences (e.g., FAQRVPP (539), QHLPRDH (540)¹¹⁰⁸) preferably interacting with murine NSCs and connected those peptide sequences to LDLK12 peptides⁸⁰² for generating functional self-assembling peptides. Confirmed by rheology, these synthesized self-assembling peptide sequences behave as classic hydrogelators to form hydrogels at a concentration of 1 wt % which consist of nanofibers of ∼12 nm width and ∼1.6 nm height. The authors found that the new functional peptide sequences, being linked to the LDLK12 peptide, have the capacity to bind to NSC-derived neural precursor cells (NPCs) and promote the proliferation and differentiation of the cells in vitro. On the basis of the high stem cell viability and neural differentiation achieved by the 539 peptide in vitro, the authors tested that peptide in acute contusive spinal cord injury in rats, and reported that the peptides foster nervous tissue regrowth and improve locomotor recovery. On the basis of these results, the authors concluded that phage-display-derived functional motifs need further investigation to elucidate their relevant molecular targets and cellular pathways. According to the authors, in vitro experiments are essential but still poorly informative for in vivo experiments. In another phage display panning, the same laboratory identified KLPGWSG (541)¹¹⁰⁹ as the NSC binding peptide and linked it to LDLK12 peptides for the differentiation of NSCs. The authors concluded that the enhancement provided by the peptide conjugate still required the presence of the growth factors (e.g., epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF)) and differentiative substrates. Again, this emphasizes the need to understand the molecular mechanisms linked to the observed neuronal phenotype. On the basis of their works on BMHP1, Gelain et al. derived a series of self-assembling peptides by connecting biotin to various mutated BMHP1 peptides. After identifying that one of the peptides (biotin-GGGAFASTKT-CONH₂, 542) is particularly effective at fostering adhesion, differentiation, and proliferation of human NSCs in vitro, the authors conducted in vivo experiments and reported that 542 causes a negligible immune response in the host nervous tissue in the short term, though its performance on nerve fibers is similar to that of saline.¹¹¹⁰

Using a β-sheet-forming peptide, PDFDFDFDFDFDP (denoted as P_FD-5, 543), to form hydrogels as the depot of tricalcium phosphate (β-TCP), Rapaport et al. tested the proliferation of human fetal osteoblasts in vitro and evaluated the performance of the hydrogels of 543 in rat bone defect models. The authors found that the hydrogels of 543 (at a concentration of 5%, w/v) are able to absorb calcium ions and to induce osteoblast differentiation in vitro. Although the TCP-loaded hydrogels exhibit an efficacy of bone generation similar to that of nonporous TCP, the in vivo results of bone defect healing in rat demonstrate that the peptide hydrogel alone induces better bone regeneration in comparison to the control (nontreated defects). This result, indeed, agrees with the observation of calcium absorption by the hydrogels due to the presence of a high density of aspartic acids in the peptides, and the hydrogels and the mineral act synergistically to enhance bone regeneration. The authors concluded that the hydrogels of 543 might act as biocompatible and biodegradable matrixes to support cellular osteogenic activity and to promote the turnover of calcium minerals, through cellular processes, into bone tissue.¹¹¹¹

Banta et al. have evaluated a series of peptides¹¹¹² consisting of β-roll peptide derivatives as the calcium-responsive motif and an α-helical leucine zipper domain (LZ) for intermolecular interactions. One of the most valuable features of these peptides is that the β-roll domain of the peptides is intrinsically disordered in the absence of calcium, while upon the addition of calcium, the peptide forms a β-roll secondary structure.¹¹¹³ The authors reported that these peptides form hydrogels only in calcium-rich environments. Recently, the same laboratory reported another class of β-roll peptides,¹¹¹⁴ but the application of these specific peptides in tissue engineering has yet to be reported. Recently, George et al. reported the use of LZ-based self-assembling peptides to form hydrogels for tissue engineering. The authors performed a quite comprehensive study of these hydrogels, from in vitro culture of human marrow stem cells (HMSCs) to in vivo evaluation of the hydrogels. Besides demonstrating that the concentration of the LZ peptide is able to tune the pore size of LZ hydrogels by altering the peptide concentration from 7 to 12 wt %, the authors functionalized the LZ polypeptide by the incorporation of the RGD domain for creating a suitable microenvironment for cell adhesion. According to the results reported by the authors, the incorporation of the canonical RGD domain has drastically improved the performance of the LZ hydrogels in many aspects. For example, an increase of the percentage of RGD in the hydrogels not only improves the proliferation of the HMSCs, but also allows the HMSCs to travel long distances within the LZ-RGDS hydrogels. The in vivo implantation of the LZ-RGDS scaffolds in a mouse model also significantly reduces the foreign body reaction to the scaffold. In vivo experiments with HMSCs also show that LZ-RGDS hydrogels have a better ability to support neovascularization than the LZ hydrogels do. On the basis of these results, the authors concluded that it should be possible to generate a functional and stable LZ scaffold for tissue engineering applications in vivo.¹¹¹⁵

Galler and D’Souza et al. reported the use of the hydrogel of a self-assembling, multidomain peptide for dental pulp tissue engineering.¹¹¹⁶ The authors used a peptide with a sequence of K(SL)₃RG(SL)₃KGRGDS (420; with a final peptide concentration of 1 wt %) to interact with heparin (with a final concentration of 0.1 wt %) to form a hydrogel for incorporating growth factors (e.g., vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor β 1 (TGFβ1)) and then tested the use of the hydrogels for encapsulating dental pulp stem cells in vitro and in vivo. The authors observed that the proliferation of the cells increases in the FGF-containing hydrogel, but decreases in the TGFβ1-containing hydrogel and that the dental pulp stem cells spread and form a collagenous matrix in the peptide hydrogel. One important observation is the formation of a vascularized soft connective tissue similar to dental pulp subcutaneously after transplantation of the hydrogel within dentin cylinders into immunocompromised mice. Although the authors concluded that the multidomain peptide is a highly promising candidate for regenerative endodontics, the requirement of dental pulp stem cells, various growth factors, and dentin highlights the complexity of this regenerative process and underscores the importance of mechanistic understanding. Tirrell et al. reported an innovative branched peptide amphiphile (544) that forms a hydrogel upon changing the pH from acidic to neutral. In addition, at 1% (w/v) 544, the hydrogel is capable of achieving a storage modulus of 10 kPa. By modulating the concentration of 544, the authors were able to regulate the viscoelastic properties of the hydrogels, thus broadening their versatility for complying with the mechanical requirement of a wide range of tissues. The authors tested the culture of NIH/3T3 fibroblast cells on the hydrogel of 544 for evaluating the biocompatibility of the hydrogel (Figure 20). After seeding the fibroblasts in the absence of serum to ensure the cells attach to the hydrogels through nonspecific interactions, the authors observed that these previously attached cells spread when serum was added. On the bais of LIVE/DEAD staining, the authors determined that the fibroblasts are predominately alive and suggested that the hydrogel is a viable biocompatible nanofiber-based tissue scaffold for supporting 3D cell growth.¹¹¹⁷

Figure 20.

Nanofibrous hydrogels are reported to be compatible with NIH/3T3 fibroblasts. In the presence of serum, fibroblasts spread by 28 h. At 72 h, spreading appeared to be spindle-like, resembling the natural morphology of the cell type. The fibroblasts proliferated for a minimum of 96 h. These images are from a single hydrogel of 544. Adapted with permission from ref (1117). Copyright 2012 Royal Society of Chemistry.

Akiyama et al. have designed a synthetic peptide hydrogel which consists of a 16-amino acid peptide (281)¹¹¹⁸ and is called PuraMatrix.^1119,1120 The authors used 281 to assess the feasibility of transplantation of isolated mucosal cells to repair a damaged middle ear. The authors collected middle-ear bullae with mucosa from rats, transfected the cells with enhanced green fluorescent protein (EGFP), encapsulated the cultured middle-ear mucosal epithelial cells into PuraMatrix hydrogels (1%, w/v), and then transplanted the cells into the immunosuppressed rats (Figure 21). Besides validating that primary cultured cells retain the character of middle-ear epithelial cells, the authors found that a high proportion of EGFP-expressing cells reside in the recipient middle ear after the transplantation using the hydrogel, but not without the hydrogel. These extensive studies demonstrated the feasibility of transplantation of cultured middle-ear mucosal epithelial cells encapsulated within 281 for regeneration of surgically eliminated mucosa of the middle ear in Sprague Dawley (SD) rats.¹¹²¹ However, the authors also observed that the proliferation rate depends on the seeding density and suggested that it might be due to contact inhibition or the limitation of the nutrient supply. In another related study, PuraMatrix served as the carrier for recombinant human bone morphogenetic protein-2, which significantly enhances bone regeneration in a bone augmentation rabbit model.¹¹²²

Figure 21.

(A–L) Analysis of transplanted mucosal epithelial cells in recipient tissues at postoperative days 14 and 28. Serial frozen sections of middle-ear bullae after transplantation (0.5 × 10⁶ cells/mL) at (A–F) postoperative day 14 and (G–L) postoperative day 28. (A, B, G, H) Fluorescence images at several time points. Enhanced green fluorescent protein (EGFP)-expressing cells were detected on the internal surface of recipient middle-ear bullae (green, EGFP; blue, 4′,6-diamidino-2-phenylindole) (A, G). Results of immunostaining with (C, I) antipancytokeratin, (D, J) antivimentin, (E, K) anticollagen III, and (F, L) anticollagen IV antibody. EGFP-expressing cells were positive for pancytokeratin (C, I, arrows), but not for vimentin (D, J). Collagen III-positive regions were detected mainly in the subepithelium (E, K). Collagen IV-positive regions were detected under the monolayer structure of donor cells at 14 and 28 days after transplantation (F, L, arrowheads). The scale bars represent 50 μm. Adapted with permission from ref (1121). Copyright 2013 Dove Medical Press Ltd.

Wang et al. tested the hydrogels made of the self-assembling peptide 281(1118) or 281 containing the laminin epitope IKVAV (545) at the C-terminal to act as a functional peptide-based scaffold to repair injured brain tissue. They found that 545 self-assembles to form nanofibers with a bilayer β-sheet structure and affords a hydrogel with mechanical stiffness similar to that of brain tissue, which makes the hydrogel suitable for encapsulating NSCs in an animal model study. The authors reported that the in vitro results showed that 545 serves as a guiding cue to promote the adhesion of the encapsulated NSCs and to bias the neuronal differentiation of these cells. Using the injected peptide solution to form the 3D hydrogel immediately in situ for filling up the cavity and bridging the gaps in the wound created in the brain, the authors demonstrated that the hydrogel of 545 enhances the survival of the encapsulated NSCs and reduces the formation of astrocytes. Although the authors reported enhanced neuronal differentiation and an improvement in brain tissue regeneration after 6 weeks post-transplantation,¹¹²³ the functional recovery of the damaged brain remained to be evaluated. In addition, the authors also linked other functional groups derived from fibronectin and laminin (e.g., GRGDS (546) or YIGSR (547)) to the 281 motif to evaluate the capability of these functionalized self-assembling peptides for the purpose of maintaining hemostasis and liver tissue regeneration.¹¹²⁴ After developing responsive α-helical peptide hydrogels,⁶³² Woolfson et al. tested these hydrogels for cell culture.¹¹²⁵ They reported that the cell viability is high and the α-helical gel network is stable in tissue culture conditions over 14 days.

5.6. Drug Delivery

Besides macroscopic properties, such as soft and wet, the majority of the volume of supramolecular hydrogels is micropores filled with water. These interstices allow the hydrogels to serve as a carrier or medium of other bioactive molecules^1126−1128 for a relatively straightforward application, such as drug delivery.^{28,60,890,1091,1093,1129−1139} To describe the applications of supramolecular hydrogels for drug delivery, we arrange the following section in two parts: first, we mainly describe various hydrogelators used for encapsulating drugs;^1140−1146 second, we focus on hydrogelators covalently conjugated with therapeutics.^1147−1153

5.6.1. Hydrogels Encapsulating Drugs

As shown in Scheme 71, van Esch et al. reported the use of the classical small molecular hydrogelator N,N′-dibenzoyl-l-cystine (DBC, 1) for the release of small molecules 8-aminoquinoline (AQ, 548) and 2-hydroxyquinoline (HQ, 549) as model molecules of drugs. Using self-assembly of 1 to form stable, clear hydrogels in 150 mM NaCl solution and PBS buffer, the authors tested this kind of hydrogel for the release of certain small molecules. The authors concluded that the release profiles depend on the interactions of the hydrogelator with the entrapped molecules because they observed that the release of 549 from the gels of 1 was 7 times faster than that of 548 due to acid–base interactions between 548 and 1. As suggested by the authors, the judiciary combinations of the hydrogelator and the drug molecules should be able to control the release of the drugs.⁷⁸ Xu et al. reported the combination of two simple Fmoc-amino acids (533 and 201) to form semitransparent hydrogels at pH 9.1 with a minimum concentration of 533 or 201 of 10 mM. After the addition of 2 molar equiv of Na₂CO₃, the mixture of 533 or 201 forms a clear hydrogel consisting of entangled irregular fibers with widths of 120–500 nm. Besides the inclusion of 201, an anti-inflammatory drug candidate,¹¹⁵⁴ the hydrogels act as carriers for other bioactive agents, such as 5-fluoro-2A-deoxyuridine (5-FU), an antineoplastic agent, or pamidronate, an osteoporosis drug,²⁸² by the simple mixing of the drugs in the solution of 533 or 201 prior to hydrogelation.¹¹⁵⁵ Haldar et al. reported that the addition of γ-aminobutyric acid (550) to the solution of Fmoc-lysine (202) leads to the formation of a hydrogel at pH 6.9 without any heating–cooling cycle at a concentration of 2 wt %. The authors suggested that this hydrogel could be used for the recognition and release of the anti-inflammatory agent 202.¹¹⁵⁶

Scheme 71. Representative Molecular Structures of Hydrogelators Encapsulating Drugs.

John et al. synthesized a hydrogelator made of amygdalin–fatty acid conjugates (551). In this type of hydrogelator, the sugar moiety facilitates intermolecular hydrogen bonding, the phenyl ring enhances intermolecular aromatic–aromatic interactions, and the hydrophobic hydrocarbon chain decreases the solubility in water and increases the molecular association through the van der Waals interactions. The authors used the networks made of helical ribbons and fibers (∼50 nm) in the hydrogel of 551 for encapsulating curcumin, a chemopreventive hydrophobic drug, to demonstrate the potential application of the hydrogel of 551 in drug release.¹¹⁵⁷ On the basis of l-phenylalanine and l-tyrosine with subtle variation in the structure of the head group, Das et al. designed and synthesized 10 structurally correlated amino acid-based amphiphiles for screening hydrogelators, and found 3 of them to confer pH-responsive hydrogels at room temperature. Forming at a CGC of 4 wt %, the hydrogel of 552 exhibits remarkable sensitivity to pH, which makes the hydrogels suitable for the release of vitamin B₁₂ and cytochrome c. The authors found that, at pH 7.4, all three hydrogelators form suitable hydrogels to release the entrapped biomolecules via diffusion. At endosomal pH (∼5.5) or a further lower pH, the release rate of biomolecules from the hydrogel of 552 increases by about 10-fold compared to that observed at pH 7.4, largely due to the dissociation of the gels.⁷⁴

Lehn et al. reported a guanosine derivative, guanosine-5′-hydrazide (347), that forms tetramers (i.e., G-quartet (G4)^1158,1159) in the presence of cations such as Na⁺, K⁺, and NH₄⁺. The authors used the hydrogel of 347 to entrap acyclovir, vitamin C, or vancomycin for controlled release.¹¹⁶⁰ Besides the physical trap and release of biological molecules, Lehn et al. later reported that the G-quartet structure of 347 acts as a delivery system for the slow release of bioactive carbonyl derivatives since aldehydes or ketones can reversibly react with the free hydrazide functions at the periphery of the G-quartet to form acylhydrazones.¹¹⁶¹ Barthelemy et al. reported the first example of the use of a glycosyl-nucleoside lipid for the delivery of oligonucleotides into cells (human Huh7 cells). They linked a simple monosaccaride, a lipidic chain, and a nucleoside together by 1,2,3-triazole bridges to obtain 339, which self-assembles to form nanofibers roughly 20–30 nm in diameter and results in hydrogels at a concentration above 2.5 wt %. They reported that 339 is compatible with Huh7 cells after 5 days of incubation and the nucleic acid–339 complex enhances the cellular uptake of the nucleic acids in the presence of serum.¹¹⁶² Yi et al. designed and synthesized three amphiphilic 3,4,5-trihydroxybenzoic derivatives with alkyl chains of different lengths, among which 553 and 554 gel aqueous ethanol in the presence of the water-soluble drug tetracycline hydrochloride. They found that a small amount of small molecules (10 mg/mL 554 or 3.3 mg/mL 553) are able to entrap a large amount of tetracycline, up to 91.5%. In addition, the release studies of tetracycline in various solutions indicate that the release rate of tetracycline for a bovine serum albumin (BSA) solution (10 mg/mL) is faster than that with the other solutions because of the strong interaction between tetracycline and BSA.¹¹⁶³

Schneider and Pochan have designed a class of self-assembling peptides that undergo triggered hydrogelation in response to physiological pH and in salt conditions (pH 7.4, 150 mM NaCl) to form mechanically rigid, viscoelastic hydrogels.²⁸ Among the β-hairpin peptides, 251 and 257 are two peptide sequences with different charge states for directly encapsulating and controllably releasing model fluorescein isothiocyanate (FITC)–dextran macromolecules of varying size and hydrodynamic diameters.¹¹⁶⁴ Using fluorescence recovery after photobleaching (FRAP), the authors studied the self-dissociation of the hydrogels and bulk release of model FITC–dextran macromolecules. The authors reported that the mobility of the macromolecules or the probes within and release of these hydrogels depended on the sizes of the probes, the peptide sequences, and the mesh size of the hydrogel.^1165,1166 Later, the authors also found that the self-assembling 257 peptide hydrogel is an effective vehicle for the local delivery of curcumin.¹¹⁶⁷ Hamachi et al. reported a single-component, multiple-stimulus responsive hydrogelator, 555, containing a phosphate group that displays a macroscopic gel–sol response toward four distinct input stimuli (temperature, pH, Ca²⁺, and light). The authors suggested that the hydrogelator confers gel-based supramolecular logic gates displaying AND, OR, NAND, and NOR functions. By using these logic-gate-like functions, they found that the hydrogel is able to hold and release bioactive substances (e.g., vitamin B₁₂ or the protein Rh-Con A) in response to various input triggers.¹¹⁶⁸

Several labs evaluated the Ac-(RADA)₄-CONH₂ (281) peptide hydrogel to act as an efficient slow release carrier of a variety of proteins,^1169,1170 such as lysozyme, trypsin inhibitor, BSA, and immunoglobulin G (IgG), which differ in physicochemical properties and morphologies. The results of the fluorescence correlation spectroscopy (FCS) analysis indicated that the peptide hydrogel of 281, at a concentration of 1 wt %, can encapsulate the proteins and release the proteins when the hydrogel disintegrates due to the degradation of the peptides by proteolytic enzymes in vivo. Furthermore, Zhang et al. found that the protein diffusion through the hydrogel of 281 depends primarily on the size of the proteins and the encapsulation and release barely affect the protein conformations and functions.¹¹⁷¹ Later, Zhang et al. reported the use of the peptide hydrogels for facilitating slow and sustained release of active cytokines related to many areas of regenerative medicine. However, the release of negatively charged VEGF from the hydrogel of 281 is slower compared to that of cytokines of somewhat similar molecular weight but opposite charge, suggesting that the positive guanidinium interacts with VEGF and hinders the diffusion of VEGF. The authors also found that the release of functional human βFGF and VEGF occurs over 2–3 weeks within the hydrogel of 281 and two other hydrogels formed by peptides with net positive or negative charges located at the C-terminal.¹¹⁷² Shibata et al. have recently found that the aqueous solution of 281 and insulin form a hydrogel, in vitro and in vivo, with an increase of the ionic strength by phosphate ion and an increase of the pH. The in vitro experiments indicated that the release rate of insulin depends on the concentration of 281 and the controlled release of insulin occurs at final concentrations of 281 between 0.1 and 2.0 wt %. Furthermore, the authors found that 281 forms a hydrogel in vivo for a sustained-release insulin, which also depends on the concentration of 281.¹¹⁷³ Tan and Kinoshita modified the 281 peptide scaffolds by positioning a phenylalanine residue at the C-terminal to generate 556 and 557 and studied the entrapment and the release of certain enantiomers (e.g., d-, and d-phenylalanine) of amino acids. They found that the amount and chirality of the guests tailor the network nanostructures, thus affecting the release of the enantiomers. In addition, the release rate of the enantiomers from the hydrogels containing one phenyl group (556 and 557) is much slower than from the hydrogels without a phenyl group (281), agreeing with the aromatic interactions between the hosts and the guests. The concentration of the trapped enantiomers after the diffusion¹¹⁷⁴ matches with the release kinetics controlled by Fickian diffusion, which depends on both the rational design of the peptides used for making the hydrogels and the choice of the size and lipophilicity of the entrapped molecules.¹¹⁷⁵

Xu et al. reported the first in vivo imaging for investigating the drug release properties of the supramolecular hydrogel formed by hydrogelators 558 and 559 consisting of naphthalene (Nap) and a d-peptide of diphenylalanine (Figure 22). TEM images show that the hydrogels consist of nanofibers with a length of over tens of micrometers, a width of about 50 nm, and an average mesh size of about 200 nm. Since the hydrogels resist hydrolysis catalyzed by proteinase K and offer long-term biostability, the hydrogel of 559 is suitable for the controlled release of drugs in vivo.¹¹⁷⁶

Figure 22.

(a) Profiles of the mean blood concentration of ¹²⁵I-NaI vs time after subcutaneous (sc) administration to rats (160 μCi/kg) (■, control, ¹²⁵I-NaI solution, AUC (area under the curve) = 1213.3 μCi·h/L; ●, experimental, ¹²⁵I-NaI in gel II, AUC = 1453.5 μCi·h/L). (b) Dynamic (upper two lines) and static (lower line) single-photon emission computed tomography (SPECT) images of rats with ¹³¹I-NaI (500 μCi/rat; left, in solution; right, in gel II) administered sc. (c) Profiles of the mean blood concentration of ¹²⁵I-epidepride vs time after sc administration to rats (160 μCi/kg) (■, control, ¹²⁵I-epidepride solution, AUC = 645.5 μCi·h/L; ●, experimental, ¹²⁵I-epidepride in gel II, AUC = 693.6 μCi·h/L). (d) Dynamic SPECT images of rats with ¹³¹I-epidepride (500 μCi/rat; left, in gel II; right, in solution) administered sc. Adapted from ref (1176). Copyright 2009 American Chemical Society.

As shown in Scheme 72, Banerjee et al. reported two synthetic self-assembling tetrapeptides, GAIL (560) and GFIL (561), that form thermoreversible and pH-sensitive hydrogels which consist of long, interconnected nanofibrillar network structures with diameters of 15–30 and 10–25 nm, respectively. These hydrogels entrap doxorubicin to allow its slow release at physiological pH, and achieve almost 85% (for peptide gel 560) and 90% (for peptide gel 561) release of the drug molecules after 45 h.¹¹⁷⁷ Adams et al. reported the hydrogels of Fmoc-Phe (200) and Fmoc-Tyr (15) formed by careful adjustment of the pH of the solution using GdL. They found that the hydrogels of 200 and 15 entrap and release certain dye molecules under the control of Fickian diffusion. On the basis of the similar diffusion coefficients of the dyes of different radii from the hydrogel of 200, the authors concluded that the networks in the hydrogel of 200 only restrict molecules larger than 5 nm.¹¹⁷⁸ Banerjee et al. reported two N-terminally protected dipeptides (562 and 563) with a β-amino acid residue that form hydrogels at physiological pH (7.46) and temperature (37 °C). Having different CGCs (0.85 wt % for 562 and 1.21 wt % for 563), the hydrogels consist of nanofibers of different widths (45–130 nm for 562 and 30–60 nm for 563). In addition, these two hydrogels can encapsulate and sustainably release two vitamins (vitamin B₂ and vitamin B₁₂) over 3 days.⁹⁰ Banerjee et al. reported that a designed tripeptide-based hydrogelator (564) having both 11-aminoundecanoic acid and Phe-Phe residues forms hydrogels that entrap vancomycin and vitamin B₁₂ for sustained release at physiological pH and temperature for about 2 days (Figure 23). According to an MTT-based cell viability assay at 24 h, the authors suggested that this peptide gelator, 564, is innocuous to cells.¹¹⁷⁹

Scheme 72. Representative Molecular Structures of Hydrogelators Encapsulating Drugs.

Figure 23.

(A) Illustration of the injectable nature of the hydrogel and its vitamin release phenomenon with vitamin B₁₂. (B) Percentage release plot of some important biomolecules from hydrogel 564 at physiological pH (7.46) and temperature (37 °C), where the concentration of the drugs loaded into the hydrogel was 1.14 mg/mL for cyanocobalamin (vitamin B₁₂) and 0.24 mg/mL for vancomycin. Adapted from ref (1179). Copyright 2014 American Chemical Society.

Zhang and Jiang et al. reported a new peptide comprised of a peptide backbone containing an Arg-Gly-Asp (RGD) sequence and a hydrophobic Fmoc tail. The peptide derivative 565 self-assembles to form a transparent hydrogel and exhibits biocompatibility in rabbit eyes. The authors found that this peptide hydrogel, acting as an implanted carrier, delivers an antiproliferative model drug (5-fluorouracil, 5-FU) in rabbit eyes and inhibits postoperative scarring formation. According to the in vivo experiments reported by the authors, the 5-FU-loaded peptide hydrogel releases 5-FU to inhibit scleral flap fibrosis efficiently after the surgery.¹¹⁸⁰ Later, Castelletto et al. reported another functionalized peptide, 401, which also contains RGD. They found that 401, at a concentration of 10 wt %, formed homogeneous hydrogel monoliths that are stable in water for nearly 40 days. Consisting of a rigid porous structure made of the peptide fibers, the hydrogel monoliths are able to encapsulate and release various molecules, including model hydrophilic dyes and drug compounds (e.g., bioactive riboflavin and hydrophilic pseudodrug salicylic acid).¹¹⁸¹

Diaz et al. reported the supramolecular coassembly of complementary structures followed by controlled thiol–ene coupling as a new strategy for fine-tuning the drug release kinetics of self-assembled hydrogels made of small molecules 1. TEM and SEM images indicate that 1 self-assembles into fibers 30–150 nm in diameter at a concentration of 0.2 wt %. Using in vitro experiments, the authors showed that the hydrogel of 1 entraps and releases small drugs, such as 2-hydroxyquinoline (549), a model of water-soluble and UV-active drugs.¹¹⁸² Jung and John et al. reported the formation of a coordination polymeric hydrogel made of a simple pyridine derivative (516) and Cu²⁺ ions. Consisting of a fibrillar network of fibers several micrometers in length and 45–65 nm in width, the hydrogel of 3 can encapsulate curcumin. Being pH-triggered at physiological temperature, the hydrogel dissociates and releases the encapsulated curcumin.¹¹⁸³ Shimizu et al. designed and synthesized a simple amphiphile, 566, consisting of a photoresponsive azobenzene and a hydrogen-bonding glycine to construct self-assembled nanotubes. TEM showed that the self-assembled morphologies strongly depend on the pH conditions; that is, the self-assembly of 566 at pH 6.1 gives fibers, but at pH 9.2 gives sheets. Upon UV-light irradiation, the trans–cis photoisomerization of the azobenzene within the tubular wall results in a morphological change from nanotubes to cylindrical nanofibers to release the pre-encapsulated guests (e.g., carboxyfluorescein (CF)) in the hollow cylinder of the nanotubes.¹¹⁸⁴ Miravet and Escuder et al. reported a hydrogelator bearing a nucleophilic reactive site that reacts with aldehydes to cause the disassembly of the hydrogel network. 567 forms stable hydrogels at a concentration above 2 mM. The authors found that the hydrogels of 567 entrap and release dyes or drugs (e.g., methylene blue or ketoprofen) in response to the presence of specific aldehydes. In addition, 567 is highly biocompatible, which may present a protective effect against toxic aldehydes.¹¹⁸⁵ Rapaport et al. have developed amphiphilic β-sheet peptides P_FD-5 (543) decorated by acidic amino acids. 543 self-assembles to form ordered monolayers at the interfaces as well as hydrogels near physiological pH. The authors found that the mildly amphiphilic doxorubicin can be entrapped within the amphiphilic matrix of the peptide hydrogel, due to electrostatic forces and hydrophobic interactions. The peptide–doxorubicin interactions may affect the release of doxorubicin from the peptide hydrogels as less doxorubicin was released from the hydrogels with a higher loading of doxorubicin.¹¹⁸⁶

As shown in Scheme 73, Zhao et al. designed a self-assembling peptide, P4 (568), containing 16 amino acids that forms stable β-sheet nanofibers with a diameter of 25 nm and a length of micrometers. The authors found that the hydrogel of 568 is capable of stabilizing hydrophobic anticancer agents, such as ellipticine, a natural plant alkaloid. SEM images showed that the state of ellipticine in the complexes relies on the concentration of 568, which also affects the size and morphology of the complex. It was found that the complexes of ellipticine and the peptide significantly reduce the viability of two cancer cell lines (SMMC7721 and EC9706 cells).¹¹⁸⁷ Miller et al. reported three octapeptides, VEVEVKVE (VEK1, 569), VEVKVEVK (VEK2, 570), VKVKVEVK (VEK3, 230), which carry a net charge of −2, 0, and +2 at neutral pH, respectively. The author found that all three peptides form transparent and self-supporting hydrogels. The hydrogels of 570 and 230 encapsulate two hydrophilic model drug molecules (naphthol yellow and martius yellow) and release them following Fickian diffusion and depending on the fibrillar network and the overall charges of the complex molecules.¹¹⁸⁸

Scheme 73. Representative Molecular Structures of Hydrogelators Encapsulating Drugs.

Nachtsheim et al. reported the simple and remarkable small molecular hydrogelators of phenylalanine-containing cyclic dipeptides (diketopiperazines, DKPs, 571 and 572), which only contain proteinogenic amino acids (serine, cysteine, glutamate, histidine, or lysine) as building blocks. 571 and 572 form stable and self-healing hydrogels with a porous network or dense lamellar sheets with bundled nanofiber connections between the sheets, respectively. Furthermore, the authors found that the mixture of 571 and 572 forms heterotypic hydrogels and demonstrated their use for the release of BSA and tetracycline.¹¹⁸⁹ Liu et al. designed a supramolecular hydrogel based on a peptide dendron (573) and found that metal ions can trigger a continuous shrinkage after the gels have been annealed for several hours. It is reported that the metal ions (e.g., Mg²⁺, Cu²⁺) significantly promote the gelation capacity, and decrease the CGC from 0.3 to 0.08 wt % or below. In addition, the reversible shrinkage property of the hydrogels allows the controlled release of small molecules such as vitamin B₁ after addition of divalent metal ions (such as Mg²⁺) into the gel.^1190,1191 Saiani et al. focused on an octapeptide, FEFEFKFK (228), which self-assembles into antiparallel β-sheet-rich fibers and forms hydrogels at concentrations above 2 wt % in water. The authors used the hydrogel of 228 for the delivery of two commercial drugs, lidocaine and flurbiprofen. They found that the addition of lidocaine to the hydrogel stiffens the samples without affecting the overall peptide release, while the hydrogel encapsulating flurbiprofen exhibits improved resistance erosion and enhances drug retention.¹¹⁹²

Kumar et al. reported the use of a class of phenylalanine (Phe)-containing self-assembling peptide nanofibrous material (RATEA-F8, 574) for the delivery of 5-fluorouracil (5-FU) and leucovorin (LV), which shows synergistic action against colon cancer cells. The study of the gelation properties indicated that 574 self-assembles to form nanofibers in water and Tris–HCl buffer at a concentration of 0.78 wt % with a diameter of 5–20 nm and a length of 60–80 nm. The in vitro experiments indicated that the hydrogel of 574 slowly releases 5-FU, LV, and Phe.¹¹⁹³ Koutsopoulos et al. designed a class of lipid-like peptides with an aspartic acid or lysine hydrophilic head and a hydrophobic tail composed of six alanines (Ac-A₆K-CONH₂ (575) or Ac-A₆D-COOH (576)). The authors found that the addition of this kind of lipid-like peptide into water or an electrolyte solution results in formation of a turbid suspension due to self-assembly of the peptide monomers to minimize the interaction between the hydrophobic domains and polar environment. In addition, it was found that 576 is more suitable for the encapsulation and release of carboxyfluorescein and Nile red.¹¹⁹⁴ In a related study, Qiu et al. reported the use of 575 to transfer pyrene into living HepG2 cells.¹¹⁹⁵

5.6.2. Hydrogelators Conjugated with Drugs

As shown in Scheme 74, van Esch et al. reported the gelation properties of a class of cyclohexanetrisamide-based hydrogelators with an l-phenylalanylamidoquinoline (l-Phe-AQ, 577) moiety as well as two ethylene glycol chains.¹¹⁹⁶ According to this design, α-chymotrypsin (α-chy) can enzymatically cleave the 577 moiety to release the fluorogenic model “drug” 6-aminoquinoline (6-AQ, 578). Moreover, the cleavage of the two ethylene glycol chains increases the hydrophobicity of the overall structure to improve the gelation properties. The hydrogelator forms fibers with identical diameters of 4.2 nm and a thermoreversible hydrogel with a CGC of 0.03 wt % at room temperature. The authors envisioned that the hydrogel might act as a two-stage enzyme-mediated drug release system. That is, AQ (548), in the gel fibers, is initially protected from enzymatic cleavage; after the temperature is increased, more hydrogelators become available for enzymatic cleavage to result in a dramatic increase in the rate of release of 548.¹¹⁹⁷ Kim et al. synthesized a class of (S)-(+)-ibuprofen-based hydrogelators, among which 579 forms hydrogels consisting of entangled irregular fibers with widths of 60–100 nm and with a CGC of 0.9% (w/w). Two hours after the addition of an enzyme (carboxypeptidase Y), the hydrogel is able to release the drug (S)-(+)-ibuprofen.¹¹⁹⁸

Scheme 74. Representative Molecular Structures of Hydrogelators Conjugated with Drugs.

Wang et al. reported a simple drug candidate, 4-oxo-4-(2-pyridinylamino)butanoic acid (580), which self-assembles to form hydrogels at a 4 wt % concentration under various conditions. They found that this hydrogel with different backbone structures releases drug molecules at different speeds.¹¹⁹⁹ Polonelli et al. reported a synthetic decapeptide KP (581) derived from the variable region of the light chain of a recombinant antibody. They found that, in nonreducing conditions, the solubilized 581 molecules easily dimerize due to the formation of disulfide bridges and spontaneously and reversibly self-assemble to generate an organized network of fibril-like structures. Furthermore, this self-assembled network, likely being resistant to proteolysis, slowly releases the active dimeric form of 581, which acts as a microbicide in vitro against a number of pathogenic microorganisms, such as Candida albicans.¹²⁰⁰ John et al. reported the synthesis of small hydrogelators 582 and 583 containing amphiphilic prodrugs, such as acetaminophen. The prodrugs self-assemble to form branched or entangled fibrous/sheetlike gel networks with a fiber thickness of 50–400 nm and fiber lengths of several micrometers. The hydrogels are able to encapsulate a second drug such as curcumin. Using the in vitro experiments with MSCs, the authors showed that the hydrogels of 582 and 583 release single or multiple drugs under physiological conditions and upon enzyme catalysis, and retain certain features of the MSCs.¹²⁰¹ Odriozola et al. reported the use of coordination for converting N-acetyl-l-cysteine (584), a mucolitic agent or an antidote in paracetamol intoxication, into a hydrogelator. 585 is a metal–thiolate that self-assembles to form metallophilic hydrogels in the presence of Au(III), Ag(I), and Cu(II) salts with a microporous structure in the form of flakes. Although these specific hydrogels only form at pH < 4 to be able to act as drug delivery systems,⁴⁸⁴ the concept demonstrated by Odriozola et al. should be applicable to other systems that form metallophilic hydrogels at physiological pH.

On the basis of the serendipitous discovery of a supramolecular hydrogelator made of vancomycin,¹⁸ Xu et al. reported the first example of a hydrogelator (586) derived from an aminoglycoside antibiotic (e.g., kanamycin) and the resultant hydrogel for sequestering 16S rRNA selectively via divalent interaction (Figure 24, Scheme 75). 586 self-assembles to form a transparent hydrogel containing small fibrils with diameters from 50 to 60 nm at neutral pH with a CGC of 0.3 wt %. Kanamycin A in the hydrogel of 586 likely binds to the A-site of 16S rRNA. The association constant between 586 and 16S rRNA is much higher than the binding constant of kanamycin A with 16S rRNA, suggesting cooperative binding.¹²⁰² Xu et al. designed and synthesized another hydrogelator (587) by using a tripeptide derivative that consists of a naphthyl group, two phenylalanines, and one modified lysine residue carrying an olsalazine moiety (a clinically used anti-inflammatory prodrug) in the side chain of the lysine residue. They found that 587 self-assembles to form supramolecular hydrogels under mildly acidic conditions with a CGC of 0.8 wt %. In addition, the reduction of olsalazine not only leads to a gel-to-sol phase transition but also controls the release of 5-aminosalicylic acid as the anti-inflammatory agent, which potentially provides a way to encapsulate the prodrug and release the active ingredients upon biological cues.¹²⁰³

Figure 24.

Supramolecular nanofibers sequester the potential targets (e.g., 16S rRNA (in red)) in the gel phase. Adapted with permission from ref (1202). Copyright 2012 Royal Society of Chemistry.

Scheme 75. Representative Molecular Structures of Hydrogelators Conjugated with Drugs.

Xu et al. designed another hydrogel precursor (588) based on paclitaxel, a well-established antineoplastic agent that binds specifically to the β-tubulin subunit of microtubules to arrest mitosis and result in apoptosis. Upon the action of alkaline phosphatase, 588 turns into a hydrogelator (589) that self-assembles to form nanofibers with a uniform width of 29 nm and affords a supramolecular hydrogel of the paclitaxel derivative. The MTT cell viability assay indicated that, after 48 h of incubation with HeLa cells, 588 exhibited an IC₅₀ value of 25 nM, which is comparable to that of paclitaxel (13.5 ± 2.2 nM). In addition, 589 itself also exhibited an IC₅₀ of 25 nM, which is also comparable to that of paclitaxel and 588.¹⁵⁵ Miravet et al. reported a class of innovative hydrogelators (590) by connecting a gel-forming lysine moiety with model drugs (e.g., benzylamine and phenethylamine) through a self-immolating spacer (p-aminobenzyloxycarbonyl). TEM images showed that 590 self-assembles to form hydrogels containing fibers with dimensions ranging from hundreds of nanometers in width and several micrometers in length. The authors found that trypsin catalyzes the hydrolysis of the amide linkage between the gelator moiety and the spacer of 590, thus releasing the model drug.¹²⁰⁴

As shown in Scheme 76, Yang et al. designed and synthesized the first example of a folic acid (FA)–paclitaxel conjugate,¹²⁰⁵ FA-G_pYK-paclitaxel (591). This innovative precursor contains paclitaxel, an effective, clinically used anticancer drug, folic acid, a ligand targeting cancer cells, and tyrosine phosphate, an enzymatic trigger for self-assembly.¹²⁰⁶ Upon dephosphorylation catalyzed by phosphatases, 591 forms a transparent hydrogel in PBS buffer at a concentration of 0.2 wt % with a nanosphere morphology. In addition, 591 also acts as a prodrug for releasing paclitaxel upon ester cleavage.¹²⁰⁷ Later, Yang et al. designed and synthesized another precursor (592) that contains two complementary anticancer drugs, dexamethasone (Dex), an anti-inflammatory and immunosuppressant, and paclitaxel or hydroxycamptothecin (HCPT). Upon the reduction by GSH and DTT, 592 turns to 593, which self-assembles to form hydrogels with CGCs of about 0.25 and 0.75 wt %, respectively, in PBS buffer. Four hours after the formation of the hydrogels, the authors added an equal volume of fresh PBS buffer solution and observed the original drug molecules of Dex, paclitaxel, and HCPT releasing from the gels due to the hydrolysis of the ester bond.¹²⁰⁸ They also developed a series of hydrogelators based on paclitaxel and short peptides/amino acids with simple synthetic strategies and high yields that could be used for the release of paclitaxel without any burst releases.¹²⁰⁹

Scheme 76. Representative Molecular Structures of Hydrogelators Conjugated with Drugs.

As shown in Scheme 77, Yang et al. also designed a precursor (594) by using a releasable disulfide carbonate linker to form stable molecular hydrogels. Although 594 is unable to be completely converted from the gelator terminated by a free thiol group to the gelator terminated by a hydroxyl group, the authors succeeded in generating a stable molecular hydrogel mainly formed by the hydrogelator terminated with a hydroxyl group. In addition, upon the endosomal reduction of the disulfide bond, a self-cyclization process results in the unmodified drug.¹²¹⁰ Yang and Zhao et al. combined a galactose-caged nitric oxide (NO) donor and a short peptide of Nap-FFGGG to generate a hydrogelator (595) that forms hydrogels in PBS buffer at a concentration of 0.5 wt %. Using the β-galactosidase to remove the protective galactose from the NO donor, the authors demonstrated enzyme-triggered release of NO from the hydrogel of 595. Furthermore, the in vivo experiments showed that the two-component hydrogels of 595 improve wound healing of mice (Figure 25).^{943,1211−1214}

Scheme 77. Representative Molecular Structures of Hydrogelators Conjugated with Drugs.

Figure 25.

(A) Percentage of wound area left in different groups at day 7 compared to the original wound area (mean ± SEM) at day 0. (B) Photographs of wounds in animals treated with PBS buffer, 3 (hydrogel containing 1.0 wt % 3), free NO + GAL (solution containing 0.2 wt % NO donor with daily addition of 1.5 × 10^–4 U of β-galactosidase), NO gel (hydrogel containing 1.0 wt % 3 and 0.6 wt % 595 without the addition of β-galactosidase), and NO gel + GAL (hydrogel containing 1.0 wt % 3 and 0.6 wt % 595 with the addition of 1.5 × 10^–4 U of β-galactosidase each day). Adapted with permission from ref (1211). Copyright 2013 Royal Society of Chemistry.

Kim et al. designed and synthesized an amphiphile (596) containing riboflavin, an essential biomolecule (vitamin B₂) that is involved in various biochemical processes. 596 forms hydrogels at a concentration of 1.6 wt % in acidic (pH 5) and neutral (pH 7.4) buffer solution with mild heating. The authors found that the hydrogel of 596, being cell compatible, helps deliver VEGF-siRNA efficiently into human cells.^307,1215 Xu et al. conjugated anti-HIV reverse transcriptase inhibitor 2′,3′-dideoxy-3′-thiacytidine (3TC) or azidothymidine (AZT) to a versatile self-assembly motif of hydrogelators 597 to form supramolecular nanofibers as the matrixes of hydrogels in weak acidic conditions. In the presence of prostatic acid phosphatase (PAP), the hydrogels exhibit drastically enhanced elasticity, which should help to match the change of the physiological environment. In addition, the hydrogelators are biocompatible, and are able to release the HIV inhibitors under physiological conditions.¹²¹⁶ Cui et al. reported the synthesis and assembly of a type of innovative amphiphile (598) containing a very bulky anticancer drug, paclitaxel, and a short peptide. With a relatively high loading (41%) of paclitaxel, 598 self-assembles to form nanofibers typically a few micrometers in length and with a diameter of 11.8 ± 1.3 nm in PBS buffer. The addition of GSH induces the release of paclitaxel, and the release depends on the concentration of 598. In addition, the in vitro cytotoxicity of 598 showed that the amphiphile inhibited the growth of cancer cells (e.g., MCF-7, A549, and PC3-flu).¹²¹⁷

5.7. Immunological Modulation

As adjuvants are crucial components of vaccines, safer and more potent adjuvants are gaining increasing interest and attention. As injectable biomaterials for drug delivery and tissue engineering, supramolecular hydrogels made of peptides or peptide derivatives are excellent candidates as adjuvants because of their low cost of production, ease of being produced in large quantities, and relatively high activity and stability. These merits of peptides or peptide derivatives promise their applications in cancer immunotherapies and vaccination against infectious diseases, particularly for enhancing the potency of vaccines or for delivery of vaccines. For example, Sun et al. reported a self-assembling peptide, FLIVIGSIIGPGGDGPGGD (599) (Scheme 78), consisting of two native sequences from an elastic segment of spider silk and a transmembrane segment of the human muscle L-type calcium channel. They prepared supramolecular hydrogels with 599 either by changing the pH of its solution or by adding the cation Ca²⁺ at a concentration of 0.5 wt % and found that the two resulting hydrogels formed by 599 through different approaches have distinct physical properties. They also found that the shear-thinning, rapid-strength-recovering hydrogel made of 599 and Ca²⁺ can be used as an H1N1 influenza vaccine adjuvant which is biologically safe and improves the immune response by 70% compared with an oil-based commercial adjuvant.¹²¹⁸ They also evaluated the potential of 599 to act as an adjuvant for the porcine reproductive and respiratory syndrome virus (PRRSV) attenuated live virus (MLV) vaccine. Their studies suggest that the supramolecular hydrogel of 599, when combined with the PRRSV MLV vaccine, can enhance the vaccine efficacy against two different PRRSV strains by modulating both the host humoral and the host cellular immune responses.¹²¹⁹ Jiang and Yang et al. also reported a nanovector composed of a peptide-based nanofibrous hydrogel formed by NapGFFY-NMe (600) with a CGC value of 0.01%. According to their report, 600 can condense DNA to lead to a strong immune response against HIV by activating both humoral and cellular immune responses in mice. Their results indicate that the peptide-based nanovector promises biocompatibility and may provide a safe, straightforward, and effective approach for HIV DNA vaccines⁹⁴³ if HIV DNA vaccines are effective in humans (Figure 26).¹²²⁰ Moreover, the impressive potency of 600 indicates its applications in other immunotherapies such as its use as an adjuvant in cancer immunotherapy.

Scheme 78. Representative Molecular Structures of Hydrogelators for Immunological Modulation.

Figure 26.

Process of a peptide-based nanofibrous hydrogel enhancing the immune responses of HIV DNA vaccines. Adapted from ref (943). Copyright 2014 American Chemical Society.

Besides being immunomodulating adjuvants, synthetic peptides or peptide derivatives are excellent candidates as antigens due to their precise chemical definitions, which allow one to specify the exact epitopes against an immune response. However, most peptides, despite being antigenic, are poorly immunogenic by themselves, thus requiring the assistance of strong adjuvants. Thus, the unique properties of self-assembling peptides motivate the pursuit of self-adjuvanting or adjuvant-free systems derived from peptides. Cao et al. developed a vaccine with mutant Aβ peptides 601–606 that avoid the use of an adjuvant. They demonstrated that these adjuvant-free vaccines with different Aβ peptides are able to induce a good antibody response without stimulating an unwanted inflammation reaction, thus acting as a safe vaccination approach against Alzhheimer’s disease.¹²²¹ Another example of a self-adjuvanting vaccine is derived from the understanding of MUC1 (mucin 1, cell surface associated). MUC1 proteins are key targets of the vaccines for epithelial tumors, which have a variable number of tandem repeats bearing tumor–tumor-associated carbohydrate antigens. However, short MUC1 peptides usually exhibit low immunogenicity, which remains a major obstacle in cancer vaccine development. To overcome this problem, Li et al. synthesized and evaluated a class of synthetic self-adjuvanting vaccine candidates (607–610) comprising a B-cell epitope with different glycosylation patterns and a nonimmunogenic self-assembling domain. They found that all of the peptide derivatives self-assemble into fibers over 200 nm long in aqueous solution at a concentration of 400 μM and display B-cell epitopes on the fiber surfaces. They demonstrated that the vaccine with Tn glycosylation in the PDTRP domain (610) after intraperitoneal injection elicits a significant immune response in mice.¹²²²

Collier and Rudra et al. investigated the molecular determinants and immunological mechanisms leading to the significant immunogenicity of the self-assembling peptide OVA-Q11 (611), which elicits strong antibody responses in mice.^1223−1226 Their results showed that the deletion of amino acid regions in the peptide recognized by T cells or the mutation of the key residues in the self-assembling domain to prevent fibrillization could diminish or attenuate the immunogenicity of the peptides. Using a different self-assembling sequence to make OVA-KFE8 (612), which also self-assembles to form nanofibers and elicits a strong immune response, they demonstrated that 407 and KFE8 themselves are unrelated to immumogenicity while it is the peptide assembly that matters. Collier et al. concluded that a key strategy for modulating the immunogenicity appears to center on the effective T cell epitope, and this appears to be broadly applicable to fibrillar peptide assemblies.¹²²⁷ Collier et al. also utilized the same self-assembling domain 407 to design and develop another self-adjuvanting supramolecular vaccine which carries a folded protein antigen. They first synthesized pNP-Q11 (613), having a pNP (p-nitrophenyl phosphonate) ligand⁹¹⁹ on the N-terminal of the 407 domain. In parallel they designed and expressed a fusion protein (cut-GFP) containing cutinase and GFP domains separated by a flexible linker of glycine and serine residues. They found that 613 self-assembles to form nanofibers, with a morphology similar to that of the nanofibers of 407, and the resulting nanostructure remains unchanged after reaction with cutinase fusion proteins. The cutinase–pNP interaction allows antigens to be conjugated without destroying their tertiary structures. Their results demonstrate that the nanofibers bearing GFP elicit robust anti-GFP antibodies, which indicates that the supramolecular assemblies can act as self-adjuvanting vaccines for whole-protein antigens.¹²²⁸

In addition to boosting the immune response, supramolecular hydrogels formed by peptide or peptide derivatives also suppress immunity. Xu et al. designed and synthesized a conjugate, 614, of a self-assembling motif and l-rhamnose to examine its immunomodulatory properties (Scheme 79). They found that 614 self-assembles in water to form a weak hydrogel (0.4 wt %) which allows the encapsulation of a fluorescent model antigen, (R)-phycoerythrin (PE). Surprisingly, they found that the resulting hydrogel, in contrast to the properties of monomeric l-rhamnose, suppresses the antibody response of mice to PE.¹²²⁹ Several laboratories also explored supramolecular hydrogels as anti-inflammatory agents. Xu et al. demonstrated that the covalent conjugation of d-amino acids to naproxen (i.e., a nonsteroidal anti-inflammatory drug (NSAID)) not only affords supramolecular hydrogelators (e.g., 615, 616, 617, and 618) for potential topical anti-inflammatory gels but also significantly raises the selectivity toward COX-2 about 20-fold at little expense of the activity of naproxen.¹²³⁰ Similarly, Dastidar et al. conjugated naproxen with β-amino acid to generate a variety of supramolecular conjugates such as 619, 620, 621, and 622, all of which are able to gel pure water, NaCl solution, or PBS buffer with CGC values of 0.80–2.0 wt %. They found that all of the hydrogelators display an anti-inflammatory response comparable to that of the parent drug.¹²³¹

Scheme 79. Representative Molecular Structures of Hydrogelators for Immunological Modulation.

5.8. Wound Healing

Although many wound dressings have entered clinical use, these wound dressings still are unable to fully satisfy the requirement of wound healing. An ideal wound-healing therapeutic should offer an optimal microenvironment to achieve a rapid wound closure, a functionally satisfactory recovery, and minimal scar formation.^1232,1233 Hydrogels are of great interest as wound dressing or its component because hydrogels preserve the gaseous permeability, provide a hydration environment, absorb wound exudate, and serve as the matrixes for drug delivery.¹²³⁴ However, current wound-healing hydrogels have only limited functions and are still unable to adequately match the complexity of wound-healing processes. Despite the enormous challenges in the development of hydrogels for wound healing, the exploratory works described in the following have provided useful insights for further development of supramolecular hydrogels for wound healing.

On the basis of the biological functions of glucosamine in the wound-healing process,¹²³⁶ Xu et al. designed a hydrogelator (623) containing glucosamine. 623 is able to form a hydrogel at a concentration of 0.2 wt %. The preliminary animal model study found that the application of the hydrogel of 623 to the mice with a skin wound promotes wound healing and reduces the formation of scars, compared to the results for the control mice without the treatment (Figure 27).¹²³⁵ One intriguing observation is that the use of l-Phe in 623 fails to afford a wound-healing hydrogel, which indicates that the subtle structure change in the glycopeptide may have profound impacts on both hydrogelation and their biological functions. It would valuable to understand the underlying mechanisms of this kind of observation. In another experiment on wound healing, Xu et al. used disodium pamidronate (624) (Scheme 80), a clinically used drug that binds with UO₂²⁺, to generate a supramolecular hydrogel with Fmoc-Leu (533) and Fmoc-Lys (201). Four equivalents of 624 plus 533 and 201 yields a transparent hydrogel at pH 10.4. The resulting hydrogel is able to reduce the uranyl ion poisoning in the wound on mice because the pamidronate remains active in the form of a hydrogel.^139,283 Later, the authors conjugated pamidronate with the motif of naphthelene-l-Phe-l-Phe to generate hydrogelator 625, which self-assembles to form nanofibrils and induces hydrogelation. The hydrogel of 625 significantly reduces the amount of uranyl nitrate in the kidney and enhances the survival rate of the wounded mice.²⁸⁴

Figure 27.

(A) Molecule 623 self-assembles to form a hydrogel. Gross appearance of the wound site treated without (B) or with (C) the gel on day 6. Adapted with permission from ref (1235). Copyright 2007 Royal Society of Chemisty.

Scheme 80. Some Supramolecular Hydrogelators for Wound Healing.

The synthetic self-assembling peptide 248a, initially discovered by Zhang et al.,¹⁸¹ is able to form nanofibers under physiological conditions and results in a hydrogel with a CGC of 0.1 wt %. Serving as a wound dressing, the hydrogel of peptide 248a can reduce the edema of burn wound, advance the beginning and disappearance of eschar, and speed wound contraction. Zhao et al. suggested that the hydrogel of 248a provides an optimal hydration microenvironment and simulates various cytokines and growth factors in the extracellular matrix to confer a beneficial effect.¹²³⁷ In another experiment, Ellis-Behnke and So et al. found that 248a is able to immediately stop bleeding of a wound in the brain, spinal cord, femoral artery, liver, or skin of mammals.^1238,1240 Zhao et al. reported a molecular mechanism for such an observation.¹²³⁹ Zhang et al. designed another peptide, EAK16, for rapid homeostasis.¹²⁴¹ On the basis of a similar principle, Hauser et al. developed two supramolecular hydrogels based on the short peptides Ac-ILVAGK-NH₂ (626) and Ac-LIVAGK-NH₂ (627). 626 and 627 form rigid and transparent hydrogels in PBS buffer at the concentrations of 0.5 and 0.75 wt %, respectively. Compared to the standard-of-care wound dressing Mepitel,¹²⁴² the authors reported that the hydrogels of these two peptides, in a rat model, result in earlier onset and completion of autolytic debridement, and promote epithelial and dermal regeneration without the exogenous growth factor.¹²⁴³ It would be important to correlate the in vivo stability of these peptides with this exciting result. After mixing a dendron, 628, and the polymer 629 in PBS buffer, Grinstaff et al.¹²⁴⁴ prepared a thioester hydrogel within several seconds at a concentration of 30 wt %. This resulting hydrogel is transparent, adhesive, and cell compatible, and exhibits strong mechanical properties even after swelling 4-fold in PBS buffer. As a hydrogel sealant for wound closure, this hydrogel could be washed away from the skin by simply using a thiolate solution during surgical care.

5.9. Unique Biological Functions of Supramolecular Hydrogelators

Compared to the conventional polymeric hydrogels,^1245,1246 self-assembly is a ubiquitous feature of supramolecular hydrogels. Considering self-assembly of proteins to generate assemblies that are crucial for cellular functions (e.g., actins and tubulins to form cytoskeletons¹²⁴⁷), the biological functions of supramolecular assemblies of small molecules are scientifically intriguing and increasingly significant in biology and medicine. The development of supramolecular hydrogelators, thus, provides a new frontier for scientists to explore molecular self-assemblies at the intersection of supramolecular chemistry and cell biology.¹⁹⁰ In the following section, we discuss how the development of supramolecular hydrogelators and hydrogels leads to the interplay between supramolecular assemblies of small molecules and proteins (Figure 28) as a new paradigm in chemistry and in biology. We first introduce enzyme-instructed self-assembly (EISA),³⁴ a process that allows the control of the formation and the location (extra- or intracellular) of the supramolecular assemblies and hydrogels. Second, we highlight several examples of enzyme-instructed self-assembly in a cellular environment. Third, we illustrate the use of the hydrogels formed by the assemblies of small molecules to bind proteins, including the molecular hydrogel protein binding (MHPB) assay^883,884,1248 and the assemblies of small molecules promiscuously interacting with proteins inside cells to control the cell fate.

Figure 28.

Interplay between supramolecular assemblies and proteins. (I) Enzyme-instructed self-assembly (EISA): the enzyme transforms a precursor to the self-assembling small molecules (i.e., hydrogelator) to form the supramolecular assemblies (in the form of nanofibers/hydrogel). (II) Molecular hydrogel protein binding (MHPB) assay: the hydrogels formed by the supramolecular assemblies bind proteins for proteomic analysis and identification of the protein targets of the assemblies.

5.9.1. Enzyme-Instructed Self-Assembly To Form Supramolecular Hydrogels

To investigate the biological functions of the supramolecular assemblies of small molecules, one has to generate supramolecular assemblies in a cellular environment. Enzyme-instructed self-assembly (EISA)—the integration of enzymatic transformation and self-assembly—of small molecules, which usually results in the formation of supramolecular hydrogels, has provided a facile approach to examine and to create supramolecular assemblies in a cellular environment. In general, there are two strategies of enzyme-instructed self-assembly for generating supramolecular nanofibers—making or breaking bonds. Both routes allow the enzyme to convert a precursor to a hydrogelator which self-assembles in the aqueous phase to form nanofibers and results in hydrogelation (Figure 29). Such a relatively simple design permits enzymatic formation of supramolecular nanofibers to be applicable on (almost) any gelators^{9,226,1157,1249−1253} because the attachment of a hydrophilic segment to a hydrogelator easily generates a precursor that is soluble in the aqueous phase. The removal of the hydrophilic segment by the enzyme-catalyzed bond cleavage converts the precursor back to the hydrogelator, which self-assembles into nanoscale assemblies (e.g., nanofibers) and affords the hydrogel.^34,153 Similarly, an enzyme can catalyze bond formation to link two precursors together to create a hydrogelator that self-assembles into nanofibers.¹⁶² One obvious advantage is the self-assembly of the hydrogelator to exhibit a selective response to the biological environments because the expression of enzymes in a living organism usually is highly specific in a spatiotemporal manner.

Figure 29.

Illustration of EISA to form supramolecular nanofibers via bond formation or bond cleavage and the macroscopic outcomes (i.e., viscosity change or hydrogelation).

As discussed earlier (Scheme 6¹⁵³), Xu et al. demonstrated enzyme-instructed self-assembly of small molecules based on an alkaline phosphatase and an amino acid derivative.¹⁵³ Catalyzing the removal of phosphate groups from a variety of substrates containing the phosphate group, phosphatase can control the balance between hydrophilicity and hydrophobicity, thus converting a precursor to a hydrogelator which self-assembles in water to form supramolecular nanofibers. As shown in Figure 30, the commercially available Fmoc-tyrosine phosphate (14) dissolves in a weak alkaline aqueous solution. The addition of alkaline phosphatase to the solution converts 14 to a hydrogelator, 15, which self-assembles into a three-dimensional network of nanofibers and affords a hydrogel.¹⁵³ Because phosphatases, prevailingly existing in the cellular environment and constituting a large family of enzymes, act as the integral component of the canonical phosphatase/kinase enzyme switch that dictates cellular signaling, this seemingly simple process promises many possibilities and has opened a new paradigm of molecular biomaterials, as evidenced by the subsequent research.^{30,34,140,152−154,156,288,883,888,1254−1259}

Figure 30.

(A) Molecular structures of the precursor 14 and its corresponding hydrogelator 15 and the enzymatic transformation. (B) Transmission electron microscopy (TEM) image of the nanofibers made by the self-assembly of 15. Optical images of (C) the solution of 14 in alkali buffer (pH 9.8) and (D) the hydrogel formed by adding the phosphatase to the solution of 14 to produce the nanofibers of 15. Adapted with permission from ref (153). Copyright 2004 Wiley-VCH Verlag GmbH & Co. KGaA.

Unlike phosphatases that catalyze hydrolysis to trigger molecular self-assembly, thermolysin catalyzes the formation of a covalent bond via reverse hydrolysis. Ulijn et al.¹⁶² reported the use of thermolysin to couple two peptide derivatives (200 and 208) to make a hydrogelator (447) which self-assembles to form a three-dimensional network of nanofibers and affords a hydrogel (Figure 31). Since thermolysin catalyzes the reverse hydrolysis of many substrates, especially hydrophobic amino acids and peptides, it greatly expands the scope of enzyme-instructed self-assembly and the formation of supramolecular nanofibers/hydrogels. Although bond formation may serve as a useful route to produce hydrogels as scaffolds for tissue engineering,⁹⁶ the low solubility of the hydrophobic precursors in the aqueous phase likely limits their application (especially in vivo).

Figure 31.

(A) Structures of the precursors 200 and 208 and the hydrogelator 447 and the enzymatic transformation. (B) SEM image of the corresponding nanofibers (scale bar 500 nm). Inset: optical image of the hydrogel. Adapted from ref (162). Copyright 2006 American Chemical Society.

Many advances have taken place in the development of enzyme-instructed self-assembly.^{14,890,922,1260−1263} On the basis of the enzyme switch in the cellular process,¹⁷⁷ Xu et al. designed a substrate that undergoes phosphorylation and dephosphorylation catalyzed by an alkaline phosphatase and a tyrosine kinase, and examined the use of the kinase/phosphatase switch to regulate the formation of nanofibers/hydrogels.¹²⁵⁴ As shown in Figure 32A, a pentapeptide derivative, Nap-FFGEY (630; Nap = 2-(naphthalen-2-yl)acetic acid; F = phenylalanine (Phe); G = glycine (Gly); E = glutamic acid (Glu); Y = tyrosine (Tyr)), self-assembles into nanofibers and results in a hydrogel at 0.6 wt %. The addition of a kinase to the hydrogel in the presence of adenosine triphosphates (ATPs) phosphorylates 630 to give the corresponding precursor 631, thus disrupting the self-assembly to induce a gel–sol phase transition and produce a solution; treating the resulting solution with a phosphatase converts the precursor 631 to the hydrogelator 630, again, thus repeating the self-assembly of the hydrogelator to form the network of nanofibers and afford the hydrogel. Besides illustrating a general way of using enzymes to instruct the formation or disassembly of supramolecular nanofibers, this work demonstrates that enzyme-instructed self-assembly generates more ordered self-assembling nanostructures than a simple adjustment of the pH does (Figure 32B).¹²⁵⁴

Figure 32.

(A) Structures of the precursor 630 and the hydrogelator 631 and the corresponding transformations catalyzed by phosphatase and kinase. (B) TEM images showing (I, left) the nanofibers of 630 formed by adjusting the pH, (II, middle) the absence of nanofibers due to enzymatic phosphorylation of 630, and (III, right) the restored nanofibers of 630 by enzymatic dephosphorylation of 631. (C) Optical images of (I) the hydrogel of 630 formed by changing the pH, (II) the solution obtained by treating the hydrogel with a kinase and ATP (at 50% conversion), and (III) the hydrogel of 630 restored by adding phosphatase. Adapted from ref (1254). Copyright 2006 American Chemical Society.

To serve as therapeutic agents, the supramolecular nanofibers have to be innocuous to normal tissues. Therefore, it is essential to evaluate the biochemical properties (e.g., biocompatibility, biodurability, and toxicity) of enzyme-instructed supramolecular nanofibers in vivo. After confirming that 630 is biocompatible, Xu et al. injected the solution of 631 into mice to evaluate enzymatic formation of the nanofibers and the hydrogel of 630 in vivo. They observed that the hydrogel forms at the location of subcutaneous injection (Figure 33A). HPLC analysis of the hydrogel reveals that 80% of precursor 631 turns into hydrogelator 630. On the basis of the weight change of the mice after being injected with 631 (Figure 33B), subcutaneous administration of 631 at the experimental dosage results in little acute toxicity to the mice. Moreover, because the enzyme-catalyzed reaction quickly converts 631 to the biocompatible molecule 630, there is hardly long-term in vivo toxicity of 631.¹²⁵⁴

Figure 33.

(A) Optical image of the hydrogel formed 1 h after subcutaneously injecting the solution of the precursor 631 into the mice. (B) Weight gain of the mice (n = 6, initial body weight 20 ± 2 g) after subcutaneously injecting 0.5 mL of 631 at 0.8 wt % concentration. A saline solution (0.5 mL) served as the control. Adapted from ref (1254). Copyright 2006 American Chemical Society.

To generate supramolecular nanofibers that resist hydrolytic enzymes (e.g., proteases) in challenging biological conditions (e.g., biological fluids), Xu et al. designed a precursor based on a β-amino acid.¹²⁵⁵ As shown in Figure 34, tyrosine phosphate attaches at the C-terminal of a β-amino acid derivative to afford a precursor (632) which serves as a substrate of phosphatase. After being treated with a phosphatase, 632 hydrolyzes to give a hydrogelator (633) which self-assembles to afford nanofibers. Moreover, this enzymatic formation of nanofibers proceeds in complex and challenging biofluids (e.g., blood and cytoplasm) that contain a variety of proteases and results in the hydrogelation of these fluids (Figure 34C,D).¹²⁵⁵ The β-peptide-based nanofibers exhibit a longer half-life than the α-peptide nanofibers do.¹²⁵⁵ The excellent biostability renders β-amino acids and other non-natural amino acids as potential candidates for creating supramolecular hydrogels for long-term biomedical applications.

Figure 34.

(A) Structures of the precursor 632 and the hydrogelator 633 and the enzyme-catalyzed transformation. (B) TEM image of the nanofibers of 633. (C) Hydrogel formed by mixing blood, PBS buffer, and alkaline phosphatase. (D) Gel formed by mixing the solution of 632 (1.0 wt % in PBS buffer, pH 7.4), alkaline phosphatase, and the cytoplasm collected from 1.0 × 10⁶ broken HeLa cells. Adapted with permission from ref (1255). Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

β-Lactamases are an important family of bacterial enzymes that catalyze the hydrolysis of β-lactam antibiotics and have caused widely spread antimicrobial drug resistance.^1264,1265 Xu et al. explored enzyme-instructed self-assembly by β-lactamases.¹⁵⁹ As shown in Figure 35, the precursor 634, consisting of a cephem nucleus as the linker coupling a hydrophilic group and a proper hydrogelator, is too soluble to self-assemble in water and bacteria cell lysates. Upon the action of a β-lactamase, the lactam ring opens to release the hydrogelator 635, which self-assembles to form nanofibers and to afford a hydrogel. This facile process allows the detection of β-lactamase in the lysates of bacteria. Specifically, β-lactamase in a bacterial lysate could convert the precursor 634 to its corresponding hydrogelator 635, resulting in the formation of supramolecular nanofibers (Figure 35C–F). Without β-lactamase, no nanofiber was observed. This result not only confirms the stability of the nanofibers of 635 in bacteria lysates consisting of a wide range of enzymes, but also suggests that one may use β-lactamase to control the self-assembly of small molecules as a general platform to target antimicrobial-drug-resistant Gram-negative bacteria since only bacteria express β-lactamases.

Figure 35.

(A) Structures of the precursor 634 and the hydrogelator 635 and the β-lactamase-catalyzed transformation. (B) TEM images showing the enzymatic formation of nanofibers of 635: top, the solution, bottom, the gel. (C–F) Images showing formation of nanofibers of 635 in the lysates of E. coli that express different β-lactamases (C, CTX-M13; D, CTX-M14; E, SHV-1; F, TEM-1). Adapted from ref (159). Copyright 2007 American Chemical Society.

Xu et al. also reported the use of β-galactosidase to remove galactose from precursor 637 in water to trigger the self-assembly of the corresponding hydrogelator and to result in a hydrogel at a concentration of 0.1 wt % at pH 7.5.¹⁶⁸ The synthetic difficulty of saccharides is apparently the limiting factor for the exploration of enzymatic hydrogelation using glycolytic hydrolases. In another experiment, Xu et al. designed and synthesized a short peptide with a sequence of FFFFCGLDD (638) as a substrate of matrix metalloprotease-9 (MMP-9). After successfully cleaving the hydrophilic residue of LDD off 638, the resulting hydrogelator (FFFFCG) self-assembles to form nanofibrils and affords a hydrogel at a concentration of 0.4 wt %. Since MMP-9 is an important enzyme related to the invasiveness and metastatic potency of human malignant tumors, this work may lead to a new strategy for making biomaterials or therapeutics for cancer therapy or other diseases.¹⁶⁴ Later, Xu et al.¹¹¹ reported an esterase-based approach to generate supramolecular hydrogels. By using esterase to hydrolyze the precursor 7, the resulting hydrogelator 8 self-assembles to form a hydrogel at a concentration of 0.8 wt % and pH 7.4. One intriguing feature of this work is that the hydrogel of 8 is stable over a wide pH range, likely due to the presence of the alcohol group instead of a carboxylic group at the C-terminal. Moreover, breaking the ester-bond apparently is the only path to make hydrogel of 8. Relying on both aromatic–aromatic interactions and enzyme catalysis, Xu et al. reported that enzymatic dephosphorylation of the precursor 639 generates a hydrogel consisting of spontaneously aligned supramolecular nanofibers as the matrix of the gel.¹⁵² As shown in Scheme 81, Xu et al. also designed a new class of conjugates (640) containing a nucleobase, amino acids, and a saccharide which afford a supramolecular hydrogel (0.5 wt %) upon addition of phosphatase. Furthermore, 640 is able to inhibit the proliferation of HeLa cells.¹²⁵⁷ In another experiment, Xu et al. reported that 641 affords a supramolecular hydrogel at a concentration of 0.5 wt % in the presence of acid phosphatase (AP). The immobilized AP in the hydrogel shows higher activity and stability compared with free AP in the same solvent.¹⁵⁴ Integrating enzymatic catalysis and self-assembly, Xu et al.⁷⁰² reported a feasible way to prepare the supramolecular hydrogel of an adenosine derivative (642). This exploration may lead to a new type of biomaterial because of the ubiquitous importance of adenosine 5′-monophosphate (AMP) in bioenergetics, metabolism, and transfer of genetic information. Meanwhile, Xu et al. also prepared a series of nucleopeptides by connecting a nucleobase with Phe-Phe which self-assemble to form nanofibers and trigger hydrogelation at a concentration of 2.0 wt % and pH 5.0.⁹⁶⁵ It is worth noting that the nucleopeptides exhibit better proteolytic resistance than their corresponding peptides.

Scheme 81. Molecular Structures of Representative Hydrogelators Formed via Enzymatic Transformation.

Ulijn et al. further explored the use of thermolysin to catalyze the bond formation of a series of peptides for self-assembly and hydrogelation.^1266,1267 In one case, they reported that thermolysin catalyzes the condensation of Fmoc-Ser and phenylalanine to form 643 (Scheme 82). The favorable product 643 of the condensation self-assembles to form nanosheets that result in an opaque hydrogel.¹²⁶⁸ Later, they used subtilisin to hydrolyze Fmoc-peptide methyl esters (e.g., 644) to obtain the corresponding hydrogelator 645,¹²⁶⁹ which self-assembles to form nanotubes and affords a hydrogel.¹⁶⁰ They also reported a case in which enzyme-catalyzed dephosphorylation of precursor 646 affords a hydrogel at a concentration of 0.55 wt %. This work provides useful insights into the mechanism of 646 transforming from micelles to nanofibers.¹²⁷⁰ By incorporating a metalloproteinase substrate of PVGLIG into self-assembling peptides, Langer et al. prepared an enzyme-sensitive hydrogel of 647 at a concentration of 1.0 wt % for eliciting cell and tissue remodeling activities. Further studies found that enzyme-mediated degradation occurred on the gel surface.¹²⁷¹ While Schneider et al. reported the use of MMP-13 to degrade β-hairpin self-assembled hydrogels by proteolysis,¹²⁷² Collier et al. designed a series of depsipeptides containing ester bonds within the peptide backbone which are able to self-assemble into β-sheet fibrillar materials and degrade via ester hydrolysis with rates controllable by the amino acid proximal to the ester bonds.¹²⁷³ Among them, 648 forms a very stiff hydrogel (G′ > 10⁵ Pa) which becomes soft and dissociates over time. Unexpectedly, C3H10T1/2 cells were encapsulated in the hydrogel of 648 and exhibit better spreading and proliferation than the peptide without an ester bond. Williams et al. reported a new approach in which thermolysin catalyzes the reverse hydrolysis to produce Fmoc-Leu-Leu-Leu (649) efficiently in the presence of laminin. Hydrogelator 649 self-assembles to form nanofibrils that interact with laminin to result in a hydrogel. The authors reported that the immobilized laminin is more stable after being microinjected into a disease site of zebrafish.¹²⁷⁴

Scheme 82. Molecular Structures of Small Representative Hydrogelators Formed via Enzymatic Transformation.

McNeil and Soellner et al.¹⁶⁵ reported a generalizable method to detect protease activity via hydrogelation. They designed a recognition sequence for targeting the protease of interest (e.g., MMP-9 and prostate-specific antigen (PSA)); meanwhile an aminopeptidase removes the residues to release the hydrogelator 650 at physiological conditions. Assuming that the MMP-9 and PSA are potential biomarkers for cancer, the authors suggested that this simple visual assay might be useful for early cancer detection. This innovative two-enzyme approach may find broader applications than the detection of a specific hydrolase. In addition, Ulijn et al. examined the effect of the concentration of alkaline phosphatase on the gelation time, mechanical properties, and molecular arrangement of the enzymatic hydrogelation of 15, and reported that an increase of the enzyme concentration enhances the elastic modulus and the apparent order of the resulting hydrogel.¹⁴⁸ Recently, employing enzymatic dephosphorylation of Fmoc-tyrosine-phosphate (14),¹⁵³ Mann et al. prepared a supramolecular hydrogel to act as a matrix for calcium phosphate mineralization.¹²⁷⁵ SEM suggested that the mineralization occurred along the smooth fiber surface of 15. The authors suggested that this approach may produce biomaterials for tissue engineering, wound treatment, and drug release. Meanwhile, Yang et al. reported the methylation of 14 to afford a slightly more hydrophobic precursor, 651. Although 651 is more hydrophobic than 14, phosphatase still dephosphorylates 651 to result in a supramolecular hydrogel.¹⁵⁷ Yang and co-workers reported a short peptide (Nap-GFFY_P, 652) that results in a hydrogel of Nap-GFFY¹²⁷⁶ at a minimum gelation concentration of 0.08 wt % after dephosphorylation, and its methylation form Nap-GFFY_P-OMe has a minimum gelation concentration of 0.01 wt % after enzymatic conversion, which is one of the most efficient small molecular hydrogelators.^1277,1278 As a group pioneering the study of the Nap-GFFY motif, Yang et al. also extensively explored the properties and applications of the supramolecular hydrogels based on Nap-GFFY derivatives.¹²⁷⁹

5.9.2. Enzyme-Instructed Self-Assembly in a Cellular Environment

5.9.2.1. Intracellular Formation of Supramolecular Nanofibers To Control the Cell Fate

Enzyme-instructed self-assembly allows the exploration of molecular self-assembly in a wide range of biological processes involving enzymes, thus providing abundant opportunities to evaluate intracellular molecular self-assembly of small molecules, a previously unexplored subject. The ability to form supramolecular assemblies inside cells offers a new way to examine the emergent properties of small molecules at a new level of complexity—supramolecular and intracellular, thus providing a multiple-step process to control the fate of cells. As shown in the following sections, these studies have confirmed (at least) two aspects of enzyme-instructed intracellular supramolecular nanofibers: (i) the supramolecular nanofibers change the viscosity of the cytosol and result in selective cell death and (ii) intracellular enzyme catalysis plays the key role.

To form supramolecular nanofibers within a cell, an intracellular enzyme should convert a soluble precursor, which does not self-assemble outside cells, into a hydrogelator that self-assembles to generate the nanofibers inside the cells. To meet this requirement, Xu et al. designed and synthesized precursor 653 as an esterase substrate.¹²⁸⁹ Mammalian cells uptake 653; the endogenous esterases in the cells convert 653 to a hydrogelator, 654. The molecules of 654 self-assemble to form nanofibers, resulting in hydrogelation when a threshold concentration is reached, and thus changing the viscosity of the cytoplasm to cause cell death. As shown in Figure 36, most HeLa cells died at day 3 after the addition of 653 to the culture medium, while most of the NIH/3T3 cells remained alive and dividing. According to the esterase activity assays,¹²⁸⁹ the levels of expression of esterase between these two cell lines are different. With higher esterase activities, HeLa cells likely convert more 653 to 654 than NIH/3T3 cells do. More nanofibers form in HeLa cells than in NIH/3T3 cells, and the resulting nanofibers/hydrogel in HeLa cells cause the cell death. The kinetics of the formation of intercellular nanofibers of 654 is specific to different types of cells, which opens a new paradigm for controlling the fate of cells by enzyme-instructed self-assembly of small molecules.

Figure 36.

(A) An esterase to convert the precursor 653 to the hydrogelator 654. (B) TEM image of the nanofiber formed by 654 (inset: optical image of the hydrogel). MTT assays of (C) NIH/3T3 cells and (D) HeLa cells treated with 653 at concentrations of 0.08, 0.04, and 0.02 wt %. Adapted with permission from ref (1289). Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

To further demonstrate intracellular enzyme-instructed self-assembly to control the cell fate, Xu et al. used two types of E. coli strains: the wild-type BL21 (as the control) and a BL21 strain (BL21(P+)) that overexpresses human tyrosine phosphatase (hPTP). Since the only difference between the two strains is the expression of the phosphatase, any discrepancy in the uptake of the precursor is minimized. After the precursor 656 diffuses into the E. coli, the phosphatase converts the precursor 656 into the hydrogelator 657. 657 self-assembles to form nanofibers and results in hydrogelation. The BL21(P+) bacteria stopped growing upon the addition of 656 (IC₅₀ = 20 μg/mL), but the wild-type BL21 bacteria grew normally (IC₅₀ > 2000 μg/mL) under the same conditions (Figure 37).¹²⁵⁹ On the basis of on HPLC analysis, the authors found significant accumulation of 657 inside BL21(P+) cells. TEM analysis on the broken BL21(P+) cells suggests the formation of nanofibers of 657 inside BL21(P+) cells.¹²⁵⁹ These results confirm that enzymatic formation of the nanofibers and the subsequent hydrogelation inside the bacteria inhibit their growth. This work illustrates that intracellular enzyme-instructed self-assembly allows enzymatic transformation rather than tight ligand–receptor binding¹²⁶⁴ to control the cell fate. Ulijn et al. reported the self-assembly of several Fmoc-protected dipeptide (e.g., Fmoc-Phe-Tyr, and Fmoc-Tyr-Asn) amphiphiles and the design of their corresponding phosphorylated precursors. All the precursors could be dephosphorylated by alkaline phosphatases, generating hydrogelators that self-assemble to form nanofibers. In addition, the peptide amphiphiles showed a similar antimicrobial response when incubated with the phosphatase-overexpressed E. coli.¹²⁸⁰

Figure 37.

(A) A schematic representation of intracellular nanofiber formation and the inhibition of bacterial growth. (B, C) Structures and graphic representations of the precursor 656 and the corresponding hydrogelator 657. (D) TEM image of the nanofibers of 657 (indicated by arrows) formed inside the bacteria after culturing with 656. (E) Concentration of 656 needed to inhibit BL21(P+) and BL21 by forming nanofibers of 657 inside the bacteria. Adapted with permission from ref (1259). Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA.

Xu et al. reported a straightforward method for studying the enzyme-instructed self-assembly of small molecules inside cells. As shown in Scheme 83, they designed a new precursor, 658a, containing a fluorophore which exhibits low fluorescence. After dephosphorylation catalyzed by phosphatases, the resulting hydrogelator 658b self-assembles to form nanofibers that display bright fluorescence. On the basis of this principle, they found that 658a is easily accumulated inside cells to form nanofibers/hydrogel, and thus exhibits a bright spot near the nucleus (Figure 38). Further experiments show that most of the self-assembly of 658b occurs in the endoplasmic reticulum (ER) due to the dephosphorylation catalyzed by a tyrosine phosphatase (PTP1B).¹⁵⁶ This work illustrates a facile approach for studying enzyme-instructed self-assembly of small molecules by other enzymes inside live cells.

Scheme 83. Some Precursors and Hydrogelators for Intracellular Self-Assembly.

Figure 38.

(A) Principle of imaging enzyme-instructed self-assembly inside cells. (B) Chemical structures of 658a. (C) TEM image of the hydrogel made of 658b. (D) Fluorescent confocal microscopy images showing the time course of fluorescence emission inside the HeLa cells incubated with 500 or 50 μM 658a in PBS buffer. Adapted with permission from ref (156). Copyright 2012 Nature Publish Group.

To address the undesired issues in the fluorescence labeling technique (e.g., toxicity and alternation of macromolecular interaction), Xu et al. reported a facile method to image enzyme-instructed self-assembly of small molecules without a high concentration of fluorescent labels inside mammalian cells via a doping method. Specifically, after incorporating a dansyl (DNS)-labeled molecule of 660a into the self-assembly of the native molecule 659a as the fluorescent dopant, they determined the formation, localization, and progression of molecular assemblies generated from the nonfluorescent small molecular hydrogelator by enzyme-instructed self-assembly. After using the cell fraction experiment to confirm that self-assembly occurs in the endoplasmic reticulum (ER), they used correlative light and electron microscopy (CLEM) to further prove that molecular assemblies localized near or inside the ER and are likely processed via the cellular secretory pathway (e.g., ER–Golgi–lysosomes/secretion) by the cells. As shown in Figure 39, CLEM directly correlates the fluorescence signal of 659b/660b molecular assemblies imaged in live cells (Figure 39B–E) with ultrastructural changes of the treated cell in EM (Figure 39F–I). In the fluorescence region, the authors observed a high accumulation of vesicles with low-electron-density material in the cytoplasmic area.²¹² Therefore, this work establishes a general strategy that may reveal the spatiotemporal profile of the assemblies of small molecules inside cells.

Figure 39.

(A) Precursor 659a self-assembles to form nanofibers/hydrogels upon the addition of ALP. (B–I) Correlative light and electron microscopy (CLEM) images of HeLa cells incubated for 48 h with 500 μM 659a and 200 nM 660a. (B–E) Differential interference contrast (DIC) and fluorescence light microscopy images of treated HeLa cells growing on an Aclar plastic film. (F–I) TEM images of the cell of interest shown in (B)–(E). Adapted from ref (212). Copyright 2013 American Chemical Society.

5.9.2.2. Pericellular Formation of Supramolecular Nanofibers To Control the Cell Fate

Unlike intracellular enzymes and the cytosolic catalytic domains of membrane enzymes, ectoenzymes (i.e., an enzyme that locates on the cell surface with catalytic domains outside the plasma membrane) are less explored. However, emerging evidence indicates the important role of ectoenzymes in cellular processes.^1281−1284 Coincidentally, several laboratories are exploring ectoenzyme-instructed self-assembly to form nanofibers on and near the cell surface (i.e., pericellular space).^{288,886,888,894,1256,1258,1285} These studies suggest that one of the promising applications of the pericellular nanofibers is selective inhibition of targeted cells (e.g., cancer cells) without harming normal cells.

Xu et al. reported the first example of enzyme-instructed self-assembly to form hydrogel/nanonets in pericellular space,^288,1256 which selectively inhibits cancer cells, including certain drug-resistant cell lines.²⁸⁸ Specifically, the ectophosphatases (e.g., placental alkaline phosphatases (ALPP)¹²⁸⁶) dephosphorylate the precursor 661a made of a small d-peptide to a hydrogelator, 661b, which self-assembles to form nanofibrils at a concentration of 280 μM and results in pericellular nanofibers/hydrogel (Scheme 84). The resulting hydrogel can prevent the diffusion of a nucleus dye (4′,6-diamidino-2-phenylindole, DAPI) into the cells. A further experiment proves that the pericellular hydrogel is able to block secretary protein/enzyme in the culture medium. Therefore, such blocking of cellular mass exchange has a profound negative effect on the critical cellular activities. An enzyme-linked immunosorbent assay (ELISA) shows that active caspase-3 and active PARP increase significantly with an increase of the incubation time, suggesting that the cells undergo caspase-dependent apoptosis (Figure 40). The authors also found that the enantiomer of 656, an l-peptide derivative, is unable to inhibit HeLa cells due to proteolysis. On the basis of this observation, they investigated how a d-amino acid affects the cellular response to the enzyme-instructed nanofibers by systematically using d-amino acid residue(s) to replace the l-amino acid residue(s) in tripeptidic precursor 656 or its hydrogelator 657. Further studies found that the enantiomeric precursors exhibit dramatically different cellular responses, while enantiomeric hydrogelators show similar cellular responses (Figure 41).¹²⁵⁶ The use of the uncompetitive inhibitor of ALPP abrogates the inhibitory effects of 661a, suggesting that the overexpressed ALPP on HeLa cells is the major enzyme responsible for enzyme-instructed self-assembly and cell inhibition.

Scheme 84. Structures of Precursors That Are the Substrates of Ectophosphatases and the Corresponding Hydrogelators.

Figure 40.

(A) Overlaid images and (B) 3D stacked z-scan image of Congo red- and DAPI-stained HeLa cells after the incubation of the HeLa cell with 661a for 12 h. (C) TEM image of the pericellular hydrogels on the HeLa cells treated by 661a (280 μM). (D) Change of the relative amount of apoptosis signal molecules over time in HeLa cells treated by 661a (280 μM). (E) Cell viability of HeLa cells incubated with 661a (280 μM), 661b (280 μM), and 661a (280 μM) plus l-Phe (1.0 mM). (F) Illustration of enzyme-instructed self-assembly to form pericellular nanofibers/hydrogel and selectively induce cell death. Adapted with permission from ref (288). Copyright 2014 John Wiley and Sons. Adapted from ref (1256). Copyright 2014 American Chemical Society.

Figure 41.

IC₅₀ values of precursors and their hydrogelators on HeLa cells. F and Y indicate phenylalanine and tyrosine. Adapted from ref (1256). Copyright 2014 American Chemical Society.

The most revealing result is that the hydrogelator itself, 661b, at 280 μM, is innocuous to the HeLa cells. This result indicates that ALPP, as the enzyme on the cell membrane, catalytically generates hydrogelators to achieve a high local concentration on the cell surface to form pericellular nanofibers/hydrogel to inhibit the cancer cells. These results further establish enzyme-instructed self-assembly as a multiple-step process to control cell fates. In another experiment, Xu et al. developed a nucleopeptide (662a) as the substrate of CD73, an ectoenzyme. The resulting hydrogelator 662b self-assembles to form nanofibrils and induces a hydrogel at a concentration of 2.0 wt %. One interesting feature of this work is that 662a inhibits HepG2 cells, likely resulting from CD73-instructed self-assembly to form nanofibers of 662b.¹²⁵⁸

In a related study, Ulijn and Pires et al. reported a novel carbohydrate amphiphile (663) that is able to self-assemble into nanofibers upon enzymatic dephosphorylation. More importantly, the authors also confirmed that the membrane-bound alkaline phosphatase expressed by the osteosarcoma cell line (Saos-2) is responsible for triggering the hydrogelation of 382 in the pericellular environment (Figure 42). The resulting pericellular hydrogel reduced the metabolic activity of the cells, which induced cancer cell death.¹²⁸⁵ An important observation by the authors is that the membrane-bound alkaline phosphatase (i.e., an ectoenzyme) is responsible for the selective inhibition of the Saos-2 cells over the ATDC5 cells.¹²⁸⁵ This work, together with the results reported by Xu et al.,^288,1256 further validates the use of enzyme-instructed self-assembly, rather than an enzyme inhibitor, for selectively inhibiting cancer cells without harming normal cells. These results, together with the earlier studies,^1259,1289 are remarkable because they firmly establish the use of a process (i.e., EISA), not simply a molecule (e.g., 661b), for targeting cancer cells.

Figure 42.

(A) Chemical structures of precursors and hydrogelators made of a carbohydrate amphiphile. (B) Enzyme-instructed self-assembly for pericellular nanofiber formation/hydrogelation on Saos-2 cells. Adapted from ref (1285). Copyright 2015 American Chemical Society.

Since it is a challenge to know how the assemblies of small molecules behave in cellular environments to affect the cells, Xu et al. developed a facile and reliable method for evaluating the spatiotemporal profiles of the assemblies of small molecules. In this work, they incorporated a series of fluorophores (e.g., NBD) into a precursor (e.g., 658a) which is transformed to a hydrogelator (658b) upon the addition of phosphatase. Except 666b, all other dephosphorylated molecules form hydrogels at a concentration of 0.6 wt % and physiological conditions.¹⁰⁶³ Cell imaging experiments show that the molecules with different self-assembly properties exhibit a distinct spatial distribution and result in different cellular responses. As shown in Figure 43, 658a enters the cells to form intracellular nanofibers and is able to curtail the effect of an F-actin toxin. Self-assemblies of 664b mainly were localized in the cell membrane, resulting in considerable cytotoxicity. The resulting 665b hardly accumulated in the cell, but self-assembles to form nanofibers outside the cells and exhibits little effect on the cell adhesion. This work not only illustrates a useful approach to visualize and modulate the spatiotemporal profiles of small molecules in a cellular environment, but also serves as a caution to the indiscriminate use of fluorescent aggregates as imaging probes because the interactions between the aggregates and endogenous proteins may interfere with the goal of molecular imaging.

Figure 43.

(A) Molecular structures of the precursors and the hydrogelators containing different fluorophores. (B) Illustration of the distinct spatial distribution of the small molecules in a cellular environment. Fluorescent confocal images of the HeLa cells incubated with 500 μM (C) 658a, (D) 664a, (E) 665a, and (F) 666a for 30 min. Adapted from ref (1063). Copyright 2013 American Chemical Society.

Recently, Maruyama et al. reported that MMP-7 generates hydrogelators made of peptide lipids to result in intracellular self-assembly of the hydrogelator, which leads to the selective inhibition of cancer cells. As shown in Figure 44, they designed a precursor (667, N-palmitoyl-GGHGPLGLAAK-CONH₂) which turns into a hydrogelator (668, N-palmitoyl-GGHGPLGL) after enzymatic hydrolysis by MMP-7. The resulting hydrogelators enter the cells and are accumulated to form nanofibers which result in cancer cell death. The likely key design is the incorporation of a lipid into the peptide because the palmitoyl chain favors the localization of the precursors on the cell surface for the cleavage catalyzed by MMP-7.⁸⁹⁴ Interestingly, the inhibition concentration is around 250 μg/mL, which also falls into the average cytotoxicity of the molecular aggregates.^894,1287 This work illustrates a promising approach that combines extracellular and intracellular self-assembly to control the cell fate.

Figure 44.

(a) Illustration of how enzyme-instructed molecular self-assembly induces cancer cell death. (b) Chemical structures of precursor ER-C16 (667) and hydrogelators G-C16 (668). Adapted from ref (894). Copyright 2015 American Chemical Society.

5.9.3. Assemblies of Hydrogelators Promiscuously Interact with Proteins

The above results^{288,894,1256,1259,1285} are remarkable because they firmly establish the use of a process (i.e., EISA), not simply a molecule (e.g., 661b), for targeting cancer cells. To further understand the details and improve the efficiency of this fundamentally new process, one also needs to elucidate how the supramolecular assemblies of the small molecules promiscuously interact with the endogenous proteins of cells. In the following, we discuss the approaches for helping address the fundamental questions about the mechanisms or the consequences of the self-assembly of small molecules.

5.9.3.1. Molecular Hydrogel Protein Binding (MHPB) Assay

Xu et al. reported the use of a supramolecular hydrogel to discover the interaction between proteins and supramolecular assemblies of small molecules. In this study, Xu et al. designed a supramolecular hydrogelator (669) containing a photoreactive motif. 669 is able to form a transparent hydrogel at a concentration of 0.6 wt % and pH 7.4. Upon UV irradiation, the resulting hydrogel can retain the proteins that bind to the nanofibers in the hydrogels. The bound proteins in the hydrogel are separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and evaluated by silver staining. Tandem MS analysis suggested that the supramolecular nanofibers interact with tubulins, actins, and several other proteins. On the basis of this observation, the authors eliminated the step of photofixation, and directly used the supramolecular hydrogel for discovering the interaction of proteins and assemblies of small molecules.⁸⁸³ Using this hydrogel protein binding assay to evaluate the interaction of cytosol protein with different morphological molecular aggregates formed by the same molecule (Nap-FF, 3), Xu et al. found that the nanofibers of 3 in the hydrogel are able to bind proteins, but the precipitates of 3 bind with few proteins. Moreover, the authors found that two types of nanofibers formed by WFF show similar morphologies and bind with a similar set of proteins. These results indicate that MHPB offers a simple and reproducible method for identifying the protein targets of the supramolecular assemblies of the small molecules.⁸⁸⁴

5.9.3.2. Promiscuous Interactions with Proteins

Xu et al. studied the gelation properties and bioactivities of hydrogelator Nap-FF (3), which contains a naphthyl group and two phenylalanine residues.⁸⁸⁴ They found that 3 is able to self-assemble in PBS buffer to form β-strand-like nanofibers with a uniform width of 24 nm below a CGC of 0.4 wt %. Furthermore, the MTT cell viability assay indicates that the nanofibers of 3 significantly inhibit the proliferation of HeLa and T98G cells at a concentration of 400 μM while showing little toxicity toward PC12 cells. Besides confirming that the nanofibers of 3 disrupt the dynamics of microtubules and consequently induce apoptosis of glioblastoma cells,¹²⁹⁰ the authors also demonstrated that the nanofibers of 3 inhibit tumor growth in the xenograft mice model.⁵⁷³ These results support the approach that uses the supramolecular nanofibrils as de novo molecular amyloids for inhibiting the growth of cancer cells.

Encouraged by the emerging results that show that the assemblies of small molecules play an important role in biology,^{1259,1288−1290} Xu et al. examined the mechanism of how the assemblies of 3 inhibit the proliferation of cells. Using the MHPB assay (Figure 45)^883,884 to investigate the interaction between nanofibers of 3 and cytosol proteins (upper panel of Figure 46a), they found that nanofibers of 3 promiscuously interact with different proteins, particularly with tubulins, vimentin, and actins, as confirmed by Western blot analysis (lane B, lower panel of Figure 46a). Specifically, the absence of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in lane B (lower panel of Figure 46a) indicates that the nanofibers of 3, indeed, bind to proteins in a rather specific manner, despite the promiscuity. Furthermore, the tubulin polymerization assay (Figure 46b) shows that assemblies of 3 significantly reduce the polymerization rate. This result, together with TEM and confocal images (Figure 46c), confirmed that nanofibers of 3 impede the polymerization of microtubules.¹²⁹¹ In addition, confocal fluorescent imaging (Figure 46d,e), together with the MHBP assay, implies that assemblies of 3 also disrupt the dynamics of actin filaments and intermediate filaments of vimentins in cells. By selectively inhibiting the endocytosis processes and measuring the intracellular concentration of 3 (Figure 46f), they determined that both the assemblies of 3 and the monomers of 3 enter the cell via micropinocytosis. Using PathScan apoptosis multitarget sandwich ELISA¹²⁹² to monitor the change of several key signaling molecules in the intrinsic pathway of apoptosis, they found that the assemblies of 3 initiate the activation of Bad and p53, which later activate the caspase cascade and downstream PARP to induce apoptosis in HeLa cells (Figure 46g). On the basis of these results, the authors proposed a partial mechanism for the biological functions of the nanoscale assemblies of 3. As summarized in Figure 46h, the assemblies of 3 enter the cell via macropinocytosis, promiscuously interact with cytoskeleton proteins, and eventually induce apoptosis via the intrinsic pathway of apoptosis. As the first case of nanoscale, supramolecular assemblies of small molecules to impede the dynamics of multiple cytoskeletal proteins, this work not only provides a mechanism for inherent cytotoxicity of hydrophobic molecular assemblies, but also illustrates a facile approach for developing nanoscale assemblies of small molecules to perform a diverse range of biological functions, including serving as a new type of anticancer agent via enzyme-instructed self-assembly.

Figure 45.

Illustration of the MHPB assay and hydrogel protein pull-down coupled with electrophoresis and tandem mass spectrometry for identifying cytosolic proteins that bind to supramolecular nanofibers. (A) Photoreaction of the hydrogelator and supramolecular nanofibers that bind with proteins. (B) Silver staining of the SDS–PAGE gel shows that different conditions alter the protein binding on the supramolecular hydrogel. Adapted with permission from ref (883). Copyright 2012 Royal Society of Chemistry.

Figure 46.

(a) Molecular hydrogel protein binding (MHPB) assay: upper panel, silver staining of SDS–PAGE reveals a major protein band at ∼55 kDa in lane B; lower panel, Western blot confirms the cytoskeletal proteins as the primary protein targets. (b) Tubulin polymerization assays with 3. (c–e) Confocal images showing the assemblies of 3 impede the dynamics of cytoskeletal proteins. (f) Cellular uptake of 3 in HeLa cells treated by endocytosis inhibitors. (g) Time-dependent activation of the apoptotic proteins of HeLa cells treated with 3. (h) Mechanism of the selective cytotoxicity of 3 toward cancer cells. Adapted with permission from ref (1248). Copyright 2014 American Society for Biochemistry and Molecular Biology. Adapted with permission from ref (1290). Copyright 2013 John Wiley and Sons.

6. Fundamental Questions Related to Supramolecular Hydrogelators

The active research and development of supramolecular hydrogels over the past 20 years have revealed a simple fact; that is, one can (almost) make any small molecule a supramolecular hydrogelator, providing proper derivatization.¹⁴ This is remarkable because it implies that self-assembly of small molecules in water is beyond the scope of lipids. Such a basic phenomenon, like the formation of liposomes, also raises many fundamental questions. We arbitrarily and briefly discuss three of them for the purpose of stimulating possible discussion rather than providing answers.

6.1. Molecular Arrangements in the Hydrogels

Like many other fields in science, the promises always bring challenges. There is no exception for supramolecular hydrogels. One persistent and still unmet challenge is how to obtain the atomistic details of intermolecular interactions in the hydrogels or in the assemblies of the hydrogelators. Since the hydrogelators self-assemble to form nanofibers, this problem is analogous to the structural elucidation of the molecular arrangement of Aβ peptide (1–42) in the amyloid fibrils. Recent studies reveal the polymorphism of β-amyloid fibrils,^1293,1294 which implies the polymorphic nature of the nanofibers of supramolecular hydrogelators. Although the current methodologies still fall short of addressing this challenge, the rapid advancements in cryo-TEM^1295,1296 and X-ray lasers¹²⁹⁷ may lead to the solution of this problem in the near future. In other words, the lack of structural details of the aggregates of the hydrogelators should not be the deterrent to the exploration of the functions and applications of the assemblies of the hydrogelators or supramolecular hydrogels, as long as the functions are important and reproducible. In fact, the successful demonstration of important applications of supramolecular hydrogels is probably the prerequisite for the initiation of the endeavor of structural elucidation.^189,1298

6.2. Self-Assembly vs Self-Organization of the Hydrogelators

The majority of the research activities on supramolecular hydrogels have largely focused on molecular self-assembly under thermodynamic equilibrium conditions.⁴ Realizing a living system is at far from equilibrium and taking advantage of the self-assembly of hydrogelators, an increased number of researchers are beginning to explore the self-assembly of hydrogelators in the context of a dynamic library^{739,826,1299−1302} or with energy input.^216,217,326 The reaction diffusion features of these cases, however, remain to be examined rigorously. Arguably, one can consider enzymatic hydrogelation accompanied by energy input, but the unanswered question in enzyme-instructed self-assembly is the role of the energy generated during bond breaking¹⁵³ or bond formation.¹⁶² Does the dissipation of the energy promote or disfavor the self-assembly or simply raise the temperature? Does the energy input in enzyme-instructed self-assembly actually result in highly ordered nanostructures (e.g., Figure 32B¹²⁵⁴)? Although this question remains to be answered, nature already provides an insightful hint by evolving the energy-dissipating self-organization that is ubiquitous in cellular processes, such as the self-organization of microtubules or actin filaments. However, none of the currently reported hydrogelators are able to mimic the unique feature of actin or tubulin proteins, such as unidirectional elongation of the filaments, but the continuing exploration of the sophisticated supramolecular hydrogelators¹³⁰³ offers an opportunity to examine the fundamental differences between self-assembly and self-organization, which ultimately may lead to a man-made molecular system that self-organizes.

6.3. Origin of Life

The origin of life remains one of the most perplexing and challenging mysteries in all of science.⁵⁶ Currently, there are three main theories on the origin of life: “RNA world first” suggests that early forms of life arise from RNA,^1304,1305 “metabolism first” argues that life began from primordial metabolism networks created by existing energy sources and nonequilibrium environments, such as found in hydrothermal vents,¹³⁰⁶ and “lipid first” proposes that life started via the compartmentalization provided by liposomes.¹³⁰⁷ While each theory has its own validity and captures certain features of modern life, they all have a crucial missing link. That is, how do molecules evolve from simple ones to greater complexity? For example, what are the molecular processes that result in RNA from simple prebiotic building blocks, produce protoenzymes for the metabolic cycles, and generate the sophisticated contents to be encapsulated by the liposomes? Since one of the undeniable facts of life is that cells are largely made of molecules noncovalently packed in a highly viscous setting, it is tempting to suggest supramolecular hydrogels may provide clues for the origin of life, as hypothesized by Pollack.¹³⁰⁸ Interestingly, Luo et al. recently reported that a clay hydrogel enhances transcription and translation.¹³⁰⁹ While it remains unknown whether the primordial soup¹³¹⁰ contains supramolecular hydrogels, we speculate that a series of serendipitous events in the exploration of more sophisticated supramolecular hydrogels or hydrogelators¹³⁰³ may offer more revealing clues about hydrogels in the context of the origin of life.

7. Conclusion and Outlook

Over the past decade, the research on supramolecular hydrogels and hydrogelators has experienced rapid growth, as evidenced by the fact that the numbers of published works on the supramolecular hydrogels in 2014 was about 10 times that in 2004, according to the Web of Science. As illustrated by the supramolecular hydrogels and hydrogelators in this review, the research focuses of hydrogelators are expanding from the curiosity for an intriguing type of soft matter to the rational development of molecular biomaterials. This trend coincides with the tremendous successes and explosive generation of data in the biological sciences. Particularly, the successful completion of the human genome project and maturation of a variety of omics projects have laid the knowledge foundations to support the molecular engineering of supramolecular hydrogels and hydrogelators for potential biomedical applications. For example, the knowledge on protein–protein interactions may provide a useful guide for developing heterotypic supramolecular hydrogels.^840,1311 On the other hand, the rapid increase of the exploration of supramolecular hydrogels for developing biomaterials itself attests to the fact that self-assembly of small molecules in water offers a facile, promising, and powerful approach for scientists and engineers to develop supramolecular hydrogelators or hydrogels that aim to improve human health. However, the current supramolecular hydrogelators or hydrogels, due to insufficient molecular engineering, are still too primitive to serve as sophisticated functional molecular biomaterials.

After billions of years of evolution, a fundamental fact is that living organisms are largely made of molecules. These molecules usually self-assemble or self-organize to perform necessary cellular functions. For example, the most common form of protein assemblies is dimers (e.g., 38% of proteins in E. coli exist as dimers¹³¹²), and the most abundant proteins in cells self-organize (i.e., actins for formation of the cytoskeleton) for many functions. Therefore, it is reasonable to take nature as the inspiration to develop supramolecular hydrogelators for generating sophisticated molecular self-assembly or self-organization in water for functions. By preserving the essence of the functions, not just the appearance of the structures, of biology systems in supramolecular hydrogelators or hydrogels, one may ultimately discover or create molecular biomaterials as a new kind of biomedicine that has prescribed functions. To achieve this goal, it is necessary to identify the problems from diseases, to define the objectives from functions, and to engineer the materials from molecules. Needless to say, this endeavor requires interdisciplinary collaborations among scientists and engineers from different disciplines of the biological, physical, and medical sciences, but it needs more than just assembling an interdisciplinary team. Since the building blocks of supramolecular hydrogels are molecules, the successful development of supramolecular hydrogelators or hydrogels as molecular biomaterials demands bioengineers or medical doctors to have a deep understanding of molecular interactions, and chemists to acquire knowledge of molecular and cell biology. For example, the completion of the synthesis of molecules and the characterization of molecular structures or supramolecular structures becomes the starting point of the research for chemists, not the ending point. If the goal of the research on the supramolecular hydrogelators is to develop molecular biomaterials, it would be beneficial to have a chemist who has the knowledge of molecular biology and cell biology and clinical medicine, is able to communicate with the language of biochemistry, and possesses the skills of bioinformatics. While these capabilities seemed to be quite demanding two decades ago, the impressive development of information technology, digitalized knowledge, and the new generation of young scientists make these prerequisites increasingly easy to meet. On the other hand, having more insights into the molecular structures and intermolecular interactions, bioengineers and medical doctors will likely more accurately and effectively identify the problems, define the objective, and devise the plan. It is our conviction that the creative exploration of supramolecular hydrogelators and hydrogels not only will bring innovative molecular biomaterials, but also may lead to new frontiers of science.

On the basis of the above rationale and optimism, we propose several prejudiced possible directions of supramolecular hydrogels and hydrogelators, including controlled drug release (e.g., autogel-like^33,1313,1314 systems), tissue engineering (e.g., organoids^1315,1316 of human immune systems), intracellular delivery,¹³¹⁷ regenerative medicine (e.g., control of stem cell^1318−1320 differentiation), immunomoduation (e.g., molecular adjuvants of vaccines or even pan-flu vaccines^1321,1322), wound healing (e.g., treatment of diabetic ulcers^1323,1324), and cell signaling (e.g., biomimetics of autocrines, paracrines, or juxtacrines). Considering the complexity of biological systems, it is unlikely that one type of hydrogelator would meet all needs in biomedical applications. Having said that, our biased view is that hydrogelators, which consist of basic biological building blocks or are able to mimic a particular biological process, are excellent starting points for exploring the biomedical applications aforementioned. The applications of supramolecular hydrogels and hydrogelators certainly go beyond biomedicine; they already have found applications in the catalysis,³¹ food, cosmetic, and art industries. We have witnessed the exciting development of supramolecular hydrogelators and hydrogels in the past decade. This astonishing versatility of supramolecular hydrogelators and hydrogels has offered scientists a fruitful playground to reinvent chemistry¹³²⁵ in the context of molecular biomaterials. By shifting the research focus from molecules to processes,^34,1326 from thermodynamics to kinetics,^1327−1329 and from molecules to cells, the research on supramolecular hydrogels and hydrogelators will lead to the integration of molecular science and bioinformatics, and contribute to the use of molecules for better human life.

Glossary

Abbreviations

2D

two-dimensional

3D

three-dimensional

5-FU

5-fluoro-2A-deoxyuridine

6-ACA

6-aminocaproic acid

ACQ

aggregation-caused quenching

AgNPs

silver nanoparticles

ALP

alkaline phosphatase

ALPPs

placental alkaline phosphatases

AQ

8-aminoquinoline

BAMB

butan-1-aminium 4-methylbenzenesulfonate

β-CD

β-cyclodextrin

β-TCP

tricalcium phosphate

Bhcmoc

[(6-bromo-7-hydroxycoumarin-4-yl)methoxy]carbonyl

BMDMs

bone-marrow-derived monocytes

Boc

(tert-butyloxy)carbonyl

BPs

bisphosphonates

BSA

bovine serum albumin

BTA

benzene-1,3,5-tricarboxamide

CAB

cholesteryl 4-(2-anthryloxy)butyrate

CAC

critical assembly concentration

CGC

critical concentration of gelation

CHO

Chinese hamster ovary

CLEM

correlative light and electron microscopy

CMC

critical micelle concentration

CPC

cetylpyridinium chloride

CSD

Cambridge Structural Database

CT

charge transfer

CTV

cyclotriveratrylene

DA

donor–acceptor

DCDs

dimeric cholesterol derivatives

Dex

dexamethasone

DFT

density functional theory

DLVO

Derjaguin–Landau–Verwey–Overbeek

DMSO

dimethyl sulfoxide

DOPA

3,4-dihydroxy-l-phenylalanine

DOPC

dioleoylphosphocholine

DSA

doxylstearic acid

DSC

differential scanning calorimetry

DTT

dithiothreitol

ECD

electronic circular dichroism

ECM

extracellular matrix

EG

ethylene glycol

EISA

enzyme-instructed self-assembly

ESCs

embryonic stem cells

FA

folic acid

FCS

fluorescence correlation spectroscopy

FESEM

field-emission scanning electron microscopy

Fmoc

(fluoren-9-ylmethoxy)carbonyl

FRAP

fluorescence recovery after photobleaching

FRET

fluorescence resonance transfer

GdL

glucono-δ-lactone

GFP

green fluorescent protein

GNFs

glycosyl-nucleoside-fluorinated amphiphiles

GNLs

glycosyl-nucleoside lipids

GSH

glutathione

GSSG

glutathione (γ-glutamylcysteinylglycine (GSH))

HaCaT’s

human keratinocytes

HCPT

hydroxycamptothecin

HDFs

human dermal fibroblasts

HEK293 cells

human embryonic kidney 293 cells

HFIP

hexafluoroisopropyl alcohol

HMSCs

human marrow stem cells

HQ

2-hydroxyquinoline

hRBCs

human red blood cells

HUVECs

human umbilical vein endothelial cells

ITC

isothermal titration calorimetry

LCA

lithocholic acid

LCST

lower critical solution temperature

LDH

lactate dehydrogenase

LSCM

laser scanning confocal microscopy

LZ

leucine zipper domain

Mb

myoglobin

MDPs

multidomain peptides

MHPB

molecular hydrogel protein binding

MIC

minimum inhibitory concentration

MMP-2

matrix metalloprotease 2

MRSA

methicillin-resistant Staphylococcus aureus

MSCs

mesenchymal stem cells

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MVECs

microvascular endothelial cells

NCL

native chemical ligation

NHSF

normal human skin flbroblast

NO

nitric oxide

NPCs

nucleus pulposus cells

NSAID

nonsteroidal anti-inflammatory drug

NSCs

neural stem cells

OC

osteocalcin

Pa

pascal

PA

peptide amphiphile

PAP

prostatic acid phosphatase

PC12 cells

pheochromocytoma cells

PKA

protein kinase A

PL

photoluminescence

PNPA

p-nitrophenyl acetate

POM

polyoxometalate

PRRSV

porcine reproductive and respiratory syndrome virus

PSA

prostate-specific antigen

PTA

1,3,5-triaza-7-phosphaadamantane

ROMP

ring-opening metathesis polymerization

SCC25

squamous cell carcinomas

SDS

sodium dodecyl sulfate

SEM

scanning electron microscopy

SHED

human exfoliated deciduous teeth

SPPS

solid-phase peptide synthesis

SUMO

small ubiquitin-related modifier

T_gel

temperature of gelation

T_m

phase transition temperature

TATP

triacetone triperoxide

UV

ultraviolet

Biographies

Xuewen Du obtained his B.S. degree in the Department for Intensive Instruction (DII) of Kuang Yaming Honors School from Nanjing University in 2010. He is currently in his fifth year as a graduate student in chemistry with Professor Bing Xu at Brandeis University. His current work focuses on nanomaterials for controlling cell fates.

Jie Zhou obtained her B.S. degree in the Department of Chemistry & Chemical Engineering at Nanjing University in 2012. During her undergraduate study, she investigated the applications of photochemistry. She is currently in her fourth year as a graduate student in chemistry with Professor Bing Xu at Brandeis University. As a Howard Hughes Medical Institute (HHMI) International Graduate Fellow, her research interest lies in designing self-assembling bioactive materials.

Junfeng Shi obtained his B.S. degree in the Department of Chemistry from Xiangtan University in 2007, followed by an M.S. degree under the supervision of Prof. Liming Zhang from Sun Yat-sen University in 2009. He recently obtained his Ph.D. degree in Bing Xu’s laboratory at Brandeis University, and will start postdoctoral research at the National Institutes of Health. His current work focuses on ligand–receptor interactions and their biological applications.

After receiving his B.S. and M.S. degrees from Nanjing University in 1987 and 1990, Bing Xu obtained his Ph.D. in 1996 from the University of Pennsylvania. Before starting his independent research at the Hong Kong University of Science and Technology (HKUST) in August 2000, he was a National Institutes of Health (NIH) postdoctoral fellow at Harvard University. He was tenured as an associate professor in January 2006 and became a full professor in July 2008 at HKUST. On the basis of his work at HKUST and Brandeis, Bing Xu is identified on the Thomson Reuters 2014 and 2015 Highly-Cited Researchers lists in chemistry. He is currently a professor in the Department of Chemistry, Brandeis University. His research focuses on the applications of molecular engineering in materials, biology, and medicine.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrev.5b00299.

• Tables giving the hydrogelation concentrations of the compounds described in the molecular design session (PDF)

Special Issue

This paper is an additional review for Chem. Rev. 2015, 115, issue (15), , “Supramolecular Chemistry”.

The authors declare no competing financial interest.