Life at the interface: Engineering bio-nanomaterials through interfacial molecular self-assembly

Abstract

Interfacial self-assembly describes the directed organization of molecules and colloids at phase boundaries. Believed to be fundamental to the inception of primordial life, interfacial assembly is exploited by a myriad of eukaryotic and prokaryotic organisms to execute physiologic activities and maintain homeostasis. Inspired by these natural systems, chemists, engineers, and materials scientists have sought to harness the thermodynamic equilibria at phase boundaries to create multi-dimensional, highly ordered, and functional nanomaterials. Recent advances in our understanding of the biophysical principles guiding molecular assembly at gas-solid, gas-liquid, solid-liquid, and liquid-liquid interphases has enhanced the rational design of functional bio-nanomaterials, particularly in the fields of biosensing, bioimaging and biotherapy. Continued development of non-canonical building blocks, paired with deeper mechanistic insights into interphase self-assembly, holds promise to yield next generation interfacial bio-nanomaterials with unique, and perhaps yet unrealized, properties.

Graphical Abstract

Interfacial biomolecular assembly is fundamental to biotic homeostasis. Exploiting and engineering these natural phenomena can produce an array of functional nanomaterials and potentiate a new generation of bio-nanotechnologies with unique biochemical and biophysical properties.

1. INTRODUCTION

Molecular self-assembly is a fundamental physiologic process across all branches of the tree of life. It is a thermodynamic counterbalance of enthalpic contributions from inter- and intramolecular forces, asymmetrically opposed to entropic penalties of order. Non-covalent interactions, including hydrogen bonding, hydrophobics, electrostatics, π−π stacking, and van der Waals forces, collectively pattern molecules into thermodynamically favorable, higher-ordered structural states. In biology, the resultant nanostructures often transform inanimate building blocks into functional arrangements that have unique biophysical and biochemical properties.

Phase-separated interfaces play an outsized role in the inception of this nanoscale order. Often, hydrophobic mismatch between opposing phases templates amphiphilic biomolecular building blocks, commonly lipids, proteins, peptides, and nucleic acids, into conformational arrangements that trigger higher order organization. Examples of interphase assembly are ubiquitous among eukaryotes and prokaryotes, and at every molecular scale. For example, several plant species, most well-known of which are lotus leaves, have an epidermal layer of assembled waxy papillary tubes that create a superhydrophobic water-repellant surface. Geckos have nanoscale hair-like structures on their feet that create a large surface area for van der Waals forces with substrates, enabling adhesive qualities that allow these animals to scale sheer surfaces. In humans, and most mammals, solid-liquid, gas-liquid, and liquid-liquid separations give rise to anatomical sub-systems that are essential to physiologic homeostasis (Figure 1).

Figure 1.

Examples of interfacial molecular assembly in human physiology. Accretion of lipid and salt secretions at the skin surface (solid-liquid) creates a protective barrier from the external environment. Surfactant assembly in alveoli (gas-liquid) prevent collapse of these structures during exhalation. Organization of bile and lipid droplets (liquid-liquid) facilitates gastrointestinal digestion and absorption.

In this review, we summarize recent advances in the mechanistic understanding of interfacial assembly of biologic building blocks to create adaptive nanomaterials. Building upon the structure-function-performance relationships derived from these studies, we present recent developments in the application of interfacial assembled materials in the fields of biosensing, diagnostics and nanomedicine. Given the cross-disciplinary literature on interfacial self-assembly, we focus our review on emerging methods in the creation of functional nano-scale materials using biologic building blocks, including lipids, amino and nucleic acids, carbohydrates, proteins, and peptides.

2. INTERFACE-DRIVEN ASSEMBLY MECHANISMS

Self-assembly is a thermodynamic and kinetic equilibrium determined by both molecular structure, environment, and temporal parameters. The vast compositional landscape available provides an array of pathways that are productive to biochemists, materials scientists, and nanotechnologists towards generating functional matter. This is juxtaposed to an exponentially greater number of non-productive pitfalls. By templating molecular interactions at bi-phasic interfaces, self-assembly is encouraged towards specific regions of the conformational space, thereby favoring useful outcomes, and limiting potential undesired interactions (Figure 2a). In this sub-section we distinguish these interphases as gas-solid, gas-liquid, solid-liquid, and liquid-liquid (Figure 2b), and summarize recent efforts exploiting these interfaces to create functional nanomaterial platforms. Several examples are discussed in each context to highlight salient properties manipulated during molecular assembly at these interfaces to develop functional nanotechnologies. A unifying theme of these examples is that changing interfacial surface energies, often related through Gibbs adsorption isotherms, can be exploited to access specific hierarchical conformations and designer nanostructures.

Figure 2.

(a) Conceptual schematic of directed molecular self-assembly at a bi-phasic interface, thereby limiting conformational outcomes to a desired region of the thermodynamic landscape. (b) Representation of the four immiscible interphases used to construct interfacial assembled biomaterials from amphiphilic building blocks.

Gas-Solid

Gas-solid interfacial assemblies can be tuned by modifying the surface energy of the solid, the chemical reactivity of the gaseous phase, and assembly propensity of the amphiphilic building block. Surface oligomerization and morphology of the final structure benefits from tuned surface tension, temperature, viscosity, and rate of evaporation by immersion media. While gas-solid nano assemblies have been an exciting area of recent exploration in the fields of fluid dynamics (Bavi & Ghadak, 2021; Lu, 2017), energy (Janghel, Saha, & Karagadde, 2020; Kumar et al., 2023; Trocino et al., 2019), and electronics (Gayathri et al., 2016; Poonia, Manjuladevi, & Gupta, 2018; Xiao et al., 2019), their application to bio-nanomaterial creation has been comparatively limited. This is largely due to the prerequisite for a liquid medium to facilitate biomolecular assembly. Consequently, many of the gas-solid assemblies discussed in the literature result from solvent evaporation or material transfer from air-liquid-solid triphasic formulations.

A notable example by Li et al. used air-solid interfaces to tune the flexibility of hair-like filaments to create superhydrophobic nanostructures (Li et al., 2023). Mimicking the morphology of setae and cilia in many organisms, these filaments exploit the surface thermodynamic mismatch between the solid-air interface of a trapped gas pocket, and the solid-liquid interface of the standing water droplet, to produce pillar, curled fibril, or flake morphologies (Figure 3). The resulting ‘hairy’ surfaces reduced the solid-liquid interfacial surface area to generate a superhydrophobic nano-surface. This interesting study highlights the potential to tailor interphase surface tensions to productively modulate nanoarchitectures.

Figure 3.

Morphology of hierarchical micro-nanostructures. (a–c) Scanning electron micrograph of micropillar, filament, and flake structures. (d) View of a single filament. (e) White light interferometer view of pillar structures. (f) Three typical structures constructed with different molds. (g) Roughness of 3 different slices across the microstructure line in panel (e). Reprinted with permission from Li et al. (2023), © 2023, American Chemical Society.

Most structural assemblies at air-solid interfaces result from Langmuir-Blodgett films, where nanostructures are formed by immersing a solid substrate into a liquid containing monolayers of molecular building blocks. A unimolecular film deposits on the solid surface through physical adsorption, and the thickness can be carefully tuned via the number of dip cycles. Mechanistically, the deposition and arrangement of compounds onto the solid substrate surface is driven by minimizing the Gibbs free energy of mixing, as was recently demonstrated for fatty acids organized on the surface of a silicon wafer (Ahmed, Haque, et al., 2018). These approaches have been particularly useful for studying the biophysics of membrane-intercalating biomolecules, including peptides (Chanci et al., 2022; Di Napoli et al., 2018; Kumar, 2019), peptoids (Murray et al., 2019), and enzymes (Saha & Chowdhury, 2020). Ahmed et al. further demonstrated that addition of chitosan, a cationic biopolymer, electrostatically complexes with surface adsorbed fatty acids to generate heterogeneously distributed morphologies with reduced film elasticity (Ahmed, Dildar, et al., 2018). As a proof of principle in biosensing applications, Caseli and coworkers incorporated graphene oxide sheets into lipid films stabilizing the enzyme penicillinase. Incorporation of graphene was found to productively alter the adsorbed enzyme’s thermodynamic, structural, and metabolic properties, ultimately preserving functional protein on a supported air-solid film (Scholl, Siqueira Jr, & Caseli, 2022). This same group has demonstrated tunable catalytic activity of the enzymes asparaginase, urease, and phenylalanine dehydrogenase when co-embedded within air-solid assembled lipid films using carbon nanotube and algal polysaccharide additives (da Silva et al., 2021; Possarle, Junior, & Caseli, 2020; Rodrigues et al., 2018). These studies highlight the ability to exploit steric crowding at the surface of air-solid interfaces to tune the bioactivity of physiosorbed proteins.

Our group recently reported the development of mechanomorphogenic thin films that arise from organization of fluorine-rich amino acids at the air-solid interface (Figure 4) (Sloand et al., 2021). Initiation of self-assembly occurs at liquid-liquid interfaces, where the fluorinated non-natural amino acid first organizes at the interface of water-perfluorocarbon solvent mixtures to create an emulsion. When exposed to air, the film partitions to the gas-liquid interface, carrying the perfluorocarbon solvent with it, and ultimately migrates to the air-solid interface. These varied morphologic responses arise from the fluorophilic nature of the non-natural amino acid, which promotes the sequestration of the assemblies away from the aqueous environment. Additional biophysical studies showed that the non-covalent interactions imparted self-healing properties to the coatings when disrupted under various mechanical stimuli.

Figure 4.

Schematic and UV optical images of mechanomorphogenic films formed via supramolecular assembly of Fmoc-pentafluoro-L-phenylalanine (Fmoc-F₅-Phe) at the perfluorohexane (PFHx)-water interface. Step i shows formation of initial fluorous organelle. Exposure to air leads to spontaneous re-organization at the air–water interface (step ii). Centrifugal agitation precipitates the film (step iii), which reverts to the assembled macrostate if re-exposed to air (iii → ii). Alternatively, sonication of the precipitated film converts the material back to the initial fluorous organelle (iii → iv). These operations can be performed cyclically ad infinitum. Reprinted with permission from Sloand, et al. (2021).

2.1. Gas-Liquid

A precursor to the deposition of Langmuir-Blodgett films onto solid substrates is the generation of Langmuir monolayers at air-water interfaces (Oliveira Jr, Caseli, & Ariga, 2022). These monolayers are traditionally composed of amphiphilic monomers that preferentially organize at the interface to embed their hydrophobic and hydrophilic regions in the air and water phases, respectively. Using a Langmuir-Blodgett trough, these monolayers can then be compacted to form multilayered films. In addition to small molecule surfactants, conjugated polymers and nanoparticles have been employed as the assembling unit (da Rocha Rodrigues et al., 2020; Swierczewski & Bürgi, 2023; Yamamoto et al., 2020). While an ever-expanding array of applications using Langmuir-Blodgett films can be found in the literature, most utilize these films to develop nanocoatings on the surface of solid substrates. In this section, we focus specifically on functional nanomaterials generated from assembly phenomena that complete at air-liquid interfaces.

Gas filled nanobubbles are nanoscopic cavities <200nm in size that exist as a spherical cap at solid surfaces (surface nanobubbles) or as a gas-filled sphere in solution (bulk nanobubbles). Bulk nanobubbles are of particular interest in biomedicine due to their long persistence lifetimes, electrostatic interfaces, and potential to serve as diagnostic and drug delivery agents (Nirmalkar, Pacek, & Barigou, 2018b). Their unprecedented longevity has garnered particular interest given that Young-Laplace calculations predict they should be unstable in solution, dissolving on the time scale of 1 – 100μs (Nirmalkar, Pacek, & Barigou, 2018a). Despite continued skepticism on their existence (Jadhav & Barigou, 2020; Nirmalkar, Pacek, & Barigou, 2018b), several recent mechanistic studies have focused on the thermodynamic nature of the gas-liquid interface to explain their stability (Tan, An, & Ohl, 2021). Ohgaki et al. reported evidence that nanobubble stability may result from strong hydrogen bonding at the interface that leads to reduced gas diffusivity (Ohgaki et al., 2010). Other work suggests that contaminants in water organize at the gas-liquid interface to hinder gas diffusion (Ducker, 2009).

It is generally accepted that, at equilibrium, external electrostatic pressure at the nanobubble interface in aqueous environments balances the internal Laplace pressure to create a net zero diffusion of gas and maintain stability of the bubble. However, ionic environments containing mono- and multi-valent salts destabilize nanobubbles by screening the electric double layer (Hewage, Kewalramani, & Meegoda, 2021; Nirmalkar, Pacek, & Barigou, 2018a). Consequently, several groups have explored developing self-assembled layers that organize at the gas-liquid interface to stabilize nanobubbles in physiologic solutions relevant to biomedical applications. The Exner lab recently reported ultrastable nanobubbles that utilized bilayer membranes composed of phospholipid and propylene glycol coatings (Perera et al., 2019). The contrasting elastic properties of these layers redistributed stress and dissipated excess energy to better enable the nanobubbles to withstand mechanical deformation by ultrasound and pulsatile flow. These favorable surface properties afforded ultrasound contrast agents that lasted roughly ten times longer than commercially available analogues. The same group also demonstrated that incorporation of the surfactant Pluronic L10 into nanobubble lipid monolayers stabilized the bubbles under ultrasound through surface tension reduction (Hernandez et al., 2018). However, these effects differed due to the nature of crystalline vs. liquid-crystalline packing at the monolayer (Figure 5), indicating that the nanostructure of the interfacial molecular stabilizer plays an important role in bubble dynamics and gas diffusion.

Figure 5.

Nanobubble membrane behavior under ultrasound. (A) Change in bubble diameter in response to ultrasound pressure. (B) Schematic of lipid membrane structure undergoing compression and expansion. (C) Schematic of theoretical Π–A isotherms and corresponding structure of a lipid monolayer. Reprinted with permission from Hernandez, et al. (2018). © 2018, American Chemical Society

Cavitation of bubble nuclei has also been used to direct molecular assembly at, or from, gas-liquid interfaces. Here, the high temperature and pressure conditions generated during bubble collapse can either initiate assembly, or deposit pre-assembled interfacial constructs into organized structures following detonation. In a recent example, multi-layered carbon nanomaterials, referred to as ‘nano-onions’, were prepared via bubble detonation to create nanoscale lubricant additives (Luo et al., 2019). Similarly, two dimensional platinum nanowires were produced using bubble cavitation to axially organize platinum nanosheets that had assembled at bubble gas-liquid interfaces (Zhu et al., 2023). These examples highlight a growing area of research in utilizing cavitating bubble nuclei to rationally deposit assemblies at gas-liquid interfaces towards the construction of designer nanostructures.

2.2. Solid-Liquid

Molecular self-assembly at solid-liquid interfaces has been exploited using an array of strategies to develop hierarchical two- or three-dimensional architectures (Hou et al., 2023). Modulating the surface chemistry of nano-colloids, as well as controlling their collective properties, offers an enormous parameter space to tune interfacial tensions (Duan et al., 2021). Consequently, solid-liquid assembly phenomena can yield a broad range of functional nanomaterials with rationally designed properties using nucleic acid, carbohydrate, peptide and protein building blocks (García-Jiménez et al., 2022; J. Li et al., 2019; Stel et al., 2019; Sun, Su, & Wei, 2020; S. Zhang et al., 2021). Biochemical diversity within these molecular classes allows for tunable chirality and tertiary assembly on the functionalized surfaces, altering the identity of the surface displayed structures.

The field of DNA nanotechnology, as a notable example, has enjoyed a rapid expansion of bioapplications by moving DNA assembly from purely aqueous solutions to solid-liquid interfaces (Wang et al., 2019). Work by the Mirkin group demonstrated that nucleic acids tethered to solid nanoparticles could potentiate the surface assembly of DNA from the liquid phase, subsequently yielding an array of polyvalent DNA-functionalized nanomaterials (Cutler, Auyeung, & Mirkin, 2012; Kapadia, Melamed, & Day, 2018; H. Li et al., 2018; Mokhtarzadeh et al., 2019). An exciting extension of early DNA nanotechnologies, which often utilized thiol-functionalized gold surfaces, is the development of protein-based spherical nucleic acids (Figure 6). Samanta et al., recently demonstrated that β-galactosidase, a glucose oxidase, could serve as the functional core and template the coordination of a radially oriented oligonucleotide shell (Samanta et al., 2020). Using a duplex-sensitive dye, this technology allowed for the detection of target analytes, while exploiting the biocompatibility, biodegradability, and well-defined surface topology of the protein template.

Figure 6.

(A) Structure of β-galactosidase with lysine and cysteine residues highlighted. (B) Structure of the forced intercalation dye thiazole orange, TO (carboxymethylated derivative). (C) Unfolded intercalation motif (i-motif) sequence with a single base replaced with TO. (D) Folded i-motif with TO intercalated between base pairs. (E) Structure of the particle at pH 7.5 and pH 5.5. The formation of the i-motif structure leads to fluorescence turn on of TO. Reprinted with permission from Samanta, et al. (2020). © 2020, American Chemical Society.

DNA origami, or the assembly of oligonucleotides into regular two- and three-dimensional lattices, at solid interfaces presents a versatile and powerful method for creating functional nanostructures (Cao et al., 2020). Kielar et al., recently showed that DNA origami lattices organize on mica surfaces due to electrostatic interactions, and that the adsorption isotherms can be tuned by the ratio of monovalent Na⁺ and Mg²⁺ ions (Kielar et al., 2018). Studies using silicon surfaces also yielded ordered DNA origami lattices that could be functionalized with metal cations towards electronic applications (Tapio et al., 2023). Additionally, DNA tiles can be constructed on the surface of lipid-based substrates, enabling a broader range of functional nanostructures that mimic the arrangement of nucleic acids at cellular membranes (Endo, 2022; Julin, Keller, & Linko, 2022). Extensions of these assembly methods have allowed for patterning of DNA-based nanomaterials in dry ambient environments (Yang et al., 2021). In this strategy, DNA tiles were used as binding frames to control the deposition of gold nanorods via cation-controlled surface diffusion. This enables the large scale assembly of gold nanorods into highly ordered, complex superstructures with utility in plasmonic and electronic devices, where material properties can be tuned by controlling counterion interactions (Chee et al., 2019; Li et al., 2020). In addition to controlling DNA nanotopology, surface adsorption also yielded more stable DNA origami structures, relative to bulk solution assembly conditions, by restricting the conformational entropy of assembled oligonucleotides via physicochemical surface interactions (Chen et al., 2018).

Other elegant demonstrations of solid-liquid interfacial assembly have yielded nanomaterials with functional topologies. An interesting example was the development of ‘nano snowboards’ by Wang and co-workers (Qiu et al., 2022). Here, molybdenum disulfide nanosheets were functionalized with phospholipids to create topologies that slid across the surface of articular cartilage, like that of a snow board in action. Forming a solid-liquid composite lubricant, these materials improved the lubricity and stability of joint cavities in murine models. Cao et al. created pillar-like nanopatterns of polyvinyl alcohol (PVA) polymers at solid-liquid interfaces by using a honey-comb patterned porous template (Figure 7) (Cao, Yabu, & Huh, 2020). The structured film was subsequently coated with CdS crystals to create a multilayered structure that mimicked the morphology of the moth eye. This has implications in the development of optoelectronic devices, solar cells and microlenses, given that these unique nanostructures allow moths to see in the dark without reflecting light that could be visible to predators. Other work has utilized surfaces that directionally seed the assembly of aqueous proteins, for example the osteogenic candidate lactoferrin, to create cell-proliferative, mineralization-stimulative, and antibacterial materials for bone regeneration (Zhang et al., 2020). A similar strategy utilized self-assembled chimeric fusions linking fibronectin and spider silk proteins to create biocompatible microsphere materials for cell culture applications (Ornithopoulou et al., 2023). An interesting link between these proteinaceous applications is the formation of β-rich amyloids as the functional nanostructure, which have been extensively studied in relation to neurologic diseases potentiated by protein misfolding and aggregation at soft matter interfaces (C. Li et al., 2018). These examples emphasize how mechanistic insights from protein aggregation pathologies can be leveraged to design functional peptide and protein interfacial nanomaterials.

Figure 7.

(a) Schematic representation showing the fabrication of PVA convex-structured film. (b,c) Typical SEM images of pristine honeycomb-patterned film template and PVA convex-structured film without functionalization, respectively. Reprinted with permission from Cao, et al. (2020). © 2020, American Chemical Society

2.3. Liquid-Liquid

Liquid–liquid phase separation is thought to have been fundamental to the emergence of life by organizing primordial compounds into bioactive higher order structures (Sarkar et al., 2020). Assembly of biomolecules within liquid-liquid phases continues to be a conserved paradigm in modern organisms as well. For example, phase separation of intrinsically disordered proteins gives rise to membraneless organelles that play fundamental roles in mammalian physiology and disease (Brocca et al., 2020; Elbaum-Garfinkle, 2019; Uversky, 2017; Wei et al., 2017). A recent, and salient, example of this is the discovery that SARS-CoV-2 nucleocapsid protein undergoes a liquid-liquid phase separation with RNA to suppress innate antiviral responses (S. Wang et al., 2021). Additionally, liquid-liquid phase separation of misfolded prion and amyloid proteins are now believed to potentiate the higher ordered self-assembled structures that underly amyloid disease pathology (Agarwal & Mukhopadhyay, 2022; Xing et al., 2021).

Inspired by this, several groups have sought to exploit liquid-liquid directed assembly phenomena to develop biomimetic nanomaterials. Here, interfacial assembly patterns can be discretely tuned by altering the interfacial tension between phases through rational selection of the two liquid media or use of an amphiphilic molecular surfactant. Work from our lab has shown that non-canonical amino acids can be directed to assemble at fluorous-water interfaces to develop mechanically responsive thin films and viscoelastic synthetic mucus (Miller & Medina, 2024; Sloand et al., 2021). Mechanistic insights gained from this work enabled the development of fluorine-rich small molecules and peptides that act as emulsifiers to stabilize perfluorocarbon nanodroplets (Kim et al.; Lawanprasert et al., 2021; Medina et al., 2017; Sloand et al., 2020; Sloand et al., 2021). By controlling the physicochemical properties of the emulsifier, we have shown that the thermodynamic stability of phase separated emulsions can be rationally tailored to enable thermally and mechanically responsive nanomaterials. In our group, we have exploited these unique properties to create ultrasound-sensitive liquid nanoemulsions for diagnostic and drug delivery applications (Medina et al., 2017; Sloand et al., 2020; Sloand et al., 2021). Through this work, we have demonstrated that controlling the assembly pathway of the peptide emulsifier at the liquid-liquid emulsion interface can yield nanoparticles with tunable acoustic sensitivity, cellular internalization, and in cellulo stability (Figure 8) (Kim et al., 2023). In particular, Pickering emulsions, which are particles stabilized by the accumulation of solid matter at the liquid-liquid interface (Aveyard, Binks, & Clint, 2003; Zhao et al., 2020), can be generated by creating peptide-based fibers and sheets at the interphase. These types of emulsions are distinct from particles prepared using monomeric surfactants, where the small molecule is in a dynamic equilibrium between the bulk solvent and interfacial assembly. As a result, peptide assembled Pickering emulsions can modulate the display of targeting ligands engineered into the peptide emulsifier sequence, enabling the optimization of formulations for efficient internalization into macrophages and stable oscillation under B-mode and Doppler ultrasound imaging.

Figure 8.

(a) Schematic representation of peptide emulsions (pEM) formulated using non-crosslinked (N-pEM) or crosslinked (pEM) peptide monomers, 1D fibrils (1D-pEM) or 2D sheets (2D-pEM). (b) Negatively stained TEM image of peptide nanofibrils; inset shows histogram of fibrillar diameter. (c) ThT fluorescence emission spectra in the presence of peptide in water (1D fibrils, blue) or in 2.5% DMSO (2D sheets, green). (d) CD spectrum of peptide emulsifier in water. (e) Negatively stained TEM image of 2D peptide sheets. (f) Size distribution of the fluorous emulsions; N-pEM, pEM, 1D-pEM, and 2D-pEM (from left to right). Negatively stained TEM images of (g) N-pEM, (h) pEM, and (i) 1D-pEM. (j) Cryo-SEM image of 2D-pEM. Yellow arrows in panel (i) denote fibrils adsorbed on the surface of the emulsion droplet. Reprinted with permission from Kim, et al. (2023).

One of the most extensively studied assembling units in liquid-liquid interfacial assembly are proteins. This has largely been driven by natural examples of liquid-liquid protein phase separation to form non-membranous organelles, cellular signaling pathways and aggregation-induced pathologies (Babinchak & Surewicz, 2020; Chong & Forman-Kay, 2016; Sun et al., 2020). Mussels, aquatic bivalve mollusks, are a particularly well-studied source of inspiration due to the role liquid-liquid protein assembly plays in their adherence to underwater surfaces. Over a decade of research has shown that mussel plaques, which function as the aquatic adhesive element of the organism, are largely composed of basic residues and the post-translationally modified amino acid 3,4-dihydroxyphenylalanine (DOPA). Together, these amino acids form a robust surface adhesion network through covalent, metal coordination and hydrogen bonding interactions. More recent work has shown that assembly of these adhesive plaques results from the formation of liquid-liquid protein coacervates and crystalline phases (Renner-Rao et al., 2022), which are potentiated due to hydrogen bonding interactions between protein subunits (Deepankumar et al., 2022; Guo et al., 2022). In addition to hydrogen-bond mediated phase separation, cationic arginine and lysine residues within proteins can also initiate liquid-liquid phase separation through electrostatic and aromatic cation-π interactions (Hong et al., 2022). Histidine-rich proteins, as well as co-formulation of proteins with carbohydrates, can execute similar functions through a combination of transient hydrogen bonding and hydrophobic interactions (Gabryelczyk et al., 2019; Q. Gong et al., 2022; T.-D. Zhang et al., 2021). The culmination of these phase separation events, and formation of a liquid-liquid interface, has been shown to modulate the antioxidant activity, metal absorption and fluorescent properties of the nano-coacervate, enabling new opportunities in bioimaging, biodetection, and water remediation. It is worth noting that many of these properties arise from interfacial crystalline phases of the assembling proteins, where a single hydrocolloid component phase separates in an entropy driven mechanism from a supersaturated isotropic bulk phase (Azzari, Bagnani, & Mezzenga, 2021). These processes generate filamentous colloids, and through understanding their thermodynamic principles provide additional opportunities to advance the design of long-range biomimetic nano-structures (Fraccia & Zanchetta, 2021). An interesting example of this from Tanaka et al. demonstrated the assembly of emulsified filamentous bacterial viruses (bacteriophages) into hierarchical nano-structures at liquid-liquid interfaces (Tanaka et al., 2020).

Liquid-liquid phase separation of oligonucleotides has been employed to develop highly ordered DNA nanotechnologies. Precision over the final assembled state is enabled by the highly regulated Watson-Crick pairing of nucleobases, which allows for the generation of programmable DNA droplet coacervates for applications in biosensing and design of molecular robots. Recently, Gong et al. designed phase separated DNA droplets that could recognize and respond to specific patterns of miRNA exposure (J. Gong et al., 2022). Additional programming of DNA liquid-liquid coacervation was realized through the use of ATP-driven T4 DNA ligases, enabling temporal control over the formation and disassembly of the phase separated systems dependent on the presence of ATP (Deng & Walther, 2020). In a further step, hybrid coacervates can be formed by functionalizing oligonucleotides with assembling peptides, creating phase separating systems that can trap guest molecules with applications in bio-catalysis (S. Yao et al., 2022). Biomimetic functionalities also arise from these hybrid interactions, as was recently demonstrated by Zhou et al., in the construction of artificial cell cytoskeletons through polyelectrolyte interactions between DNA nanotubes and cationic polymers (Zhou et al., 2022). These processes create rigid fibrillar networks that can modulate the fluidic properties of coacervate protocells, demonstrating potential of this technology in creating synthetic cell analogues.

Finally, while many of the discussed liquid-liquid technologies are currently in preclinical testing, several have been translated to human clinical and consumer use. Phase separated emulsions, for example, are standard additives in cosmetics. Polymer- and lipid-stabilized emulsions have also been used in skin care applications (Donthi et al., 2023; Motamedi et al., 2022), and transdermal delivery (Kapoor et al., 2018). These translational activities have laid the groundwork for a new generation of liquid-liquid materials as novel diagnostics, therapeutics, and consumer products.

3. BIOMEDICAL APPLICATIONS OF INTERPHASE ASSEMBLED NANOMATERIALS

The rich structure-function-performance landscape available through interfacial molecular assembly has served as fertile ground for the development of an array of nanotechnology healthcare solutions. In many cases, stimuli-responsive triggers are engineered into the nanomaterial platform through tuning interfacial thermodynamics. In other examples, interfacial assembly produces thermal and kinetic traps that retain functional biomolecules, improving their properties through steric stabilization. Here, we summarize recent efforts to leverage these interfacial phenomena in the development of biosensing platforms, diagnostic nanodevices and nanomedicines.

3.1. Biosensing

Some of the greatest breadth in application of interfacial molecular assembly has been in the field of biosensing. Biosensors pair recognition motifs (e.g., enzyme, DNA, RNA) with electrical, optical, or chemical transducers to readout analyte concentrations. Because the sensitivity and accuracy of the biosensor is directly related to the state of the interface, engineering the surface chemistry and orientation of confined biomolecular probes has been a particular area of focus. Caseli and co-workers have shown that the catalytic rate of the enzymes asparaginase (Possarle, Junior, & Caseli, 2020), and urease (Rodrigues et al., 2018), embedded within floating lipid monolayers can be increased by ~30% when co-formulated with carbon nanotubes, leading to a >50% enhancement in both sensitivity and limit of detection compared to non-carbon analogues. A similar effect was later observed for penicillinase co-formulated with graphene oxide nanosheets (Scholl, Siqueira Jr, & Caseli, 2022). These works highlight the role that interfacial self-assembly can play in preserving the molecular architecture and orientation of enzymes to promote contact between the analyte and the catalytic sites of the protein biosensor. It should also be mentioned that molecular self-assembly has been used to design the sensor itself, with Liang et al. using layer-by-layer self-assembly of polymers to create a plasmonic nanoprobe for optical label-free biosensing (Liang et al., 2019).

In many biosensing applications, DNA serves as both a structural component to appropriately orient biosensors, and/or as the recognition element itself (Rutten, Daems, & Lammertyn, 2020; Yang et al., 2018; C. Yao et al., 2022; Ye, Zuo, & Fan, 2018). Among the optical detection techniques available for these applications, surface-enhanced Raman scattering (SERS) stands out due to its rapid, real-time, and non-destructive analysis of miRNAs organized at various interfaces. With respect to the structural role of DNA in these applications, Wu et al. demonstrated that DNA could stabilize gold nanoparticles into a two dimensional lattice at an oil-water interface to enable the detection of miRNA 155 down to 1.45 fmol/L (Wu et al., 2021). Samanta et al. showed that additional nucleic acid functionalization of a protein sensor, and analyte detection via fluorophore/quencher-based gold nanoparticles, led to a dramatic improvement in the sensitivity of glucose detection (Samanta et al., 2020). Activated copper nanoparticles have also served as an optical transducer for the detection of microRNA (miRNA) 153, with implications in the detection of Parkinson biomarkers (Cui et al., 2020). Zhu et al. recently demonstrated that tetrahedral framework nucleic acid structures organized and elevated DNA probes from the detector surface, thereby enabling improved molecular recognition kinetics within a microfluidic device (Zhu et al., 2021). This allowed for the development of multi-purpose devices which could capture, enrich, and culture E. coli bacteria for antibiotic susceptibility testing. In a notable example, Yeo et al. developed mRNA spherical nucleic acids that could detect changes in connective tissue growth factor expression as a visual indicator for hypertrophic scars and keloids (Yeo et al., 2018).

Another successful example of functional assemblies in biosensing are peptide and peptoid platforms. Originally inspired by amyloid fibril formation, assembly of β-rich structures from peptide monomers have yielded new opportunities in the design of bioelectronic nanodevices and biosensors (S. Zhang et al., 2021). A recent review by Puiu et al. highlights the manifold ways redox-tagged helical peptides are being assembled into multi-dimensional structures with electron transfer capabilities to produce biosensing thin films (Puiu & Bala, 2018). Silk derived peptides are particularly attractive due to their ability to form highly ordered and stable β-sheet structures that can be used to immobilize probe molecules (P. Li et al., 2019). In an interesting, related, example, Wang et al. developed a peptide chimera containing the self-assembling unit from amyloid-β (KLVFF) fused to the matrix metalloprotease 2 (MMP-2) substrate PLGVR (Wang et al., 2022). Assembly of the β-sheet directing sequence led to the formation of ionic nanochannels, while the MMP-2 recognition site detected active proteases, for example in a tumor microenvironment, with a 10 fg/mL – 10 ng/mL detection range and a limit of detection of 6.6 fg/mL. In a similar fashion, Wang et al. functionalized titanium carbide surfaces with a chimeric peptide containing a sequence sensitive to carboxypeptidase Y mediated cleavage for detection of post-translational modifications (Wang et al., 2020).

3.2. Imaging Agents and Diagnostics

Nano contrast agents have transformed nearly all modalities of medical diagnostics, including functional MRI, PET and SPECT, and ultrasound. Regarding the latter, the development of microbubble contrast agents spurred new opportunities for ultrasound diagnosis and treatment of several human diseases, particularly in oncology (Exner & Kolios, 2021; Favvas et al., 2021). In an acoustic field, these bubbles oscillate in response to pressure changes to provide echogenic nuclei that are distinct in their responses from the surrounding physiologic medium, thereby providing increased signal contrast. Consequently, several phospholipid microbubble contrast agents have been approved for use in humans to enhance echocardiogram signals. Continued study of these agents recently identified that monodispersity of the imaging nuclei can significantly improve signal intensity (Helbert et al., 2020; Yusefi & Helfield, 2022).

While these bubbles have transformed echocardiography and diagnosis of liver disease, they are limited in application due to their inherent large size (~1 – 10μm) that limits tissue penetration. Nanobubbles have demonstrated an ability to overcome these size-dependent limitations, and as such have enjoyed a rapid growth in research progress since 2010 (Exner & Kolios, 2021; Xiong et al., 2021). The Exner group has been at the forefront of this field, producing several seminal advances in nanobubble technology. Notable recent work in the manufacturing of nanobubbles has reported on the use of propylene glycol to modulate the stiffness of the lipid bubble shell, thereby tuning bubble stability and echogenic response (Sojahrood et al., 2021). This has been complemented by the development of extrusion methods to produce monodisperse formulations at scale (Counil et al., 2022). Parallel biophysical studies have elucidated how nanobubble concentration and interaction with blood components modulates signal strength in vitro and in vivo (Abenojar et al., 2019; Cooley et al., 2023). In sum, this mechanistic groundwork has paved the way for near-term clinical translation of nanobubble contrast agents.

Development of these contrast agents into tissue-specific imaging nuclei often requires the design of targeted formulations. Perera et al. showed that nanobubbles functionalized with prostate specific membrane antigen (PSMA) resulted in an approximate 4-fold enhancement in signal 30 minutes after administration to mice bearing PC3-pip prostate tumor xenografts (Figure 9). Follow up studies demonstrated similar targeting-specificity and imaging stability in an orthotopic PC3-pip tumor model (Y. Wang et al., 2021). Subsequent mechanistic studies found that imaging persistence was likely the result of endocytic uptake of the nanobubble contrast agents into prostate cancer cells, prolonging the retention of the perfluorocarbon gas nuclei in tissues relative to non-targeted controls (Perera et al., 2022).

Figure 9.

Prostate specific membrane antigen (PSMA)-targeted nanobubble ultrasound contrast agent (PSMA-NB) shows four fold higher accumulation and extended retention in PSMA positive PC3-pip tumors at 30 min post injection, compared to untargeted nanobubbles and Lumason microbubbles. Reprinted with permission from Perera, et al. (2020).

An analogue to nanobubble imaging nuclei are liquid-liquid phase-change contrast agents (PCCAs). These particles arise from assembly of a surfactant at a liquid-liquid interface, typically a water-perfluorocarbon interphase, to develop a stable nano-scale emulsion. During the rarefaction (expansion) phase of an ultrasound pulse, the liquid core of PCCAs are designed to undergo a liquid-to-gas phase change to generate echogenic microbubbles in situ (Durham & Dayton, 2021; McHugh et al., 2022; Rojas, Borden, & Dayton, 2019). This produces imaging nuclei at the site of diagnosis, while maintaining a small size during distribution to facilitate cardiovascular perfusion (Rojas & Dayton, 2019b). These particles also display longer half-lives relative to gaseous bubble nuclei due to the extremely low miscibility of the liquid perfluorocarbon core in aqueous environments. Hadinger et al. demonstrated that cell-targeted PCCAs could be generated by developing folate-functionalized particles that specifically home to the folate receptor on MDA-MB-231 breast cancer cells (Hadinger et al., 2018). Follow up studies utilized PCCAs functionalized with the cyclic RGD peptide, a motif that binds to α_Vβ₃ integrins, to locate the imaging nuclei to fibrosarcoma tissues in rodent models and permit ultrasound contrast imaging of malignant tissue (Rojas & Dayton, 2019a)

Our lab has also developed targeted PCCAs stabilized by a de novo peptide surfactant, permitting incorporation of the targeting ligand directly into the emulsifier sequence itself (Medina et al., 2017; Sloand et al., 2020). We demonstrated these particles could be internalized into immune cells to enable real-time B-mode and Doppler ultrasound imaging of cell migration in situ within tissues (Figure 10) (Kim et al., 2023). Additional development of platelet-targeting formulations allowed for contrast enhanced imaging of blood clots in deep vein thrombosis models (Sloand et al., 2021). Serendipitously it was found that, in addition to functioning as imaging nuclei, oscillation of platelet-bound nanoemulsions competitively inhibited secondary platelet–fibrinogen binding to disrupt further clot growth and dissolve the embolism. This example demonstrates the potential of many interfacial assembled diagnostic nanomaterials to serve as theranostic platforms, exerting additional therapeutic activity in a spatiotemporally controlled manner.

Figure 10.

Real-time monitoring of emulsion-laden RAW 264.7 macrophage cells within tissues. (a) Top: schematic of immune cell tracking within porcine vasculature. Bottom: photograph of ex vivo experimental setup. (b) Representative time-dependent B-mode images of functionalized RAW cells during vessel perfusion. Vessel walls are demarcated by white dashed lines. Colored dashed circles track individual cells during flow. (c) Quantification of B-mode contrast for emulsion-loaded cells compared to background. (d) Color Doppler signals superimposed on B-mode images collected during cell vessel perfusion Reprinted with permission from Kim, et al. (2023).

3.3. Therapeutics

The small size of self-assembled nanomaterials holds promise for creating minimally invasive therapeutic strategies for various human pathologies. In these contexts, gas-liquid systems (bubbles) and liquid-liquid condensates (emulsions) have played an outsized role as delivery vehicles due to their thermodynamic stability, ability to be engineered with stimuli-responsive cargo release triggers, and versatility of surface chemistries (Exner & Kolios, 2021). These properties have yielded the emerging field of sonodynamic therapy, where therapeutic activity of acoustically sensitive nanoparticles is triggered via an ultrasound pulse. For nanobubble delivery agents, the therapeutic cargo must be loaded onto the particle surface, or within the self-assembled shell itself, to enable its delivery upon acoustic cavitation of the gaseous nuclei (Nittayacharn et al., 2020). Nittayacharn et al. demonstrated this approach recently through using doxorubicin-loaded nanobubbles (Nittayacharn et al., 2019). When activated by therapeutic ultrasound, nanobubbles were able to deliver more drug into LS-174T human colorectal adenocarcinoma cells in vitro and in vivo compared to the free chemotherapeutic alone. In another, more recent example, Nittayacharn et al. delivered an iridium(III) complex that could produce reactive oxygen species under ultrasound stimulation (Nittayacharn et al., 2022). When paired with sonication, iridium-loaded nanobubbles enhanced reactive oxygen generation, and led to a >60% increase in apoptosis of human ovarian and breast cancer cells relative to uninsonated controls. Parallel studies have shown that the nanobubble itself can serve as a therapeutic warhead, where acoustically driven implosion of nanobubbles leads to mechanical fractionation of cancer cells and physical ablation of tumor tissue in vivo (Bismuth et al., 2022). Similar mechanical phenomena have been employed for PCCA based therapies. Durham et al. reported that ultrasound activation of PCCAs permeabilized biofilms of the bacterial pathogen methicillin resistant S. aureus (MRSA) to enhance the efficacy of co-delivered antibiotics (Durham et al., 2021). Our group, as well as Zhang et al., similarly reported sonothrombolysis upon cavitation of phase-changing nanodroplets (Sloand et al., 2021; B. Zhang et al., 2021). Many of these mechanically driven therapeutic strategies are a consequence of the poor loading efficiency of hydrophilic therapeutics into the hydrophobic perfluorocarbon PCCA liquid core. To address this limitation, we recently reported methods that can be used to functionalize biologics with a fluorine-rich coating to enable their efficient loading within, and ultrasound delivery from, PCCAs without compromising the structure or function of the biologic (Lawanprasert et al., 2023; Sloand et al., 2020).

Finally, interfacial assembled spherical nucleic acids have also leveraged the therapeutic functionality of oligonucleotides to develop stimuli-responsive pharmaceuticals. For example, Callmann et al. developed immunostimulant spherical nucleic acids loaded with the oligonucleotide adjuvant CpG-1826, as well as triple negative breast cancer cell lysates as the antigen, to stimulate potent anti-tumor responses in vitro and in vivo (Callmann et al., 2020). Further, oxidizing the antigenic cellular component before formulation was found to enhance the population of cytotoxic CD8+ T cells, and decrease the population of pro-tumor myeloid derived suppressor cells, within the tumor microenvironment. In other work, incorporation of a photosensitizer into spherical nucleic acid constructs, or utilizing the photothermal effects of the gold template itself, enabled the release of loaded bioactives under the control of near-infrared light irradiation to create spatiotemporally controlled therapies (Chen et al., 2021; Li et al., 2021). Clinical trials involving spherical nucleic acids are ongoing. A 2021 clinical report showed successful reduction in target protein levels after treatment with a spherical nucleic acid therapy against Bcl2L12 in recurrent glioblastoma, indicating potential for translation of this technology to clinical cancer treatments (Kumthekar et al., 2021).

Conclusion

Inspired by natural processes of interfacial self-assembly, engineers, chemists, and materials scientists have sought to exploit these strategies to construct next generation nanomaterials. In these contexts, driving assembly at phase boundaries dictates specific hierarchical structures, and limits off pathway organization, to create designer biofunctional materials. Examples discussed here show how small changes in the biophysical and biochemical properties of the assembling unit can dramatically alter intra- and intermolecular interactions to create a highly tuned surface isotherm and thermodynamic equilibria. We envision that continued development of non-canonical amino acids and nucleotides will further expand the molecular self-assembly tool kit available to nanotechnologists. Pairing these synthetic advances with additional mechanistic insights into molecular assembly at gas-solid, gas-liquid, solid-liquid, and liquid-liquid interfaces will enable the rational design of nanomaterials with potentially unprecedented properties. Additional improvements in manufacturing consistency will accelerate these platforms towards clinical translation. Accordingly, new manufacturing methods, such as microfluidic platforms, will be required to create monodisperse formulations at scales relevant to clinical application to translate these novel biosensing, diagnostic and therapeutic nanotechnologies into healthcare settings.