Method for forming a porous peptide material

Abstract

Tripeptides (sequence isomers of K / Y / W) and a method for using the same. The tripeptides, upon a process of evaporation-driven self-assembly, undergo two-stage phase separation to form highly porous particles upon drying. These particles readily re-dissolved upon reintroduction of water. The particles can trap molecular payloads that can be readily released in aqueous media.

Description

METHOD FOR FORMING A POROUS PEPTIDE MATERIAL CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and is a non-provisional of, U.S. Patent Applications 63 / 514,002 (filed July 17, 2023) and 63 / 591,342 (filed October 18, 2023), the entirety of which are incorporated herein by reference. STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant number N00014-21-1-2967 awarded by the United States Office of Naval Research and grant number FA9550-21-1-0091 awarded by the United States Air Force Office of Scientific Research. The government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] The subject matter disclosed herein relates to encapsulation of biological payloads with 3D particles comprising tripeptides.

[0004] Peptide materials usually contain patterns of backbone hydrogen-bonding derived from protein secondary structures, typically giving rise to one-dimensional (1D) or two-dimensional (2D) structures. These H-bond patterns may be further stabilized by aromatic stacking, giving rise to architectures that are remarkably stiff and stable. This is exemplified by ‘dry’ diphenylalanine (FF) zippers that have remarkable mechanical, optical and electronic properties. Aromatic tripeptides with polar groups, such as lysine- tyrosine-phenylalanine (KYF), retain strong directional self-assembly tendencies complemented with favorable solvent interactions, leading to hydrogelation.

[0005] In addition to 1D and 2D structures, the notion of non-directional assembly is also common in biology, e.g., in liquid condensates typically leading to spherical morphologies dictated by surface tension. In these systems, side-chain interactions dictate the assembly complementing flexible backbone interactions. Short peptide motifs that display liquid-liquid phase separation have been reported, including through flexible linkers separating aromatic dipeptides to disrupt directionality.

[0006] It would be desirable to provide 3D structures that would be useful to encapsulate payloads, such as proteins. To date, no such 3D structures have been found that are entirely satisfactory. An improved methodology is therefore desired. SUMMARY

[0007] This disclosure provides tripeptides (sequence isomers of K / Y / W) and a method for using the same. The disclosed tripeptides, upon a process of evaporation- driven self-assembly, undergo two-stage phase separation to form highly porous particles upon drying. These particles are readily re-dissolved upon reintroduction of water. The particles can trap molecular payloads that can be readily released in aqueous media.

[0008] The tripeptides provide a biocompatible, versatile, and instrument-free material for drying and stabilizing biomolecules, aiming to extend the shelf life of key biomolecules crucial for different applications. The product is useful for encapsulating, preserving, and storing biomolecules. Can you stabilize and extend the shelf-life of biologics at room temperature? The disclosed peptides provide, in some embodiments, a composition that can preserve biologics in powder form at room temperature and provide an alternative to the freeze-drying process. .

[0009] In a first embodiment, a method for forming a porous peptide material is provided. The method comprising steps of: dissolving a tripeptide in water, the tripeptide selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW-NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; and waiting for the water to evaporate, thereby causing the tripeptide to form a porous material.

[0010] In a second embodiment, a porous peptide material formed by a particular method is provided. The method comprising steps of: dissolving a tripeptide in water, the tripeptide selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW-NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; and waiting for the water to evaporate, thereby causing the tripeptide to form a porous material.

[0011] In a third embodiment, a method for delivering a payload to an aqueous medium is provided. The method comprising: dissolving a tripeptide in water, the tripeptide selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW-NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; dissolving a payload in the water, the payload being an organic molecule; waiting for the water to evaporate, thereby causing the tripeptide to form a porous material while exposed to the payload, thereby encapsulating the payload in the porous material to form an encapsulated payload; and adding the encapsulated payload to an aqueous medium.

[0012] This brief description of the invention is intended only to provide a brief overview of subject matter disclosed herein according to one or more illustrative embodiments, and does not serve as a guide to interpreting the claims or to define or limit the scope of the invention, which is defined only by the appended claims. This brief description is provided to introduce an illustrative selection of concepts in a simplified form that are further described below in the detailed description. This brief description is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. The claimed subject matter is not limited to implementations that solve any or all disadvantages noted in the background.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the invention encompasses other equally effective embodiments. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. Thus, for further understanding of the invention, reference can be made to the following detailed description, read in connection with the drawings in which:

[0015] FIG.1A depicts chemical structures of tripeptides KFF, KYF, KYY, KYW and representation of the interaction space of sidechains.

[0016] FIG.1B shows selected snapshots of computer simulations for KFF (only backbone shown for clarity) and KYW, with predominant H-bond interactions highlighted.

[0017] FIG.2A is a Representation of evaporation-driven self- assembly in sessile droplets.

[0018] FIG.2B is an image of evaporation patterns over time with differential assembly of KYW clearly visible. Scale bar = 1 mm.

[0019] FIG.2C shows microscopy time course for evaporation-driven assembly of KYW (scale bar = 50 ^m) and KFF and KYF (scale bar 20 ^m).

[0020] FIG.2D depicts optical microscopy images showing 1D assembly for KYYand space-filling (3D) assemblies for KYW, WKY and WYK (left) (right). Scale bar = 10^m.

[0021] FIG.2E are graphs of fluorescence emission spectra at two different excitation wavelength (280 and 318 nm) of the different sequences in solution, dried foams (visual appearance shown in FIG.2D) and rehydrated foams reveals similar packing in dried particle film, suggesting comparable evaporation-driven assembly regardless of sequence and recovery of sequence dependent conformation selection in hydrated state.

[0022] FIG.3A is a schematic representation of evaporation-driven assembly of buoyant liquid droplets that settle at the droplet interface and upon drying form half- dome shaped and spherical particles.

[0023] FIG.3B is an SEM image showing half-dome and spherical particles at the interface and also showing surface pores.

[0024] FIG.3C shows FIB-SEM analysis confirming half-dome shape and spherical particles and revealing the porous structure inside of foam particles. Note that the fine granular structures on the surface are from sputtered-coated gold.

[0025] FIG.3D is a TEM analysis of glutaraldehyde crosslinked KWY dissociated particles reveals that non-surface particles are highly porous spheres.

[0026] FIG.3E, FIG.3F and FIG.3G depict the results of an AFM analysis showing porous half-dome and spherical particles.

[0027] FIG.3H is a plot of Young’s Modulus stiffness of fibers or particles formed by indicated peptides. A total of 15-36 particles or fibers were profiled for each sequence.

[0028] FIG.4A shows images indicating temperature controls the size distribution of particle films. The example of KWY shows reduction in particle size and more homogeneous size distribution, which correlated with the increased temperature during evaporation. Scale bar = 10 ^m. Total 368 - 1456 particles at each temperature were analyzed.

[0029] FIG.4B is a graph of KWY particle diameter as a function of temperature.

[0030] FIG.4C is a graph of particle diameter for a WKY peptide solution that was degassed followed by evaporation assay. Imaris 3D rendering demonstrated reduction in particle size.

[0031] FIG.4D is a graph of particle diameter for a WKY peptide solution that was degassed followed by evaporation assay. Imaris 3D rendering demonstrated increase in sphericity.

[0032] FIG.4E are images showing fully reversible formation of porous peptide foams upon addition of water and re-evaporation. Representative time-lapse images of KWY were shown. Scale bar = 50 ^m. The macroscopic images of 5 ^L peptide solution drop at each stage were shown in the insertion.

[0033] FIG.4F confocal live imaging of KWY peptide particles incorporating EGFP shows significant enrichment of EGFP protein in peptide droplets. Scale bar = 20 ^m.

[0034] FIG.4G shows the results of a fluorescence intensity analysis of the pre-load solution, the droplet, and the post-load solution at the frontier of emulsification. A total of 20 regions of each indicated groups from 6 time-lapse frames were quantified, and the average intensity of pre-load solution was set as 100 arbitrarily. Error bar represents s.d.

[0035] FIG.4H are confocal and bright field images of dried peptide particles reveal that the green fluorescence signals of EGFP retain in WKY peptide particles under ambient condition for 5 days after solidification.

[0036] FIG.4I and FIG.4J schematically depict an experiment to test the storage and re-dispersion ability of WKY with a schematic representation of the experiment and the fluorescence emission at 510 nm (^^௫^= 488 nm) of the corresponding solution (EGFP, EGFP+KYF and EGFP+WKY) over time.

[0037] FIG.4K are images for WKY and WYK showing non-directional self- assembly resulting in differential tryptophan environments and water binding with zoomed areas showing a dry (top) and water bound (bottom) tryptophan

[0038] FIG.5A is a graph showing sequence-dependent aggregation propensity, AP=(SASAinitial / SASAlast50ns) and (Bottom) distribution of hydrogen bond interactions between simulated peptides from MD trajectories. Backbone and sidechain as an indication of directionality of self-assembly.

[0039] FIG.5B is a graph showing Circular Dichroism (CD) spectra of KYF, KYY and KYW measured at 5 mM concentration at pH 7.5.

[0040] FIG.5C is a graph showing AP score, W Solvent accessible surface area (SASA) and hydrogen bonding interaction distribution analyses for K / Y / W sequence isomer MD trajectories.

[0041] FIG.5D shows 3D fluorescence spectra showing the differential polarity and water-interactions of tryptophan residues in solution.

[0042] FIG.6A shows a confocal live imaging of KWY peptide particles incorporating Alexa 488 shows significant enrichment of the dye in peptide droplets. Scale bar = 20 ^m.

[0043] FIG.6B is a graph showing the result of a fluorescence intensity analysis of a pre-load solution, the foam coacervate, and the post-load solution at the frontier of emulsification. A total of 20 regions of interest of each group from 6 time-lapse frameswere quantified, and the average intensity of pre-load solution was set as 100 arbitrarily. Error bar represents s.d.

[0044] FIG.6C depicts a confocal image showing that the dye Alexa 488 remains encapsulated upon solidification. Scale bar = 20 ^m. DETAILED DESCRIPTION OF THE INVENTION

[0045] Addressing the pivotal challenge of biomolecule stability holds far-reaching implications and unveils significant commercial potential across various industries, including pharmaceuticals, cosmetics, healthcare, and food production. Solving this issue not only underscores the importance of stable biomolecules but also highlights the vast opportunities for innovation and growth within these sectors.

[0046] This disclosure provides a biomolecule stabilization technology. The disclosure provides a short peptide-based material capable of encapsulating and preserving biomolecules, ensuring their stability and functionality at room temperature. Upon air-drying and water evaporation, the peptide creates a protective shield around the protein, thus preserving its structure and activity. The dry powder can then be stored without the need for cold temperatures.

[0047] This disclosure provides a composition and method to create porous particles that encapsulate payloads and are readily dissolved in aqueous media. Unlike existing emulsification methods, the method does not require mechanical energy, instead relying on an evaporation process to achieve phase separation and encapsulation. Unlike existing peptide-based materials that are frequently highly directional (one- or two-dimensional structures such as fibers, tubes sheets), the disclosed particles grow in three dimensions and are space-filling. Unlike existing polymer-based systems, these foams are made from tripeptides that are biocompatible and biodegradable.

[0048] Without wishing to be bound to any particular theory, systematically modulating aromatic interaction strength and directionality through variation of sidechains (F, Y, W) leads to motifs with richer and dynamic sidechain interactions (FIG. 1A, FIG.1B). Exchanging F or Y for tryptophan (W) removes the strong directionality from minimalistic self-assembling peptide systems. Compared to phenylalanine, tryptophan’s non-covalent interaction space is expanded to include not only ^ í^^, cation-^, but also H-bonding (distinct from tyrosine due to the indole NH), NH- ^ and dipole-dipole interactions, including with water (FIG.1A). Tryptophan supports precise roles in proteins enabled by its unique heteroaromatic nature, such as specific interfacial functions in proteins, e.g., near lipid bilayers and ion channels. Thus, the self-assembling behavior of KFF, KYF, KYY, KYW was initially examined.

[0049] Referring to FIG.2A, upon investigation of the fluorescence of KYW peptide assemblies using confocal microscopy, a remarkable phase separation behavior was observed that was triggered by localized evaporation during drying.

[0050] Macroscopically, when observing tripeptide assembly in drying droplets starting from 20 mM peptide dispersions, surprisingly contrasting behaviors were observed. A strongly scattering ring moving inward upon drying for KYW in contrast to KFF, KYF, and KYY that give rise to heterogeneous semi-transparent structures (FIG.2B, PBS buffer). When observed by microscopy (FIG.2C), the differential morphologies (1D fibers versus 3D spherical objects) are evident after drying is complete (KFF and KYF shown in comparison). When observed over time, KYW displays a remarkable interfacial phase separation behavior, giving rise to formation of films composed of densely packed spherical particles (FIG.2D). This behavior strongly contrasts observations for KFF, KYF, KYY giving rise to 1D structures with varying degrees of bundling, resembling previous observations for evaporation-driven assembly of a fluorinated phenylalanine derivative inside drying solvent droplets. The multitude of possible interactions and the flexibility of the backbone enable the K / Y / W peptides to more effectively reconfigure and adapt to thenewly formed interfaces that form upon water evaporation. The observed (lack of) directionality initiates at the molecular level, and clearly translates hierarchically across the nano to macroscopic scales. Complex drying patterns were observed, related to complex gradients in drying of droplets with solute. The phase separations occur at the moving drying front due to concentration gradients inside the drying droplet.

[0051] In one embodiment, the tripeptide is selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW-NH2, YWK- NH2, WKY-NH2, WYK-NH2, and combinations thereof.

[0052] The tripeptide may be dissolved in water at a concentration that is greater than 0 mM and less than 70 mM (e.g., 0.1 mM to 70 mM, 0.1 mM to 50 mM, 0.5 mM to 20 mM, 0.1 mM to 10 mM). The water may be at a pH between 2 and 7.4 (e.g., 3 to 7.4, 4 to 7.4, 6 to 7.4, 6.6 to 7.4, 7.1 to 7.3). In some embodiments, a dilute salt solution is used, especially at low pH (e.g. pH 2) to screen charges. The salt concentration may be, for example, from 0M to 2M (e.g., 1M). In some embodiments a buffer is present in the water. For example the water may include phosphate buffered (PBS) at a concentration of 0.1-1000 mM, 0.1-300 mM, 0.5-300 mM, 0.1-100 mM (e.g., 100 mM).

[0053] One then waits for the water to evaporate to form the porous material. The evaporation may occur at a temperature, for example, from 20^ to 80^, from 25^ to 60^, from 20^ to 40^, from 20^ to 30^ or from 20^ to 25^. In some embodiments, the evaporation is accelerated using laser excitation (e.g. at 405 nm, at 488 nm, at 561 nm, etc.).

[0054] In some embodiments, a payload is present in the water that is encapsulated within the porous material. In one embodiment, the payload is an organic molecule such as a protein, a biomolecule, a dye, a fluorescent dye, a fragrance molecule, a vitamin, a nucleic acid (e.g. siRNA, mRNA, etc.) or a drug.

[0055] As one specific example, porous peptide materials may be prepared by dissolving the tripeptide in 100 mM of phosphate buffer (pH 8) to a final concentration of 0.5-20 mM. The pH was adjusted to 7.5 using 0.5 M HCl solution. In one embodiment, the solution is heated for 5 minutes at 70 ^ and aged for 1 hour at room temperature (i.e., 21-23^) to ensure homogenous distribution. Evaporation-driven assembly was performed by depositing 5 ^L droplets on a glass microscope slide and left to evaporate (temperature range 25-80^) at around 40-50% humidity. Alternative evaporation-driven methods include the use of vacuum pumps, desiccators and temperature adjustments to vary dry times.

[0056] The sequence-dependent tryptophan exposure is believed to lead to interfacial aggregation and reduction in surface tension. The dynamic tension profile was measured via the pendant drop method (Langmuir 19, p8436–8442, 2003) of tripeptides WYK, WKY, and KYW, which have contrasting W solvent-exposure and environments. Surface adsorption of peptide aggregates reduced surface tension in a way that correlates with both increase W-accessibility in supramolecular aggregates (concentration dependence) and the differences in molecular packing and W-solvent exposure (sequence dependence). Despite these differences, upon evaporation-driven assembly inside drying droplets, each of the sequences gave rise to similar sequential phase separation behavior, forming droplets followed by solidification to form solid particle films, similar to KYW (FIG.2E). Upon analysis of the tryptophan emission in the dried films at 5% relative humidity (RH), a loss of emission from the 318 nm band in the 3D excitation / emission spectra was noted with spectra of the dried films appearing similar (FIG.2E). The band was recovered by exposing the solid film to high humidity (95% RH). These data highlight the adaptive nature of the assemblies where tryptophan partitioning reversibly changes upon cycling the water environment via weak multivalent and dynamic interactions.

[0057] Size control, reversibility, and encapsulation of payloads

[0058] Systematic increase of the temperature gives rise to reduction in size and a much more homogeneous size distribution (FIG.4A, FIG.4B). Without wishing to be bound to any particular theory, a combination of reduced buoyancy, due to lower gas content at elevated temperature, with acceleration of phase transition and solidification, yields particles that are monodisperse.

[0059] Buoyancy

[0060] Upon studying the dried particulate films using various microscopy techniques, the expected spherical objects were observed. Additionally, a significant numbers of porous, disk-shaped particles of varying sizes with remarkably flat surfaces were observed (shown schematically in FIG.3A, panel i). These structures are found at the top of the sample, as seen by SEM (FIG.3B, FIG.3C), suggesting that they are formed at the air-water interface. The observation suggests that, in contrast to typical dense coacervate droplets that sediment, the structures are low-density, buoyant condensates. The observation of flattened 2D hemi-spherical objects suggests that these particles are initially liquid and due to their low density, they rise to the interface in their liquid state, where they deform, flatten, and solidify. This observation is confirmed using FIB SEM (FIG.3C), which shows a part-circular cross-section. The pore size distribution ranged from 30-500 nm.

[0061] Without wishing to be bound to any particular theory, the observed buoyancy is believed to be related to the formation of gas bubbles inside phase separated droplets (FIG.3A, panel i). This may be a consequence of a secondary phase separation event where the condensate interface nucleates gas bubble formation and is stabilized by adherence of indoles to newly formed gas / aqueous interfaces (FIG.3A, panel ii). Notably, upon degassing of the peptide dispersion prior to evaporation-driven assembly, the average particle size becomes smaller, and the morphology is more spherical (FIG. 4D). This provides further evidence that buoyant nature of the droplets relates to theirgas content, and it can be regulated. Non-surface particles are smaller and spherical, indicating fusion and flattening at the air-water interface (FIG.3B). TEM analysis of glutaraldehyde crosslinked KWY particles reveals highly and fine porous and spherical structure of the non-surface particles (FIG.3C, FIG.3D). The hemispherical porous structure was further confirmed by AFM (FIG.3E, FIG.3F, FIG.3G). Young’s Modulus analysis demonstrated that the solid particle formed by the peptide sequence isomers show similar stiffness around 6 GPa compared to softer KYF and KYY fibers (2.5 GPa) (FIG.3H) and in line with previously reported mechanically stiff peptide fibers based on FF derivatives.

[0062] Gas content

[0063] The air was removed from solution through sonication under vacuum (e.g., 20^ for 15 min, or sonication at 4^ with a cycle of 30 seconds on / 30 seconds off for 20 cycles) followed by storage under argon gas. The degassed solution was subjected to the evaporation assay in a chamber filled with argon. Live confocal time-lapse imaging of the particle forming process revealed smaller droplets forming in degassed peptide samples as exampled in WKY. Further Imaris 3D reconstruction and analysis demonstrated a 20- fold reduction in particle volume and increase in sphericity in the degassed samples compared to prevalence of disk-shaped particles non-degassed samples (FIG.4C, FIG. 4D). Consistent with the role of air in pore formation, degassed peptide particles show fewer and smaller pores.

[0064] Reversibility

[0065] Upon re-introduction of 5 ^l droplet of water on top of a dried film, the structures near-instantaneously re-dispersed as shown in FIG.4E. When this droplet was left to dry again, the particle film reappeared, demonstrating that the process is fully reversible. These observations strongly contrast previously reported peptide materials, where formation of 1D and 2D structures are highly stable due to large enthalpic gain from cooperative H-bonds which are not readily reversed. Peptide ensembles withmultivalent, weak, and dynamic interactions offer more reversible assemblies as water solvation can compete with the energetics of the interaction space.

[0066] Encapsulation

[0067] Considering the highly porous nature of the particle films, the disclosed particles can be used for encapsulation. The small organic fluorophore ALEXA FLUOR® 488 was used as a test payload. ALEXA FLUOR® 488 has been demonstrated to give rise to a non-covalent conjugation through the sulfonate group and lysine side chains, observable by confocal microscopy. Upon introduction of the dye, it is encapsulated during droplet formation and remains there upon solidification (see FIG. 6A, FIG.6B and FIG.6C). Analysis of the fluorescence intensity of the pre-load mixture of peptides and dye, the formed liquid droplets, and the post-load solution, revealed efficient enrichment of dye inside particles (FIG.6B).

[0068] The ability to encapsulate and stabilize a biomacromolecule, Enhanced Green Fluorescent Protein (EGFP), was demonstrated. Efficient encapsulation inside the structures was observed for the dynamically assembling peptide, WKY (FIG.4F, FIG. 4G). As EGFP reports on its conformation through fluorescence, the preservation of structure was evaluated in the dried state (FIG.4H). After 4h of drying consequent rehydrating the EGFP solution loses its fluorescence (FIG.4I, FIG.4J). The control KYF+EGFP (fibrous assembly) reveals a decrease in the fluorescence suggesting a level of stabilization through protein / peptide interactions, whereas WKY+EGFP sample shows retention of fluorescence emission after drying, storage in dried form for five days, followed by instantaneous re-dissolution. These results highlight that the supramolecular peptide particles provide an environment where proteins are stabilized in the dried state, possibly through mechanisms analogous to dynamic interfacial complexation through self- organization of side chains with protein surface observed in random polymer / enzyme complexes. These data suggests that K / Y / W isomer tripeptides offer potential as universal and minimalistic modalities for encapsulation, drying, storage, and redispersion of proteins. FIG.4K depicts snapshots for WKY and WYK showing non-directional self-assembly resulting in differential tryptophan environments and water binding with zoomed areas showing a dry (top) and water bound (bottom) tryptophan

[0069] Molecular Dynamic Simulations

[0070] Atomistic MD simulations were used to reveal differences in molecular interactions (FIG.1B, FIG.5A). Exchanging F with Y and subsequently with W leads to an increase in the aggregation propensity (AP) (FIG.5A). The contribution of the backbone-backbone (as a proxy for 1D self-assembly), backbone-sidechain, and sidechain-sidechain hydrogen bonds were then analyzed (FIG.5A). Consistent with the increased AP scores, a concomitant increase in the total number of hydrogen bonds in F՜Y՜W variants was observed. Of these hydrogen bonds, backbone-backbone interactions decreased, while participation of the sidechain-backbone and sidechain- sidechain hydrogen bonds increased in the Y- and W-containing sequences (FIG.5A). The data show that the introduction of H-bonding capabilities from the rotationally flexible sidechains can favor non-directional assembly, especially for KYW (FIG.5A).

[0071] The relative contributions of sidechains and backbone interactions when substituting F՜Y՜W are also evident in the circular dichroism (CD) spectra of the KYF / Y / W peptides (FIG.5B). At a concentration of 5 mM, which is just above the critical aggregation for KYF and KYY show a carbonyl ^ ՜ ^כtransition peak at 203 nm which is a consequence of backbone-backbone interactions. For KYW, this transition is less intense, while the appearance of the 229 nm positive signal confirms the dominating influence of tryptophan sidechains interactions. The CD was also monitored for the three tripeptides (KYF, KYY and KYW) at concentrations ranging from 0.5 mM up to respectively 10, 5 or 20 mM. KYF and KYY eventually undergo gelation and phase separation, but KYW remarkably retains the appearance of a clear solution up to 20 mM. The CD data was in agreement with previously reported characterization using TEM, where KFF, KYF and KYY images show nanoscale fibers whereas KYW displayed amorphous aggregates. Thus, while KFF, KYF, KYY show directional assembly because of backbone H-bonds, KYW forms soluble ensembles that are stabilized by dynamic sidechain interactions. Aggregates could not be observed by observed by microscopy, indicating that these ensembles are dynamic and have less defined boundaries. The presence of dynamic soluble ensembles in the micron size range were confirmed by dynamic light scattering (DLS).

[0072] Sequence-dependence in tripeptide dispersions

[0073] Without wishing to be bound to any particular theory, in a system that is stabilized by a multitude of possible side-chain interactions, the system is not defined by one dominant conformation, but instead represented by an ensemble of conformers. Accordingly, the six K / Y / W sequence isomers were studied by MD to determine the sequence-dependent aggregation (FIG.5C) and found comparable AP scores and similar ratios of sidechain / backbone interactions, consistent with formation of non-directional ensembles. The solvent exposure of tryptophan was analyzed, measured through Solvent-Accessible Surface Area (SASA) (FIG.5C) of the indole sidechain that was found to be sequence-dependent. The most contrasting sequences in terms of AP and tryptophan solvent exposure were WYK and WKY. This aspect could be experimentally verified by measuring the tryptophan emission, which is widely used as a probe to detect the tryptophan’s’ environment and solvent exposure in proteins, with red-shifted emissions indicating an increase in local polarity and blue-shifts indicating hydrophobic environments. The emission spectra of all sequence isomers were measured at 20 mM (FIG.5D). Each of the sequences resulted in formation of (non-scattering) soluble dispersions that did not show any features when observed by optical microscopy and displayed a faint blue emission under UV light. The results found that emission maxima (^^௫= 280 nm) range from 357 nm to 370 nm with the most red-shifted emission corresponding to the most hydrated tryptophan (WYK) and the most blue-shifted emission corresponding to the most hydrophobic tryptophan environment (WKY). The 3D excitation / emission spectra of contrasting sequences (FIG.5D) differed by the appearance of a new band in WYK’s spectrum at ^^௫= 318 nm, which is also prominentin YKW and YWK. The intensity and absence / presence of the band is concentration dependent, suggesting its origin from an emissive state of a supramolecular arrangement.

[0074] The contrasting sequences WKY and WYK were analyzed by DLS, and similar diameter particles sizes were observed with respectively 460 and 500 nm but with a distinct narrower size dispersion for WYK. To further confirm differential environments experienced by tryptophans, the1H-NMR spectra of WYK and WKY solutions at variable concentrations show contrasting concentration-dependent chemical shifts of W protons. The indole ring protons in WYK shift downfield as the concentration increases from 1 to 5 to 20 mM indicating their positioning in an electron poor environment and formation of a polar network / hydrogen bonding while (minimal) upfield shifts were observed for WKY, in line with the formation of ^ െ ^ stacking driven aggregation.

[0075] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Claims

What is claimed is:

1. A method for forming a porous peptide material, the method comprising steps of: dissolving a tripeptide in water, the tripeptide selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW- NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; and waiting for the water to evaporate, thereby causing the tripeptide to form a porous material.

2. The method as recited in claim 1, wherein the tripeptide is present in water at a concentration between 0.1 mM and 1000 mM prior to the step of waiting.

3. The method as recited in claim 1, wherein the tripeptide is present in water at a concentration between 0.5 mM and 300 mM prior to the step of waiting.

4. The method as recited in claim 1, wherein the tripeptide is present in water at a concentration between 0.1 and 100 mM prior to the step of waiting.

5. The method as recited in claim 1, wherein the step of waiting occurs at a temperature between 20^ and 80^.

6. The method as recited in claim 1, wherein the step of waiting occurs at a temperature between 25^ and 60^.

7. The method as recited in claim 1, wherein the step of waiting occurs at a temperature between 20^ and 40^.

8. The method as recited in claim 1, wherein the tripeptide is selected from a group consisting of WKY, WYK, KYW, WKY-NH2, WYK-NH2 and KYW-NH2.

9. The method as recited in claim 1, wherein the tripeptide is selected from a group consisting of WKY, WYK, WKY-NH2 and WYK-NH2.

10. The method as recited in claim 1, wherein the tripeptide is selected from a group consisting of WKY and WKY-NH2.

11. The method as recited in claim 1, wherein the tripeptide is selected from a group consisting of WYK and WYK-NH2.

12. A porous peptide material formed by a method comprising steps of: dissolving a tripeptide in water, the tripeptide selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW- NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; and waiting for the water to evaporate, thereby causing the tripeptide to form a porous material.

13. A method for delivering a payload to an aqueous medium, the method comprising: dissolving a tripeptide in water, the tripeptide selected from a group consisting of KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW- NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; dissolving a payload in the water, the payload being an organic molecule; waiting for the water to evaporate, thereby causing the tripeptide to form a porous material while exposed to the payload, thereby encapsulating the payload in the porous material to form an encapsulated payload; and adding the encapsulated payload to an aqueous medium.

14. The method as recited in claim 13, wherein the payload is selected from a group consisting of a protein, a dye, a florescent dye, a fragrance molecule, a vitamin, a nucleic acid and a drug.

15. The method as recited in claim 14, wherein the payload is a protein.

16. The method as recited in claim 14, wherein the payload is a nucleic acid.

17. The method as recited in claim 13, wherein the tripeptide is selected from a group consisting of WKY, WYK, KYW, WKY-NH2, WYK-NH2and KYW-NH2.

18. The method as recited in claim 13, wherein the tripeptide is selected from a group consisting of WKY, WYK, WKY-NH2and WYK-NH2.

19. The method as recited in claim 13, wherein the tripeptide is selected from a group consisting of WKY and WKY-NH2.

20. The method as recited in claim 13, wherein the tripeptide is selected from a group consisting of WYK and WYK-NH2.

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