Photoactive properties of supramolecular assembled short peptides

Abstract

Bioinspired nanostructures can be the ideal functional smart materials to bridge the fundamental biology, biomedicine and nanobiotechnology fields. Among them, short peptides are among the most preferred building blocks as they can self-assemble to form versatile supramolecular architectures displaying unique physical and chemical properties, including intriguing optical features. Herein, we discuss the progress made over the past few decades in the design and characterization of optical short peptide nanomaterials, focusing on their intrinsic photoluminescent and waveguiding performances, along with the diverse modulation strategies. We review the complicated optical properties and the advanced applications of photoactive short peptide self-assemblies, including photocatalysis, as well as photothermal and photodynamic therapy. The diverse advantages of photoactive short peptide self-assemblies, such as eco-friendliness, morphological and functional flexibility, and ease of preparation and modification, endow them with the capability to potentially serve as next-generation, bio-organic optical materials, allowing the bridging of the optics world and the nanobiotechnology field.

Introduction

Self-assembly provides a source for the design of diverse hierarchical structures with functional properties at different scales.^1–7 Peptides and proteins are widely used self-assembling building blocks, reflecting their role as the central structural elements of the biological world.^8–14 The unique physical properties of ordered peptide and protein assemblies at the macro- and nano-scale allow living systems to conduct various biological functions and metabolic processes.^11,15–19 Specifically, short peptides have gained increasing interest due to their structural simplicity, flexibility and variability in molecular design, compared to polypeptides or proteins.^20–25 In the past few decades, many natural and synthesized short peptides have been assembled into a variety of nanostructures and widely utilized in technological applications.^26–30

Short peptide assemblies exhibit several remarkable physico-chemical characteristics such as high thermal and chemical stability, metal-like rigidity, hydrophobicity, electrical features, and optical properties.^{12,15,31–36} In fact, many photoactive phenomena previously observed in inorganic nanocrystals and quantum dots have also been revealed in short peptide supramolecular assemblies.³⁶ Due to the intrinsic biocompatibility, their optical properties, such as photoluminescence and optical waveguiding characteristics, show intriguing potential for biomedical and nanotechnological applications.^22,37 Moreover, inspired by protein-based natural systems, short peptide-modulated self-assemblies with photoactive moieties have been explored for the design of biomimetic systems for photosynthetic reactors and antitumor therapy.^38,39

In this review, we focus on the optical properties of short peptide self-assembly systems and their applications in diverse technologies. We first highlight the mechanisms of quantum confinement-based intrinsic photoluminescence of short peptide structures and the effects of various factors. Next, the optical waveguide properties of the short peptide structures are reviewed, as well as the methods aimed at obtaining aligned short peptide assemblies with optical waveguide properties which bring new prospects for oriented optical devices. Then, the co-assembly or covalent conjugation of short peptides with other photoactive agents for photocatalysis and phototherapy applications are summarized. We highlight the ways the structural diversity and unique optical properties of the short peptide entities can reflect their biological functions and potentially help to understand the mechanisms of disease-related structures.

Photoluminescent properties

Quantum confinement-induced inherent photoluminescence

Self-assembled short peptide nanostructures possess inherent optical absorption and photoluminescence, both in the UV and visible regions, due to the formation of nano-sized quantum confined structures in the hydrogen bonding and aromatic interaction regions inside the assemblies.^40,41 The excitons in the quantum confined regions mediated by the strong Coulomb interaction of ‘electron–hole’ lead to these unique optical characteristics of short peptide assemblies.⁴² The most prominent examples are aromatic dipeptides which were found to self-assemble into supramolecular semiconductors.³⁶ Density functional theory (DFT) calculations were used to investigate the electron band structures of self-assembled diphenylalanine (FF), dityrosine (YY) and phenylalanine–tryptophan (FW) nanotubes.⁴³ The results demonstrated that FW and FF formed wide, direct bandgap semiconductors with electron transitions at the Z and Γ points, respectively, thus behaving like GaN (gallium nitride), while YY assembled into wide, indirect bandgap semiconductors similar to SiC (silicon carbide) (Fig. 1).⁴³

Fig. 1.

Band structures along the tube axis for FF (left panel), YY (central panel), and FW (right panel) nanotubes calculated using the optPBE function. The conduction band minima and valence band maxima are shown in red. The dashed lines serve as a guide to indicate the borders of the bandgaps. Reproduced from ref. 43 with permission from the [AIP Publishing], copyright [2015].

Quantum confinement structures generate remarkable changes in the optical properties of semiconductors and have wide applications in photonic devices.⁴⁴ Two-dimensional (2-D) quantum wells are the most common quantum confinement structures characterized by a step-like behaviour.^45–47 Highly ordered alignments of FF nanotubes were fabricated by vapour deposition methods (Fig. 2a).⁴¹ As shown in Fig. 2b, compared to a narrow absorption peak for FF monomers at 257 nm, the absorption spectrum of the aligned FF nanotubes showed two distinct steps located at 245–264 and 300–370 nm. This typical step-like optical absorption behaviour indicates the 2-D quantum wells’ crystalline structures formed in the FF nanotubes. This is also in accordance with the semiconductive properties of these peptide nanostructures.^17,48 The photoluminescence of the 450 nm peak under excitation at 370 nm is very intense, 5-fold higher than the 305 nm peak under excitation at 260 nm (Fig. 2c). Under excitation at 340–380 nm, the FF nanotube patterns showed green photoluminescence, compared to the dark purple reflection of the excitation light from the substrate (Fig. 2d).⁴¹ The photoactive FF nanotube arrays promote the use of short peptides in novel bio-organic optoelectronics.³⁶ This phenomenon was later observed in Fmoc-FF self-assembling hydrogels, thus further demonstrating the organization of the peptide structures into 2-D quantum-wells in a gel state.⁴⁹

Fig. 2.

Intrinsic optical properties of self-assembled aligned FF nanotubes. (a) Scanning electron microscopy image of the aligned FF nanotubes. (b) Optical absorption of aligned FF nanotubes (solid line) and FF monomers (dash line). (c) Photoluminescence spectra of aligned FF nanotubes upon excitation at 370 nm (solid line) and 260 nm (dashed line). (d) Fluorescence microscopy image of patterned aligned FF nanotubes upon excitation (340–380 nm), showing green emissions. Reproduced from ref. 41 with permission from the [American Chemical Society], copyright [2009].

In contrast to the arrays, discrete FF nanotubes exhibit spike-like optical absorbance and photoluminescence characteristic of quantum dot (QD) structures.⁵⁰ Interestingly, when the solvent was changed from water to methanol, the nanotubes disassembled to individual dimeric QDs, confirming that the dimers existed as single entities and behaved as elementary building blocks of the FF nanotubes. Taking this approach, QD nanoparticles of different diameters can be fabricated using FW (2.1 nm compared to 1.1–1.3 nm for FF QDs). This method allows simple modification of the peptide morphologies between larger superstructures and QDs, a key feature for the development of multifunctional photoluminescent devices.^51,52 For example, cyclic dipeptides with backbones of 2,5-diketopiperazine configurations, such as cyclo-FW and cyclo-WW, were found to dimerize into dimeric QDs which then served as building blocks to self-assemble into larger quantum confined nanostructures.⁴⁸ Notably, these nanostructures possessed intrinsic photoluminescence with the emission tuned from the visible to the near-infrared (NIR) region (420 nm to 820 nm) by various means of modulating the self-assembly process. These bioinspired nanostructures could be used as biocompatible labelling agents for in vivo imaging and as phosphors for engineering light-emitting diodes.⁵³ Furthermore, the crystals assembled by cyclo-FW showed wide-spectrum photoluminescence in the visible region, ranging from 395 nm to 550 nm, and intensity-controllable optical waveguiding.⁵⁴ In another study, a tryptophan-based simple peptide, cyclo-glycine–tryptophan (cyclo-GW), was designed to crystallize into monoclinic crystals with ultrahigh thermal stability (up to 370 °C) and mechanical strength (with elastic modulus of 24.0 GPa).⁵⁵ In particular, the extensive and directional hydrogen bonding and aromatic interaction network could decrease the optical bandgap and facilitate aggregation-induced emission in the visible light range, optical waveguiding, and especially, stable photoluminescence, rather than general aggregation-induced quenching.⁵⁵

Influence of water molecules

FF nanotubes are formed via a combination of peptide–water hydrogen-bonding, interpeptide head-to-tail hydrogen-bonding and T-shaped aromatic stacking interaction networks.^56,57 As a result, water molecules exert a crucial effect on the assembly and on the resulting photoluminescent properties.^54,58 Indeed, deficiency of water was shown to alter the bandgap of the FF nanotubes.⁵⁹ Typical hierarchical FF nanotubes with a clear hexagonal shaped head and many overlapping hexagonal nanotubes on the tail were fabricated at a relative humidity of 33% (Fig. 3a).⁵⁹ UV-visible-near infrared diffuse reflectance spectra indicated that the bandgap of the nanotubes increased with FF concentration, and was closely related to the highly-ordered subnanometer crystalline structure of FF nanotubes with weak water bonding formed by FF molecules in the channel core of the hexagonal crystalline unit cell (Fig. 3b).⁵⁹ The UV photoluminescence of these nanotubes red shifted with FF concentration or the relative humidity (Fig. 3c). When the concentration of FF was increased to 160 mg mL⁻¹, the photoluminescence peak was closely related to the number of water molecules bound to the FF nanotubes, implying that the bandgap increased with the water molecules.⁵⁹ DFT calculations revealed that significant splitting of the valence-band peak occurred with the addition of water molecules into the channel which led to the shift and splitting of the photoluminescence peak. The emission position and shift were calculated to be linear to the number of water molecules (Fig. 3d), thus allowing the use of the FF nanotubes’ photoluminescence to detect the number of water molecules or the level of humidity.⁵⁹

Fig. 3.

Influence of water molecules on the intrinsic photoluminescence of FF nanotubes. (a) Field-emission SEM image of the hierarchical FF nanotubes. The inset shows the enlarged head of the nanotube. (b) Schematic of the weak water molecule bonding (red balls) onto the FF molecule in the channel core of a hexagonal unit cell (inset) in the highly-ordered FF subnanometer crystalline structure. (c) Photoluminescence spectra of three FF nanotubes formed using FF concentrations of 30, 105, and 160 mg mL⁻¹ at a relative humidity of 0.33, 0.52, 0.67, 0.83, and 1.00, as indicated. (d) Linear dependence of the UV photoluminescence peak position versus the average number of water molecules per FF molecules. Reproduced from ref. 59 with permission from [John Wiley and Sons], copyright [2011].

The effect of temperature

Temperature can also affect the photoluminescence of short peptide assemblies.^60–62 After heating to 140–180 °C, both FF and dileucine (LL) nanotubes irreversibly converted into ultrathin amyloid-like nanofibrils due to the molecular transformations of linear dipeptide molecules into their cyclic counterparts.⁶³ This gave rise to new secondary β-sheet structures and new optical properties, namely, blue photoluminescence. Similarly, blue photoluminescence was observed for other dipeptide-based nanotubes and nanowires,^25,41,48 thus suggesting a general methodology for producing peptide nanostructures with blue luminescence by heating.

Complementary to high temperatures, low temperatures can also influence the photoluminescence of short peptide nanostructures. At room temperature, the emission of FF microtubes revealed a near-UV broad band centred at 290 nm.^41,60 However, a strong near-UV/blue/green emission of 350–500 nm appeared when the temperature was decreased below −73 °C.⁶¹ Fig. 4a shows that the photoluminescence intensity and lifetime of FF nanotubes decrease dramatically upon temperature increase from 5 °C to 65 °C. Further characterization demonstrated that non-radiative recombination of the excitons localized on the defect sites occurred and induced the thermal quenching of the photoluminescence.⁶⁰ High temperature can activate the thermally-assisted energy transfer process in FF nanotubes, accelerating the nonradiative events and eventually reducing the photoluminescence lifetime and intensity (Fig. 4b).⁶⁰ Therefore, by monitoring the photoluminescence intensity and lifetime of FF nanotubes, rapid changes of local temperature and absolute temperature between 5–65 °C could be detected in situ. In particular, this temperature-sensing capability was independent of the ionic (K⁺) concentration or pH (6.0–8.0) in a physiological microenvironment, thus implying the potential to be used for bio-imaging and monitoring of temperature fluctuations in vivo.⁶⁰

Fig. 4.

The effect of temperature on the photoactive properties of FF nanotubes. (a) Plots of the average photoluminescence intensity (blue circle) and the fitted lifetime (black square) versus temperature. (b) Schematic representation of the mechanism underlying the temperature-dependence of the photoluminescence lifetime. Reproduced from ref. 60 with permission from the [American Chemical Society], copyright [2011].

The effect of chemical conjugation

Chemical conjugation with functional moieties provides another effective approach for the fabrication of short peptide nanostructures with tuneable and functional optical properties.^64,65 For instance, spike-like absorption spectra can be observed for tertbutoxycarbonyl group-modified FF (Boc-FF) nanospheres, indicating that 0-D QDs exist in the assemblies. Calculations have demonstrated the radius of the QDs to be approximately 1.3 nm, implying that the QDs were composed of two Boc-FF molecules.⁴⁹ In contrast, the absorption spectra of fluorenylmethyloxycarboxyl-modified FF (Fmoc-FF) nanofibrillar hydrogels and Fmoc-5-aminopentanoic acid plate-like crystals exhibited a pronounced step-like absorption pattern⁶⁶ accompanied by a peak at the long-wavelength edge derived from the strengthened Coulomb interactions of the excitons, a characteristic of the formation of 2-D quantum well confinement structures.⁶⁷

Another direction proven to be useful for enhanced and controllable short peptide optics is the modification of side chains. One example is the use of peptide nucleic acids (PNAs) which combine the chemical versatility and architectural flexibility of peptides with the precise base pairing and specific molecular recognition of nucleic acids.⁶⁸ For instance, the self-assembly of diPNA–guanine–cytosine (di-PNA–GC) was driven by Watson–Crick base pairing and stacking.⁶⁸ The integration of these extensive and directional non-covalent interactions led to excitation-dependent fluorescence from 440 nm to 684 nm, covering nearly the entire visible region. In particular, when dropping the self-assemblies on a chip, the nanostructures responded to the gate voltage in the same manner as a field-emission transistor, showing lower current rates as the voltage increased.⁶⁸ Such a device could controllably emit bright electroluminescence by applying alternating voltages (5 V to −5 V), thus showing the potential for optical bio-sensing and light emission-based applications, including organic light-emitting diodes and imaging labels with tuneable emissions via optical or electrical modulations.⁶⁸

Furthermore, benzhydryloxycarbonyl (Bhoc) and Fmoc-modified PNA-G (Fmoc-G-(Bhoc)-aeg-OH) was used to self-assemble into uniform photonic crystals with an average diameter of 1.7 μm.⁶⁹ In particular, the guanine-based PNA spheres could organize into a colourful layer in solution (Fig. 5a).⁶⁹ The arranged pattern of these PNA spheres was similar to the biogenic guanine nanocrystals observed in chameleons (Fig. 5b).⁷⁰ Upon addition of NaCl to the solution, the spacing between the PNA spheres changed due to aggregation of the spheres resulting from masking of the surrounding electrostatic potential, thus leading to changes in the colour of the film and visible changes in their reflected wavelength (Fig. 5c and d).⁶⁹ These short PNA self-assembling spheres have potential applications in optical coating sensors and diffraction gratings.

Fig. 5.

Microscopic and cartoon images of the optical lattices organized by Fmoc-G-(Bhoc)-aeg-OH microspheres, showing high reflectance of the incident light. (a) A coloured layer (left) and SEM image of the PNA spheres. Reproduced from ref. 69 with permission from [John Wiley and Sons], copyright [2016]. (b) Photographic image showing the skin of a mature male panther chameleon (left) and TEM micrograph of the guanine nanocrystals lattice from panther chameleon iridophores in a relaxed state. Reproduced from ref. 70 with permission from [Springer Nature], copyright [2015]. (c and d) Schematic representation of the PNA sphere array (c) before and (d) following the addition of NaCl, demonstrating the geometrical alternation leading to the finely-modulated reflectance colour. Reproduced from ref. 69 with permission from [John Wiley and Sons], copyright [2016].

The effect of doping

Doping with other optical entities, such as fluorescent molecules, photosensitizers, lanthanide ions and quantum dots, can enhance the luminescence and extend the functionality of the short peptide assemblies. For example, pyrene and porphyrin have been shown to enhance the photoluminescence of FF assemblies by covalently conjugating to the peptide backbone.^71,72 Photosensitizers and lanthanide ions could be co-assembled into FF nanotubes or organogels to form photoactive biosystems.^73,74 In this case, the peptide assemblies served both as host matrixes and as antennas for lanthanide ions. Enhanced photoluminescence was observed as a result of the energy-transfer cascade from the peptides to lanthanide ions through the photosensitizers. Thus, various colours of FF nanotubes or short peptide organogels were obtained, and their luminescence wavelengths could be readily tuned by changing the composition of the assemblies.

QDs could also be encapsulated into short peptide assemblies to endow them with tuneable photoluminescence properties.⁷⁵ The 3-D colloidal spheres prepared by the C-terminal amidated FF (cationic dipeptide, CDP) gel with encapsulated CdSeS nanocrystals (QD523) were stably dispersed in a serum-containing cell medium and showed good biocompatibility. Confocal laser scanning microscopy (CLSM) characterization demonstrated that the 3-D colloidal spheres could be internalized by cultured cells and radiate punctate spots of luminescence, thereby validating their potential for bioimaging.⁷⁵

Based on the change of fluorescence, such as photoluminescence quenching, upon binding of specific recognizing/targeting probes, short peptide assemblies can be utilized as ultrasensitive biosensors. For example, a peptide hydrogel-based biosensing platform was prepared by incorporating enzymes (glucose oxidase or horseradish peroxidase) and QDs (CdTe or CdSe) into the Fmoc-FF self-assemblies.⁷⁶ The obtained Fmoc-FF hydrogels, combined with encapsulated enzyme bioreceptors and QDs, formed a 3-D nanofiber network and retained the photoluminescence property of the QDs. The analytes, i.e. glucose or phenolic compounds, were enzymatically converted to products acting as electron acceptors on the surface of the QDs, thus resulting in photoluminescence quenching (Fig. 6a). In particular, the quenching degree was in correlation with the concentration of the analytes, demonstrating the potential of this short peptide hydrogel to serve as a versatile platform for enzymatic detection of metabolic analytes (Fig. 6b).⁷⁶ In addition, this strategy could also be utilized for the selective detection of a neurotoxin among organophosphates and nitro-group compounds using photoluminescent FF nanotubes.⁷⁷

Fig. 6.

Influence of doping on the photoluminescence properties of short peptide self-assemblies. (a) Scheme of the photoluminescent Fmoc-FF hydrogel with QDs and enzyme bioreceptors and their photoluminescence quenching associated with the enzymatic detection of analytes. Upon the addition of the analyte, the photoluminescence from the FF hydrogel decreases as the excited electrons of the QDs are absorbed by the quenching agent produced from the enzyme-catalysed reaction. (b) Colour change of the Fmoc-FF hydrogel at different glucose concentrations under UV light (365 nm). The colour of the photoluminescent hydrogel gradually changes from red to black under UV exposure upon the increase in glucose concentration from 1 to 10 mM due to quenching of the QDs in the hydrogel triggered by glucose. Reproduced from ref. 76 with permission from [Elsevier], copyright [2011]. (c) Schematic presentation of the self-assembly of Zn²⁺-coordinated WF dipeptides into fluorescent nanoparticles. Reproduced from ref. 80 with permission from [Springer Nature], copyright [2016].

Metal ions can coordinate with the carboxyl group of amino acids and the side chain of certain amino acids (such as the imidazole group of histidine and the indole group of W),⁷⁸ thereby taking part in the self-assembly of peptides and affecting the resulting photoluminescence properties.⁵³ For example, enhanced luminescence could be detected from Zn²⁺-conjugated BFPms1, a green fluorescent protein variant.⁷⁹ Inspired by this finding, WF, an aromatic dipeptide, was designed to assemble into spherical nanoparticles with blue emission at 423 nm under excitation at 370 nm (Fig. 6c).⁸⁰ Subsequent doping of Zn²⁺ significantly stabilized the nanospheres and improved their quantum yield, thus resulting in sustainable and enhanced photoluminescence. Compared to the state-of-the-art fluorescent counterparts, including organic fluorophores (rhodamine 6G, R6G), QDs (CdSe) and green fluorescent proteins, the doped nanoparticles showed remarkably sustained visible fluorescence and biocompatibility.⁸⁰ In particular, these dipeptide nanoparticles could be easily modified with functional moieties and loaded with small molecular drugs, thus bearing the promising potential to be simultaneously used for intracellular labelling, imaging and tracking of drug delivery.⁸⁰ In addition, a cyclic octapeptide, cyclo-[(d-Ala-l-Glu-d-Ala-l-Trp)₂], was designed to self-assemble into spherical nanoparticles of approximately 28 nm (f-PNPs) in the presence of Zn²⁺ (Fig. 7a).⁸¹ The Zn²⁺ ion could complex with the carboxyl groups of the glutamic acids, thus stabilizing the nanostructures and limiting the energy dissipation. This resulted in intrinsic fluorescent emission with a high quantum yield (Fig. 7b). In particular, these f-PNPs exhibited a wide-spectrum photoluminescence covering the visible to NIR region. When excited at 370 nm, the f-PNPs were able to emit fluorescence in the visible range (Fig. 7c). Also, NIR fluorescence was observed under excitation at 760 nm.⁸¹ The emission was more stable than that of a commercially-used NIR organic dye (indocyanine green), thus showing the promising potential for in vitro and in vivo imaging applications. Therefore, after conjugation with an Arg–Gly–Asp (RGD) peptide moiety (the cell adhesion sequence of fibronectin) and embedding with epirubicin (EPI, a chemotherapeutic drug for oesophageal cancer) via π–π stacking and electrostatic interactions, the integrated RGD-f-PNPs/EPI nanoparticles could be used as a biocompatible nanoplatform for in vivo, real-time oesophageal cancer imaging and therapy (Fig. 7d).⁸¹

Fig. 7.

In vivo characterizations and targeted tumour imaging application of RGD-f-PNPs/EPI. (a) Schematic representation of the spherical nanoparticles co-assembled by cyclo-[(d-Ala-l-Glu-d-Ala-l-Trp)₂-] peptides and Zn²⁺ ions. The nanoparticles could be easily modified with RGD moieties and embedded with EPI through non-covalent interactions. (b) Emission vs. excitation profile showing the visible to NIR emissions of the nanoparticles under excitation from 370 to 790 nm. (c) Fluorescence emission spectra of the f-PNPs and RGD-f-PNPs under excitation at 370 nm. (d) In vivo targeted tumour imaging and enhanced anti-tumour efficacy of RGD-f-PNPs/EPI. Reproduced from ref. 81 with permission from [Springer Nature], copyright [2018].

Amphiphilic peptides

Amphiphilic peptides are a new class of self-assembling bioinspired molecules bearing hydrophilic peptide segments covalently coupled to hydrophobic lipid tails.⁸² They draw parallels to phospholipids and other membrane-forming amphiphiles, while combining the advantage of peptide secondary structures.^82–84 Since the first report of custom-designed self-assembling peptide amphiphile nanofibers with controllable gelation properties,¹¹ self-assembling peptide amphiphile nanostructures have been widely applied in diverse fields.⁸² Interestingly, after integrating W and pyrene chromophores in the peptide backbone, the self-assembled peptide amphiphile structures displayed fluorescence signals, thus facilitating spectroscopic examinations of the interior of the resulting supramolecular objects.⁸⁵ Following self-assembly, the chromophores were found to be constrained to a defined location within the aggregates. The W fluorescence (with emission maximum around 350 nm) demonstrated that high degrees of free volume were retained in the supramolecular aggregates. Moreover, the fluorescence quenching behaviour of W and pyrene probes based on modified Stern–Volmer formalisms indicated different degrees of quencher penetration.⁸⁵ In addition to the covalent bonding, chromophore/amphiphilic 4-helix bundle peptide complexes were also obtained utilizing axial ligation coordination of metalloporphyrins and histidyl residues (H) on F6H20 and H6F20 peptides.⁸⁶ The resulting complexes were thermally stable, thus suggesting them to be ideal candidates for nonlinear optical biomolecular materials.

Optical waveguide properties

Intrinsic optical waveguiding properties

Quantum confined organic nanomaterials are promising building blocks for next-generation, eco-friendly optoelectronic devices, such as optical waveguides, which propagate and manipulate light on the sub-wavelength scale.^87–90 Due to the facile optical tuneability, high photoluminescent properties and excellent self-assembly propensity, short peptides are expected to be effective building blocks in the fabrication of future miniaturized optoelectronics, allowing the generation and propagation of light.⁹¹ This concept was demonstrated for self-assembled hexagonal FF microtubes obtained upon solvent thermal annealing.⁹² These hollow FF microtubular crystals were obtained in the water phase and consisted of hierarchically and directionally organized nanotubes. Their optical waveguide properties were demonstrated by the incorporation of dyes, such as Nile red (NR) or R6G. Upon light excitation at one end of the FF–NR microtubes, bright photoluminescence was emitted from the other end, with the tube body emitting nearly no photoluminescence (Fig. 8a),⁹² a typical characteristic of optical waveguides.

Fig. 8.

(a) Optical waveguiding properties of short peptide self-assemblies. Optical microscopy (left) and photoluminescence (right) images of hexagonal FF–NR microtubes formed upon solvent thermal annealing. Waveguiding was observed upon local excitation at one end (515–560 nm). The light blue circle marks the excitation area and the green arrow denotes the out-coupling of photoluminescence at the other end. Reproduced from ref. 92 with permission from [John Wiley and Sons], copyright [2011]. (b) Photoluminescence image of the single FF platelets (excitation wavelength: 330–380 nm). The red circle marks the excitation area, and the green arrow denotes the out-coupling of photoluminescence at the other end. Reproduced from ref. 95 with permission from [John Wiley and Sons], copyright [2011]. (c) Cartoon model of the optical waveguiding of peptide self-assemblies based on the total internal reflection of light.

To enrich the diversity of short peptide-based assemblies showing waveguide properties, FF microtubes and microrods were further prepared via a re-precipitation approach.⁹³ The obtained FF microtubes and microrods both formed crystalline structures and exhibited excellent optical waveguide properties after doping with rhodamine B (RhB). The output light intensity showed an exponential decrease upon the increase of propagation distance.⁹³ In another work, CDP was used. Rectangular CDP microtubes and microrods fabricated in ethanol and in ethanol/water, respectively, also showed active optical waveguiding behaviours after doping with dyes, thus indicating the generality of this phenomenon for short peptide self-assemblies.⁹⁴

The effect of aldehydes

When glutaraldehyde (GA) was introduced into self-assembled FF fibrous gels, the Schiff’s base reaction between the amino groups of linear FF and the aldehyde groups of GA triggered the intramolecular cyclization of linear FF into cyclo-FF, thus leading to a structural transformation into crystalline platelets organized in 3-D order but uniaxially oriented along the longitudinal axis.⁹⁵ The FF platelets exhibited waveguiding of blue light emission upon excitation at 330–380 nm (Fig. 8b)⁹⁵ arising from the intrinsic photoluminescence of cyclo-FF and the n–π* transition of the formed Schiff base bond.⁹⁶ The optical waveguide was attributed to the total internal reflection of light inside the peptide self-assembling fibres (Fig. 8c).

Generally, the assembly process of the above-mentioned FF platelets requires at least one month at ambient temperature. However, the intramolecular cyclization of linear FF and the concomitant crystallization were significantly expedited through a solvothermal treatment, and crystalline FF nanobelts with optical waveguide properties could be obtained within only 10 min.⁹⁷ Moreover, by introduction of formaldehyde, the thickness of the FF nanobelts could be readily tuned from tens to hundreds of nanometres to form platelets with optical waveguiding properties.

Oriented optical waveguide devices

Oriented arrangement of organic materials plays an important role in enhancing their functions.^98,99 Through the dip coating process, ultralong, aligned single FF crystals could be fabricated for potential applications in large-scale optical devices.¹⁰⁰ This system allowed the growth of uniform single crystals with several centimeter in length to become continuous and oriented on a silicon wafer (Fig. 9a).¹⁰⁰ These structures of FF single crystals could be precisely controlled by modifying the process parameters and have shown active optical waveguiding properties. In addition, ultralong aligned one-dimensional FF single crystals of up to 4 cm could also be obtained using capillaries.¹⁰¹ Upon excitation at one end of the ultralong single FF crystal, photoluminescence could be observed at the other end (Fig. 9b).¹⁰¹ The development of ultralong-oriented short peptide crystals is thus expected to expand the potential applications of aligned organic nanomaterials in optical devices.

Fig. 9.

Oriented optical waveguiding properties of short peptide self-assemblies. (a) Photograph (left, upper panel) and SEM image (left, lower panel) of FF single crystals and images of an FF–RhB single crystal showing the transition of the excitation spot along the crystal upon excitation at 488 nm (right). The scale bar (right) is 10 μm. Reproduced from ref. 100 with permission from the [American Chemical Society], copyright [2017]. (b) Photographic image of an ultralong single FF–RhB crystal in capillary (upper panel) and the corresponding fluorescence image showing the optical waveguiding properties (lower panel). The circles mark the area of excitation (488 nm) and emission. Reproduced from ref. 101 with permission from the [American Chemical Society], copyright [2018].

Photocatalytic properties

Co-assembly strategies

In natural photosynthetic systems, the self-assembling protein complexes and photosensitive chromophores contribute to the highly efficient light-harvesting and energy transfer.¹⁰² This has inspired a strategy of artificially integrating the light-sensitive antennas into orderly frameworks for photoelectron transportation.¹⁰³ Short peptide self-assemblies show tremendous potential to serve as such frameworks.¹⁸ For example, through co-assembly of tetra(hydroxyphenyl) porphyrin (THPP) with FF, light-harvesting peptide nanotubes were fabricated (Fig. 10).¹⁰⁴ Followed by incorporation of platinum nanoparticles (Pt NPs) into these nanotubes, an artificial photosynthetic system of FF/THPP/nPt nanotubes was fabricated to allow harvesting of solar energy, with the THPP and Pt NPs acting as the light-harvesting unit and the mediator for efficient separation and transfer of excited electrons, respectively (Fig. 10a).¹⁰⁴ Under visible light irradiation, the FF/THPP/nPt system was shown to accelerate the regeneration of nicotinamide adenine dinucleotide (NADH) and efficiently facilitate a redox enzymatic reaction, compared to FF/THPP nanotubes, THPP monomers, or FF/nPt (Fig. 10b).¹⁰⁴ This strategy was further employed using a Fmoc-FF self-assembling hydrogel, where the J-aggregated porphyrins promoted efficient excitation energy transfer, thus facilitating increased photochemical water oxidation by iridium oxide nanoparticles.¹⁰⁵

Fig. 10.

Artificial photosynthesis applications of short peptide self-assemblies. (a) Scheme of a biomimetic photosynthetic system co-assembled by FF and THPP that can facilitate a redox enzymatic reaction. (b) Comparison of l-Glu turnover by glutamate dehydrogenase in the presence of pristine FF nanotubes, THPP monomers, THPP/nPt, FF/THPP nanotubes, or FF/THPP/nPt. Reproduced from ref. 104 with permission from [John Wiley and Sons], copyright [2012].

In addition to porphyrins, graphitic carbon nitride (g-C₃N₄) is another efficient light-sensitive chromophore for photocatalytic applications due to the adjustability of its electronic properties and the high thermal stability.¹⁰⁶ By co-assembling g-C₃N₄ and Fmoc-FF hydrogels, the effective exfoliation of g-C₃N₄ nanosheets facilitated photoinduced electron transfer. As a result, the photocurrent density was approximately two-fold higher than that of pristine g-C₃N₄, thus demonstrating the prominent biomimetic photosynthesis activity of these complexes.¹⁰⁷

To enrich the diversity of short peptide-based assemblies for artificial photosynthesis, multi-chambered microspheres co-assembled by FF and tetrakis(4-sulfonatophenyl)porphine (H₂TPPS) were prepared, consisting of an interconnected network of J-aggregates driven by electrostatic coupling of [FF]⁺ cations and [H₄TPPS]²⁻ dianions.¹⁰⁸ These porous and highly hydrated colloidal microspheres showed significant photocatalytic activity and stability in catalytic reactions involving inorganic or organic species, such as the photocatalytic reduction of Pt²⁺ and nucleation of Pt NPs on the microsphere surface, as well as the photocatalytic reduction of 4-nitrophenol into 4-aminophenol.¹⁰⁸ In addition, long-range alignments of J-aggregated nanorods were achieved via co-assembly of negatively-charged porphyrin H₂TPPS molecules and positively-charged l-Lys-l-Lys (KK) dipeptides, which further grew into long-range fibre bundles (Fig. 11a).¹⁰⁹ The fibre bundles showed red fluorescence upon excitation at 488 nm (Fig. 11b). Interestingly, the emission maximum was at 720 nm, in accordance with the detection of red fluorescence by CLSM (Fig. 11c), with a large Stokes shift of more than 200 nm due to the bundling of peptide–porphyrin J-aggregated nanorods.¹⁰⁹ In particular, these bundles displayed enhanced photostability, amplified helicity and anisotropic birefringence, and thus could be used for photocatalytic synthesis of Pt NPs in a sustainable manner.¹⁰⁹

Fig. 11.

Photocatalytic properties of short peptide assemblies upon co-assembly with photosensitive moieties. (a) Schematic representation of the co-assembly of KK and H₂TPPS into nanorods and then into long-range fibre bundles. (b) CLSM image of peptide–porphyrin fibre bundles with excitation at 488 nm and emitted fluorescence collectively detected from 680 to 760 nm. (c) Fluorescence emission spectrum of the fibre bundles showing the maximal emission at 720 nm. Reproduced from ref. 109 with permission from [John Wiley and Sons], copyright [2015].

Furthermore, stable, flexible and self-functionalized porphyrin/titanium oxide (TiO₂)/Pt/peptide-fibre bundles were fabricated by co-assembly of KK and TPPS in a recapitulated prebiotic environment (acidic, hot, mineral-containing water) under visible light illumination.¹¹⁰ After mineralization of TiO₂ and Pt NPs on the positively charged peptide fibres, TiO₂/Pt functioned as primitive reaction centres for the generation of hydrogen by utilizing the light energy harvested by the assembled porphyrins.¹¹⁰ Therefore, this system can be envisioned as a primitive photobacteria-like model for light harvesting, energy transfer, and ultimately sustainable hydrogen evolution.

The basic constituents of peptides, namely, amino acids, can also be used to mimic complex biological systems. For instance, the adhering fibres fabricated from Fmoc-l-Lys, Sn(iv)tetrakis(4-pyridyl)porphyrin (SnTPyP), 3,4-dihydroxyphenylalanine (DOPA), and Co₃O₄ NPs were used as a biomimetic cyanobacteria model for oxygenic photosynthesis, enhancing the oxygen evolution rate with excellent sustainability.¹¹¹ The hybrid microspheres fabricated using cysteine (Cys), TPPS, alcohol dehydrogenase (ADH) and metal ions (Zn²⁺) were employed as a chloroplast mimicry model for efficient photochemical production of fuels, such as hydrogen and aldehyde.¹¹² Furthermore, hybrid microspheres composed of radially aligned Cys/Zn nanorods decorated with carbonatedoped zinc sulphide (C-ZnS) nanocrystals were generated as primitive pigment models for various photochemical reactions such as carbon dioxide photoreduction, hydrogen evolution, and reduction of nicotinamide adenine dinucleotide (NAD⁺) to NADH.¹¹³

Covalent conjugation strategies

In addition to co-assembly, the light-sensitive antennas can be covalently conjugated to peptides, thereby further promoting the organization and optimizing the optics.¹¹⁴ For example, the MCpP–FF hybrid compound, produced by conjugating 5-mono(4-carboxyphenyl)-10,15,20-triphenyl porphine (MCpP) to the N-terminus of FF (Fig. 12a), was designed to self-assemble into nanofiber-based multiporous microspheres (Fig. 12b).³⁹ The notable photoelectron transfer and excitation wavelength red-shift of the microspheres, promoted by the extensive π–π interactions, resulted in broad-spectrum light sensitivity and in attenuated fluorescence decay capability (Fig. 12c). When sputtered with Pt NPs, the system presented a high turnover frequency of NADH and was applied for the biocatalytic production of l-Glu without the need for external mediators. In particular, these microspheres were stable in water, allowing their retrieval and re-usage in a sustainable manner.³⁹

Fig. 12.

Photocatalytic properties of short peptide assemblies upon covalent conjugation of photosensitive moieties. (a) Molecular structure of the MCpP–FF molecule. (b) SEM image of the MCpP–FF self-assembling multiporous microspheres. (c) Schematic electron transition profiles showing discrete excitation wavelengths absorbed by MCpP–FF in toluene solution while a broad excitation by the microspheres. Reproduced from ref. 39 with permission from the [American Chemical Society], copyright [2017].

In addition, through appending the same porphyrin moiety to the C-terminus of FF, another FF–porphyrin hybrid molecule was designed, displaying self-assembly into fibres, platelets or nanospheres, depending on the solvent polarity.¹¹⁵ Specifically, the fibres formed in a relatively apolar solvent (DCM : n-hexane at 3 : 7) showed intense excitonic couplets in the electronic circular dichroism spectra and functioned as quenched antennas. When increasing the solvent polarity (by increasing the DCM portion), the fibres transformed into nanospheres (DCM : n-hexane at 1 : 1) which showed no chiral signals. Upon further decreasing the polarity (by dilution with n-hexane), intense Cotton effects were recovered, thus providing a reversible switch between active and inactive light-harvesting. Furthermore, upon solvent drying, the hybrid building blocks could form porous spheres with tuneable size, depending on the solvent.⁷² These spherical nanostructures were found to show increased fluorescence lifetime while still maintaining the electronic properties of porphyrin, thus possessing tremendous potential for solar cell technologies and biomedical applications.

Phototherapeutic properties

The use of short peptide optics in drug release for cancer therapy emanated from the light-responsiveness of short peptide assemblies.²⁴ The inherent biological origin of short peptides makes them promising candidates for such therapy. In the wide range of cancer treatments, phototherapy, including photothermal and photodynamic therapy, has unique advantages, including reduced side effects, low toxicity for normal cells and remote controllability.^116–118

Photothermal therapy

Plasmonic gold nanostructures are widely used for photothermal therapy due to their biocompatibility and large optical coefficients in the NIR region.¹¹⁹ NIR laser-activatable microspheres were developed based on Fmoc-FF and plasmonic gold nanorods (Au NRs) via a simple freeze-quenching approach.¹²⁰ These structures exhibited a broad surface plasmon adsorption between 650 to 1000 nm, well within the ideal phototherapeutic window for tumours. When exposed to NIR laser (808 nm) illumination, a remarkable sustained doxorubicin (DOX) release from DOX-loaded Au NR-embedded microspheres was observed. Moreover, on/off (pulsatile) DOX release could be achieved by easily tuning the laser exposure, thus demonstrating the potential of the system as an effective and controlled drug delivery vehicle for cancer therapy.¹²⁰

Functional short peptides can also be used to modify other nanomaterials in photothermal therapy, allowing improvement of their selectivity and efficiency.¹²¹ For example, after attaching the RGD targeting tripeptide to high NIR-absorbed,^122,123 nanosized, reduced graphene oxide (nano-rGO) sheets, the nanomaterials exhibited selective cellular uptake into U87MG cancer cells and highly effective photoablation of cells for photothermal heating in vitro.¹²² Concurrently, through sequentially conjugating the positively-charged Arg-Arg-Leu-Ala-Cys (RRLAC) peptide on the surface, poly(ethylene glycol)–gold nanorods (PEG–GNR) were shown to form a complex with the negatively-charged photosensitizer Al(iii) phthalocyanine chloride tetrasulfonic acid (AlPcS4).¹²⁴ Compared to free AlPcS4, the GNR–AlPcS4 complexes possessed higher intracellular uptake and phototoxicity for squamous cell carcinoma (SCC7) cells in vitro. Furthermore, tumour growth in vivo was significantly reduced following this dual photothermal and photodynamic therapy, compared to the photodynamic therapy alone.¹²⁴

A hybrid molecule conjugating tetraphenylporphyrin (TPP) to the FF dipeptide, namely, TPP-G-FF, was designed to self-assemble into photothermal peptide–porphyrin nanodots (PPP-NDs) (Fig. 13a).¹²⁵ Due to the extensive π–π stacking and hydrophilic interactions, the PPP-NDs exhibited high stability and light-to-heat conversion efficiency of up to 54.2%.¹²⁵ These properties endow the PPP-NDs with the potential to comprise promising alternatives for photoacoustic imaging and photothermal therapy. For instance, after injecting the PPP-NDs into tumour-bearing mice, photoacoustic images showed that the tumours were gradually targeted and finally ablated following a 10 min irradiation treatment (Fig. 13b).¹²⁵

Fig. 13.

Photothermal therapy applications of short peptide self-assemblies. (a) Schematic illustration of photothermal nanodots (PPP-NDs) self-assembled by a peptide–porphyrin conjugate (TPP-G-FF). (b) IR thermal images of mice following intravenous injection of PPP-NDs under continuous 10 min irradiation. Reproduced from ref. 125 with permission from the [American Chemical Society], copyright [2017].

Photodynamic therapy

Photodynamic therapy employs light-active photosensitizers that generate reactive oxygen species to damage tumour tissues.¹²⁶ In this regard, short peptide-based self-assembling nanomaterials have been reported to be used for drug delivery in photodynamic therapy systems.^127,128 CDP nanoparticles were developed through GA-assisted assembly.¹²⁷ After loading of the photosensitive drug chlorine e6 (Ce6) and modification with a heparin polymer, the CDP NPs showed enhanced retention and permeability effects, as long as longer circulation lifetime.¹²⁷ As a result, these CDP/Ce6 NPs displayed an enhanced antitumor effect compared to free Ce6 both in vitro and in vivo.¹¹⁶ Furthermore, CDP and Fmoc-l-Lys were co-assembled with Ce6 to fabricate CDP/Ce6 nanoparticles (CCNPs) and Fmoc-l-Lys/Ce6 nanoparticles (FCNPs), respectively, driven by multiple weak intermolecular interactions.¹²⁸ These NPs showed improved photosensitizer-loading efficiency, preferable cellular uptake and biodistribution. Moreover, the NPs were responsive to the pH, surfactant and enzyme, thus resulting in controlled drug release. Therefore, following a single exposure to light, the enhanced photodynamic therapy efficacy of the NPs led to almost complete tumour eradication in mice.¹²⁸

However, several concerns such as limited photostability, ease of aggregation and the lack of appropriate absorption bands still hinder the extensive applications of peptide self-assemblies in photodynamic therapy.¹¹⁸ To address these problems, well-monodispersed and biocompatible dipeptide-based nanospheres (DPGNSs) were fabricated by conjugating CDP and genipin which served as the photosensitizer to convert O₂ to singlet oxygen (¹O₂) (Fig. 14).¹²⁹ When incubated with cancer cells, no obvious cytotoxicity was observed for the DPGNSs, while upon irradiation, cell viability dramatically decreased.¹¹² Spectrum analysis and electron paramagnetic resonance proved the generation of ¹O₂ in the presence of DPGNSs upon light illumination, thus indicating that the system can serve as a new type of bioinspired, efficient photosensitizer for photodynamic therapy.¹²⁹

Fig. 14.

Schematic illustration of CDP–genipin self-assembly into dimers and then into supramolecular nanospheres (DPGNSs), allowing the conversion of O₂ to singlet oxygen (¹O₂) for photodynamic therapy. Reproduced from ref. 129 with permission from [John Wiley and Sons], copyright [2016].

In photodynamic therapy, short excitation wavelength of the photosensitizers (usually below 700 nm) would result in poor tissue penetration. To increase the tissue penetration depth, an excitation wavelength of 810 nm, in the NIR region, was applied on GA-cross-linked CDP nanoparticles doped with a photosensitizer (RB) and a fluorescent dye (BP) (BP-CDPNPs-RB).¹³⁰ Upon irradiation of BP-CDPNPs-RB by a two-photon laser, the maximal emission of BP (520 nm) and RB (580 nm) was dramatically decreased and enhanced, respectively. This indicated an energy transfer from BP to RB through intraparticle energy transfer, thus leading to photogeneration of singlet oxygen for photodynamic therapy of cancer cells.¹³¹ In particular, the utilization of NIR excitation ensures high tissue penetration and negligible side effects, thus demonstrating the promising potential of short peptide nanoparticles for clinical applications. Moreover, as in photothermal therapy, functional short peptides, such as cell-penetrating peptides (transactivator of transcription (TAT) peptide), the targeting cyclic cRGDfk peptide and the proapoptosic peptide (KLAKLAK)₂, can also be used to modify the photoactive nanomaterials, thus allowing improvement of their selectivity and efficiency in photodynamic therapy.^132–134

Conclusions

Short peptide-based nanobiotechnology facilitates the fabrication of functional micro- and nano-materials self-assembled by simple building blocks. The flexible and unique optical properties of short peptide self-assemblies can be utilized for numerous nanobiotechnological applications, such as the fabrication of bio-organic optoelectronic devices and construction of photo-controlled drug delivery systems. In addition, their intrinsic biocompatibility can allow harnessing their optical properties for use in the biological world. Moreover, studies of this class of materials can not only supply next-generation, bio-inspired optical alternatives, but also extend our understanding of the roles of polypeptide or protein optics in physiology and pathology. This may allow direct, label-free, real-time monitoring and sensing of metabolic activities, as well as analysis, and possibly intervention, of biological systems. With these goals in mind, the optical short peptide systems can be appealing candidates to link chemistry, materials science, biology, optics and engineering, and provide a promising technological tool for fundamental biology, biomedicine, and nanobiotechnology studies.

Biographies

Bingbing Sun Dr Bingbing Sun joined Prof. Junbai Li’s group for her PhD studies in Physical Chemistry at the Institute of Chemistry, Chinese Academy of Sciences (ICCAS) in 2014. During her PhD research, she received the ICCAS Youth Scientific Excellent Award and the ICCAS Institute First-class Scholarship. Her current research interests include synthesis and self-assembly of peptides, and their applications in the biomedical field.

Kai Tao Dr Kai Tao studied biochemical engineering & technology at China University of Petroleum (East China) and completed his PhD in the Excellent Doctoral Dissertation Program of UPC, under the co-supervision of Prof. Jian R. Lu and Prof. Hai Xu. For his PhD studies he received several prizes including the China National Scholarship. He joined the group of Prof. Ehud Gazit for postdoctoral research in 2014. His current scientific interests mainly focus on bioorganic integration engineering and biomimetic mechanics.

Yi Jia Dr Yi Jia received her PhD degree in 2012 from the Institute of Chemistry, the Chinese Academy of Sciences, in Prof. Junbai Li’s group and then she joined his group and became an associate professor in 2015. Her research interests include layer-by-layer assembled micro/nanostructures, reconstitution of motor proteins, self-assembly of dipeptides and their related biomedical applications.

Xuehai Yan Prof. Xuehai Yan received his PhD degree in 2008 from the Institute of Chemistry, the Chinese Academy of Sciences, in Prof. Junbai Li’s group. Then he moved to the Max Planck Institute of Colloids and Interfaces in Germany for postdoctoral research as an Alexander von Humboldt fellow from 2009–2012. In 2013, he became a full professor at the Institute of Process Engineering (IPE), CAS. Currently, he is the deputy director of the State Key Laboratory of Biochemical Engineering and the Center of Mesoscience, IPE, CAS. His research interests mainly focus on peptide-modulated self-assembly, mesoscale mechanisms, supramolecular colloids and antitumor phototherapy.

Qianli Zou Dr Qianli Zou received his PhD degree in Organic Chemistry in 2013 from the Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. Then he joined Prof. Xuehai Yan’s group as an assistant professor at the State Key Laboratory of Biochemical Engineering (SKLBE) at the Institute of Process Engineering (IPE), Chinese Academy of Sciences, in 2014 and was then promoted as an associate professor in 2016. His research interests focus on synthesis and self-assembly of functional peptides for applications in antitumor therapy and biomimetic photosynthesis.

Ehud Gazit Prof. Ehud Gazit is the incumbent Chair for Biotechnology of Neurodegenerative Diseases at Tel Aviv University. He received his BSc (summa cum laude) after completing his studies at the Special University Program for Outstanding Students at Tel Aviv University and his PhD (with highest distinction) from the Weizmann Institute of Science. He has been a faculty member at Tel Aviv University since 2000, following the completion of his postdoctoral studies at Massachusetts Institute of Technology (MIT). He was funded by ERC for the project of bio-inspired self-assembled supramolecular organic nanostructures in 2016. Prof. Gazit’s research interests involve exploring the biological and bio-inspired molecular self-assembly, and using the minimalistic approach to define the smallest molecular recognition and assembly modules.

Junbai Li Prof. Junbai Li is a professor at the Institute of Chemistry, the Chinese Academy of Sciences. He obtained his BSc, MSc, and PhD degrees in Polymer Science from Jilin University. He then spent several years carrying out postdoctoral work and a joint research project at the interface department of Max Planck Institute of Colloids and Interfaces in Germany. His research interests involve molecular biomimetics-based molecular assembly, reconstitution of motor proteins, self-assembly of dipeptides, biointerfaces, bioinspired materials, and nanostructured design.