Methods of forming porous peptide materials

Abstract

Tripeptides (sequence isomers of K / Y / W) and methods of use thereof. The tripeptide is subjected to two-stage phase separation in an evaporation-driven self-assembly process, and highly porous particles are formed after drying. After water is introduced again, the particles are easily dissolved again. The particles can capture a molecular payload, which can be easily released into an aqueous medium.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Patent Application 63 / 514,002 (filed July 17, 2023) and 63 / 591,342 (filed October 18, 2023), and is a non-provisional application of those applications, the entire contents of which are incorporated herein by reference. Federally Funded Research or Development Declaration

[0002] This invention was made with government support under grant number N00014-21-1-2967 from the U.S. Office of Naval Research and grant number FA9550-21-1-0091 from the U.S. Air Force Office of Scientific Research. The Government owns certain rights to this invention. Technical Field

[0003] The subject of this article relates to the encapsulation of biological payloads in 3D particles containing tripeptides. Background Technology

[0004] Peptide materials typically contain backbone hydrogen bond patterns derived from protein secondary structures, usually resulting in one-dimensional (1D) or two-dimensional (2D) structures. These hydrogen bond patterns can be further stabilized through aromatic stacking, producing structures with exceptional rigidity and stability. For example, "dry" diphenylalanine (FF) zippers exhibit superior mechanical, optical, and electronic properties. Aromatic tripeptides with polar groups (such as lysine-tyrosine-phenylalanine (KYF)) retain a strong tendency for directional self-assembly, coupled with favorable solvent interactions, leading to hydrogelation.

[0005] Beyond 1D and 2D structures, the concept of non-directional assembly is also common in biology, for example, in liquid condensates where surface tension typically dominates the formation of spherical morphologies. In these systems, side-chain interactions dominate the assembly process, complementing the interactions of the flexible backbone. Short peptide motifs exhibiting liquid-liquid phase separation have been reported, including the separation of aromatic dipeptides via flexible linkers to disrupt directionality.

[0006] The goal is to provide 3D structures that can be used to encapsulate payloads such as proteins. To date, no completely satisfactory 3D structure has been found. Therefore, an improved approach is needed. Summary of the Invention

[0007] This disclosure provides a tripeptide (K / Y / W sequence isomers) and methods for using it. The disclosed tripeptide undergoes a two-stage phase separation during evaporation-driven self-assembly, forming highly porous particles after drying. These particles readily redissolve upon reintroduction of water. The particles can trap molecular payloads that can be readily released into an aqueous medium.

[0008] The tripeptide provides a biocompatible, multifunctional, and instrument-free material for drying and stabilizing biomolecules, designed to extend the shelf life of critical biomolecules essential for various applications. The product can be used for encapsulating, preserving, and storing biomolecules. Is it possible to stabilize and extend the shelf life of biologics at room temperature? In some embodiments, the disclosed peptide provides a composition that can preserve biologics in powder form at room temperature, offering an alternative to freeze-drying processes.

[0009] In a first embodiment, a method for forming a porous peptide material is provided. The method includes the steps of: dissolving a tripeptide in water, the tripeptide being selected from the 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 forming a porous material from the tripeptide.

[0010] In a second embodiment, a porous peptide material formed by a specific method is provided. The method includes the steps of: dissolving a tripeptide in water, the tripeptide being selected from the 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 forming the tripeptide into a porous material.

[0011] In a third embodiment, a method for delivering a payload to an aqueous medium is provided. The method includes: dissolving a tripeptide in water, the tripeptide being selected from the 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 water, the payload being an organic molecule; allowing the water to evaporate, thereby causing the tripeptide to form a porous material upon exposure to the payload, thereby encapsulating the payload within the porous material to form an encapsulated payload; and adding the encapsulated payload to the aqueous medium.

[0012] This brief description of the invention is intended only to provide a concise overview of the subject matter disclosed herein based on one or more illustrative embodiments, and is not intended as a guide for interpreting the claims or defining or limiting the scope of the invention, which is defined solely by the appended claims. This brief description is provided to introduce illustrative choices of concepts in a simplified form, which will be further described in the detailed embodiments below. This brief description is not intended to identify key or essential features of the claimed subject matter, nor is it intended to help determine the scope of the claimed subject matter. The claimed subject matter is not limited to embodiments that address any or all of the disadvantages mentioned in the background art. Attached Figure Description

[0013] This patent or application document contains at least one color drawing. A copy of the patent or application publication containing the color drawing will be provided by the Patent Office upon request and after payment of the necessary fees.

[0014] To understand the features of the present invention, specific embodiments can be obtained by referring to certain embodiments, some of which are illustrated in the accompanying drawings. However, it is important to note that the drawings only show certain embodiments of the invention and should not be considered as limiting its scope, as the scope of the invention encompasses other equally effective embodiments. The drawings are not necessarily drawn to scale, and the emphasis is generally placed on illustrating the features of certain embodiments of the invention. In the drawings, the same reference numerals are used to denote the same parts in different views. Therefore, for a further understanding of the invention, reference can be made to the following specific embodiments in conjunction with the accompanying drawings, wherein:

[0015] Figure 1A A schematic diagram depicting the chemical structures and side chain interaction spaces of the tripeptides KFF, KYF, KYY, and KYW is provided.

[0016] Figure 1B Selected snapshots of computer simulations of KFF (only the main chain is shown for clarity) and KYW are presented, highlighting the major H-bond interactions.

[0017] Figure 2A This is a schematic diagram of evaporation-driven self-assembly in sessile droplets.

[0018] Figure 2B This is an image of the evaporation pattern over time, in which the differential assembly of KYW is clearly visible. Scale bar = 1 mm.

[0019] Figure 2C Microscopic time progression of evaporation-driven assemblies of KYW (scale bar = 50 μm), KFF, and KYF (scale bar = 20 μm) is shown.

[0020] Figure 2D Optical microscope images depicting the 1D assembly of KYY (left) and the space-filling (3D) assembly of KYW, WKY, and WYK (right). Scale bar = 10 μm.

[0021] Figure 2E Different sequences in solution, dried foam (visual appearance) Figure 2D The fluorescence emission spectra of the dry particulate films (shown in the figure) and rehydrated foam at two different excitation wavelengths (280 and 318 nm) reveal similar stacking in the dry particulate films, indicating that evaporation-driven assembly is comparable regardless of sequence and that sequence-dependent conformational selection is restored in the hydrated state.

[0022] Figure 3A This is a schematic diagram of evaporation-driven assembly in which buoyant droplets settle at the droplet interface and form hemispherical and spherical particles after drying.

[0023] Figure 3B It is an SEM image showing hemispherical and spherical particles at the interface, as well as surface pores.

[0024] Figure 3C FIB-SEM analysis is shown, confirming the presence of hemispherical and spherical particles and revealing the porous structure within the foam particles. Note that the fine granular structure on the surface originates from the sputtered gold coating.

[0025] Figure 3D TEM analysis of the glutaraldehyde-crosslinked KWY dissociated particles revealed that the non-surface particles are highly porous spheres.

[0026] Figure 3E , Figure 3F as well as Figure 3G The results of AFM analysis are depicted, showing porous hemispherical and spherical particles.

[0027] Figure 3H This is a Young's modulus stiffness diagram of the fibers or particles formed by the specified peptides. A total of 15–36 particles or fibers were analyzed for each sequence.

[0028] Figure 4A Images illustrating the temperature-controlled particle film size distribution are shown. KWY's example demonstrates a reduction in particle size and a more uniform size distribution, which correlates with the increase in temperature during evaporation. Scale bar = 10 μm. A total of 368–1456 particles were analyzed at each temperature.

[0029] Figure 4B This is a graph showing the relationship between the diameter of KWY particles and temperature.

[0030] Figure 4CThis is a particle diameter diagram of the WKY peptide solution after degassing and evaporation experiments. Imaris 3D rendering shows a decrease in particle size.

[0031] Figure 4D This is a particle diameter diagram of the WKY peptide solution after degassing and evaporation testing. Imaris 3D rendering shows an increase in sphericity.

[0032] Figure 4E Images show the fully reversible formation of porous peptide foam after the addition of water and re-evaporation. A representative time-lapse image of KWY is shown. Scale bar = 50 μm. Macroscopic images of 5 μL peptide solution drops at each stage are shown in the inset.

[0033] Figure 4F This is a confocal live-cell image of KWY peptide particles incorporating EGFP, showing significant enrichment of EGFP protein in the peptide droplets. Scale bar = 20 μm.

[0034] Figure 4G The fluorescence intensity analysis results for the solution before loading, the droplet, and the solution after loading at the emulsion boundary are shown. Quantitative analysis was performed on a total of 20 regions from each specified group across 6 time-delay frames, with the average intensity of the solution before loading arbitrarily set to 100. Error bars represent standard deviations.

[0035] Figure 4H These are confocal and bright-field images of dried peptide particles, revealing that the green fluorescence signal of EGFP was retained in WKY peptide particles 5 days after curing under ambient conditions.

[0036] Figure 4I as well as Figure 4J The experiment testing the storage and redispersibility of WKY is schematically depicted, showing a schematic diagram of the experiment and the corresponding solutions (EGFP, EGFP+KYF, and EGFP+WKY) at 510 nm (λ). exc The fluorescence emission at 488 nm (λ = 488 nm) changes over time.

[0037] Figure 4K These are images of WKY and WYK, showing non-directional self-assembly that leads to differences in tryptophan environment and water binding, with magnified areas showing dry tryptophan (top) and water-bound tryptophan (bottom).

[0038] Figure 5A This indicates a tendency for sequence-dependent clustering (AP=(SASA)). 初始 / SASA 最后50ns A graph showing the distribution of hydrogen bond interactions between simulated peptides based on MD trajectories (bottom). The main chain and side chains serve as indicators of self-assembly directionality.

[0039] Figure 5B This is a graph showing the circular dichroism (CD) spectra of KYF, KYY, and KYW measured at a concentration of 5 mM at pH 7.5.

[0040] Figure 5C This is a graph showing the AP fraction, W solvent accessible surface area (SASA), and hydrogen bond interaction distribution analysis of the MD trajectory of the K / Y / W sequence isomers.

[0041] Figure 5D The 3D fluorescence spectrum is shown, which reveals the differential polarity of tryptophan residues in solution and their interaction with water.

[0042] Figure 6A Confocal live-cell imaging of KWY peptide particles doped with Alexa 488 is shown, revealing significant dye enrichment within the peptide droplets. Scale bar = 20 μm.

[0043] Figure 6B This is a graph showing the fluorescence intensity analysis results of the solution before loading, the foam coagulation layer, and the solution after loading at the emulsion boundary. Quantitative analysis was performed on a total of 20 regions of interest from each group across 6 time-delay frames, with the average intensity of the solution before loading arbitrarily set to 100. Error bars represent standard deviations.

[0044] Figure 6C A confocal image was depicted, showing the Alexa 488 dye remaining encapsulated after curing. Scale bar = 20 μm. Detailed Implementation

[0045] Addressing the critical challenge of biomolecular stability has far-reaching implications and reveals enormous commercial potential across multiple industries, including pharmaceuticals, cosmetics, healthcare, and food production. Solving this problem not only underscores the importance of stable biomolecules but also highlights the significant opportunities for innovation and development in these fields.

[0046] This disclosure provides a biomolecule stabilization technique. It provides short peptide-based materials capable of encapsulating and preserving biomolecules, ensuring their stability and functionality at room temperature. During air drying and water evaporation, the peptides form a protective barrier around the protein, thereby maintaining its structure and activity. The dried powder is then stored without the need for low temperatures.

[0047] This disclosure provides a composition and method for forming porous particles that encapsulate payloads and are readily soluble in aqueous media. Unlike existing emulsification methods, this method does not require mechanical energy but relies on an evaporation process to achieve phase separation and encapsulation. Unlike conventionally highly oriented peptide-based materials (one-dimensional or two-dimensional structures, such as fibers, tubing, and sheets), the particles of this disclosure are grown in a three-dimensional manner and possess space-filling properties. Unlike existing polymer-based systems, these foams are made from biocompatible and biodegradable tripeptides.

[0048] Without being bound by any particular theory, the systemic modulation of aromatic interaction strength and directionality through changes in side chains (F, Y, W) leads to motifs with richer and more dynamic side chain interactions. Figure 1A , Figure 1B Replacing F or Y with tryptophan (W) eliminates the strong directionality of minimal self-assembling peptide systems. Compared to phenylalanine, tryptophan has a broader space for non-covalent interactions, including not only π-π and cation-π interactions, but also H bonds (different from tyrosine due to indole NH), NH- and dipole-dipole interactions, including interactions with water. Figure 1A Tryptophan plays a precise role in proteins due to its unique heteroaromatic properties, such as specific interfacial functions, for example, near lipid bilayers and ion channels. Therefore, the self-assembly behavior of KFF, KYF, KYY, and KYW was first investigated.

[0049] Reference Figure 2A When studying the fluorescence of KYW peptide assemblies using confocal microscopy, significant phase separation behavior was observed, which was triggered by localized evaporation during the drying process.

[0050] On a macroscopic scale, surprisingly contrasting behaviors were observed when observing tripeptide assembly in droplets starting from a 20 mM peptide dispersion during drying. The strongly scattering inward-moving ring of KYW during drying contrasts sharply with the heterogeneous translucent structures produced by KFF, KYF, and KYY. Figure 2B PBS buffer). When observed under a microscope ( Figure 2C After drying, the morphological differences (1D fibers vs. 3D spherical objects) are evident (a comparison of KFF and KYF is shown). When observed over time, KYF exhibits significant interfacial phase separation behavior, resulting in the formation of a film comprising densely packed spherical particles. Figure 2DThis behavior contrasts sharply with observations of KFF, KYF, and KYY (which produced 1D structures with varying degrees of binding, similar to previous observations of evaporation-driven assembly of fluorinated phenylalanine derivatives within dried solvent droplets). Multiple possible interactions and the flexibility of the backbone allow the K / Y / W peptide to more effectively reconfigure and adapt to the newly formed interface after water evaporation. The observed (missing) directionality originates at the molecular level and clearly transitions hierarchically from the nanoscale to the macroscale. Complex drying patterns are observed, related to complex gradients during the drying process of solute-containing droplets. Phase separation occurs at the moving drying boundary due to concentration gradients within the dried droplets.

[0051] In one embodiment, the tripeptide is selected from the 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 can be dissolved in water at a concentration 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 pH of the water can be from 2 to 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, particularly at low pH (e.g., pH 2), a dilute salt solution is used to shield the charge. The salt concentration can be, for example, from 0 M to 2 M (e.g., 1 M). In some embodiments, a buffer solution is present in the water. For example, the water may comprise phosphate-buffered saline (PBS) at concentrations of 0.1–1000 mM, 0.1–300 mM, 0.5–300 mM, or 0.1–100 mM (e.g., 100 mM).

[0053] The water is then allowed to evaporate to form a porous material. Evaporation can be carried out at temperatures such as 20°C to 80°C, 25°C to 60°C, 20°C to 40°C, 20°C to 30°C, or 20°C to 25°C. In some embodiments, laser excitation (e.g., at 405 nm, 488 nm, 561 nm, etc.) is used to accelerate evaporation.

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

[0055] As a concrete example, porous peptide materials can be prepared by dissolving a tripeptide in 100 mM phosphate buffer (pH 8) to a final concentration of 0.5–20 mM. The pH is adjusted to 7.5 using a 0.5 M HCl solution. In one embodiment, the solution is heated at 70°C for 5 minutes and aged at room temperature (i.e., 21–23°C) for 1 hour to ensure uniform distribution. Evaporation-driven assembly is performed by depositing 5 μL droplets on a glass microscope slide and evaporating them at approximately 40–50% humidity (temperature range 25–80°C). Alternative evaporation-driven methods include using a vacuum pump, a desiccator, and temperature control to vary the drying time.

[0056] Sequence-dependent tryptophan exposure is thought to lead to interfacial aggregation and reduced surface tension. Dynamic tension profiles of tripeptides WYK, WKY, and KYW were measured using the pendant drop method (Langmuir 19, pp. 8436-8442, 2003), exhibiting distinctly different W solvent exposures and environments. Surface adsorption of peptide aggregates reduced surface tension in a manner correlated with increased W accessibility within supramolecular aggregates (concentration-dependent) and differences in molecular stacking and W solvent exposure (sequence-dependent). Despite these differences, each sequence exhibited similar sequential phase separation behavior during evaporation-driven assembly within dry droplets, leading to droplet formation and subsequent solidification into a solid particulate film, similar to KYW. Figure 2E When analyzing tryptophan emission in the dried film at 5% relative humidity (RH), an emission loss in the 318 nm band of the 3D excitation / emission spectrum was noted, while the spectrum of the dried film showed similarities. Figure 2E The bands were recovered by exposing the solid membrane to high humidity (95% RH). These data highlight the adaptive nature of the assembly, in which tryptophan partitioning undergoes reversible alterations during aquatic environmental cycling through weak multivalent and dynamic interactions.

[0057] Payload size control, reversibility and packaging

[0058] A systematic increase in temperature leads to a reduction in size and a much more uniform size distribution. Figure 4A , Figure 4B Not wanting to be bound by any particular theory, the lower gas content at high temperatures led to reduced buoyancy, and the accelerated phase change and solidification resulted in monodisperse particles.

[0059] buoyancy

[0060] When studying the dried particulate film using various microscopy techniques, the expected spherical objects were observed. In addition, a large number of porous, disc-shaped particles of varying sizes with very flat surfaces were also observed (in...). Figure 3A (Schematic illustration in Figure i). These structures are present on the top of the sample and are visible via SEM ( Figure 3B , Figure 3C This indicates that they form at the air-water interface. This observation suggests that the structure is a low-density, buoyant condensate compared to the dense, condensed droplets of typical precipitation. Observations of the flattened 2D hemispherical objects indicate that these particles were initially liquid, and due to their low density, they rose to the interface in their liquid form, where they deformed, flattened, and solidified. This observation was confirmed using FIB SEM. Figure 3C The FIB SEM image shows a partially circular cross-section. The pore size distribution ranges from 30 to 500 nm.

[0061] Not wanting to be bound by any particular theory, the observed buoyancy is considered to be related to the formation of bubbles inside the phase-separated droplets. Figure 3A (Figure i). This may be a result of a secondary phase separation event, in which the condensation interface nucleates the formed bubbles and stabilizes them by indole adhesion to the newly formed gas / water interface. Figure 3A (Figure ii). Notably, after degassing the peptide dispersion prior to evaporation-driven assembly, the average particle size became smaller and the morphology more spherical. Figure 4D This further demonstrates that the buoyancy of droplets is related to their gas content and is tunable. The small size and spherical shape of the non-surface particles indicate merging and flattening at the air-water interface. Figure 3B TEM analysis of glutaraldehyde-crosslinked KWY particles revealed a highly fine porous and spherical structure in the non-surface particles. Figure 3C , Figure 3D AFM further confirmed the hemispherical porous structure ( Figure 3E , Figure 3F , Figure 3G Young's modulus analysis showed that the solid particles formed from the peptide sequence isomers exhibited similar properties to the softer KYF and KYY fibers (2.5 GPa). Figure 3H Compared to a similar stiffness of about 6 GPa, and consistent with previously reported mechanically rigid peptide fibers based on FF derivatives.

[0062] Gas content

[0063] Air was removed from the solution by vacuum sonication (e.g., sonication at 20°C for 15 min, or 20 cycles at 4°C with a 30-second on / 30-second off cycle), followed by storage under argon atmosphere. The degassed solution was then subjected to evaporation experiments in an argon-filled chamber. Real-time confocal time-lapse imaging of the particle formation process revealed smaller droplets formed in the degassed peptide samples, using WKY as an example. Further Imaris 3D reconstruction and analysis showed that the particle volume in the degassed samples was reduced by 20-fold and the sphericity increased compared to the prevalent disc-shaped particles in the undegassed samples. Figure 4C , Figure 4D Consistent with the role of air in pore formation, degassed peptide particles exhibit fewer and smaller pores.

[0064] Reversibility

[0065] When 5 μl of water droplets are reintroduced onto the top of the dried film, the structure redisperses almost instantaneously, as... Figure 4E As shown. When the droplet was dried again, the particulate film reappeared, indicating that the process was completely reversible. These observations contrast sharply with previously reported peptide materials, where the formation of 1D and 2D structures was highly stable and difficult to reverse due to the large enthalpy increase brought about by the concerted H bonds. Peptide assemblages with multivalent, weak, and dynamic interactions offer more reversible assembly because water solubilization can compete with the energy relations in the interaction space.

[0066] Packaging

[0067] Given the highly porous nature of the particulate membrane, the disclosed particles can be used for encapsulation. The small organic fluorophore ALEXA FLUOR® 488 was used as the test payload. ALEXA FLUOR® 488 has been shown to exhibit non-covalent conjugation via sulfonate groups and lysine side chains, which can be observed via confocal microscopy. Upon introduction of the dye, the dye is encapsulated during droplet formation and retained therein upon curing (see [link to documentation]). Figure 6A , Figure 6B as well as Figure 6C Analysis of the fluorescence intensity of the pre-loading mixture of peptides and dyes, the formed droplets, and the post-loading solution revealed efficient enrichment of the dye within the particles. Figure 6B ).

[0068] The ability to encapsulate and stabilize biomacromolecules (enhanced green fluorescent protein (EGFP)) was demonstrated. Effective encapsulation of the dynamically assembled peptide WKY within the structure was observed. Figure 4F , Figure 4G Since the EGFP conformation has been reported via fluorescence, the retention of the structure was assessed in the dry state. Figure 4H After drying for 4 hours, the EGFP solution was rehydrated, resulting in the loss of fluorescence. Figure 4I , Figure 4J The control group KYF+EGFP (fiber assembly) showed reduced fluorescence, indicating some degree of stabilization through protein / peptide interactions, while the WKY+EGFP sample retained fluorescence emission even after drying, storing in dry form for 5 days, and then instantaneously resolving. These results suggest that supramolecular peptide particles provide an environment for protein stabilization in a dry state, a mechanism likely similar to the dynamic interfacial complexation observed in random polymer / enzyme complexes through self-organized side chains to the protein surface. These data indicate that the K / Y / W isoform tripeptide has the potential to provide a universal and minimally simplistic form for protein encapsulation, drying, storage, and redispersibility. Figure 4K Snapshots of WKY and WYK are depicted, showing non-directional self-assembly that leads to differences in tryptophan environment and water binding, with magnified areas showing dry tryptophan (top panel) and water-bound tryptophan (bottom panel).

[0069] Molecular dynamics simulation

[0070] Using atomic MD simulations to reveal differences in molecular interactions ( Figure 1B , Figure 5A Replacing F with Y and then with W results in an increase in the aggregation tendency (AP). Figure 5A Then, the contributions of main-chain-main-chain hydrogen bonds (as a representative of 1D self-assembly (proxy)), main-chain-side-chain hydrogen bonds, and side-chain-side-chain hydrogen bonds were analyzed. Figure 5A Consistent with the increase in AP scores, an increase in the total number of hydrogen bonds was observed in the F→Y→W variant. Among these hydrogen bonds, backbone-backbone interactions decreased, while the participation of side-backbone and side-side-side hydrogen bonds increased in sequences containing Y and W. Figure 5A Data shows that the ability to introduce H-bonds from rotatable flexible side chains can facilitate non-directional assembly, especially for KYW ( Figure 5A ).

[0071] In the circular dichroism (CD) spectrum of the KYF / Y / W peptide, the relative contribution of the side chain and main chain interaction during F→Y→W substitution can also be clearly seen. Figure 5B ). Figure 5BAt a concentration of 5 mM, just above the critical aggregation concentration for KYF and KYY, KYF and KYY exhibited a carbonyl π→π* transition peak at 203 nm, a result of main-chain-main-chain interactions. For KYW, this transition intensity was lower, while the presence of a positive signal at 229 nm confirmed the dominant role of tryptophan side-chain interactions. CD values ​​for the three tripeptides (KYF, KYY, and KYW) were also detected at concentrations ranging from 0.5 mM to 10 mM, 0.5 mM to 5 mM, and 0.5 to 20 mM, respectively. KYF and KYY eventually underwent gelation and phase separation, but KYW significantly retained the appearance of a transparent solution up to 20 mM. The CD data were consistent with previously reported characterization using TEM, where images of KFF, KYF, and KYY showed nanoscale fibers, while KYW showed amorphous aggregates. Therefore, while KFF, KYF, and KYY exhibited oriented assembly due to main-chain H bonds, KYW formed soluble aggregates stabilized through dynamic side-chain interactions. The inability to observe aggregates under a microscope indicates that these aggregates are dynamic and have indistinct boundaries. The existence of dynamic soluble aggregates in the micrometer size range was confirmed by dynamic light scattering (DLS).

[0072] Sequence dependence in tripeptide dispersions

[0073] Unwilling to be bound by any particular theory, in systems stabilized by a large number of possible side-chain interactions, the system is not defined by a dominant conformation, but rather by an aggregate of conformational isomers. Therefore, six K / Y / W sequence isomers were investigated by molecular dynamics (MD) to determine sequence-dependent aggregations (...). Figure 5C Furthermore, it was found that their AP scores were comparable and their side-chain / main-chain interaction ratios were similar, consistent with the formation of non-oriented aggregates. Solvent exposure of tryptophan was analyzed by measuring the solvent-accessible surface area (SASA) of the indole side chain. Figure 5C The sequence dependence was found to be significant. The most distinct sequences for AP and tryptophan solvent exposure were WYK and WKY. This can be experimentally verified by measuring tryptophan emission, which is widely used as a probe to detect environmental and solvent exposure of tryptophan in proteins; a redshift indicates increased local polarity, while a blueshift indicates a hydrophobic environment. Emission spectra of all sequence isoforms were measured at 20 mM. Figure 5D Each sequence resulted in the formation of a (non-scattering) soluble dispersion that showed no characteristics when observed under an optical microscope, but exhibited a weak blue emission under UV light. The results showed that the emission maximum (λ) ex= 280 nm) in the range of 357 nm to 370 nm, where the maximum redshift emission corresponds to the most hydrated tryptophan (WYK), and the maximum blueshift emission corresponds to the most hydrophobic tryptophan environment (WKY). Comparison of 3D excitation / emission spectra of the sequence ( Figure 5D The difference lies in the WYK spectrum at λ ex A new band appeared at 318 nm, which is also prominent in YKW and YWK. The intensity and presence of this band are concentration-dependent, indicating that it originates from the emission state of supramolecular arrangement.

[0074] The sequences of WKY and WYK were analyzed by DLS, and similar particle sizes of 460 and 500 nm were observed, respectively. However, WYK exhibited a significantly narrower particle size distribution. To further confirm the differentiated environments experienced by tryptophan, solutions of different concentrations of WYK and WKY were analyzed. 1 H-NMR spectra revealed distinctly different concentration-dependent chemical shifts in W protons. As the concentration increased from 1 mM to 5 mM and then to 20 mM, the indole ring protons in WYK shifted to a lower field, indicating that they were in an electron-deficient environment and formed a polar network / hydrogen bond, while a (weak) shift to a higher field was observed for WKY, consistent with the formation of π-π stack-driven aggregations.

[0075] This written description uses examples to disclose the invention, including the best mode, and also enables any person skilled in the art to practice the invention, including making and using any apparatus or system and performing any of the included methods. The patentable scope of the invention is defined by the claims and may include other embodiments that may occur to those skilled in the art. Such other embodiments should be within the scope of the claims, provided that they have structural elements that are no different from the literal expression of the claims, or that they include equivalent structural elements that are no substantially different from the literal expression of the claims.

Claims

1. A method for forming a porous peptide material, the method comprising the following steps: The tripeptide is dissolved in water, wherein the tripeptide is selected from the 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 Wait for the water to evaporate, thereby allowing the tripeptide to form a porous material.

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

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

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

5. The method of claim 1, wherein the waiting step is performed at a temperature of 20°C to 80°C.

6. The method of claim 1, wherein the waiting step is performed at a temperature of 25°C to 60°C.

7. The method of claim 1, wherein the waiting step is performed at a temperature of 20°C to 40°C.

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

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

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

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

12. A porous peptide material formed by a method comprising the following steps: The tripeptide is dissolved in water, wherein the tripeptide is selected from the 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 Wait for the water to evaporate, thereby allowing the tripeptide to form a porous material.

13. A method for delivering a payload to an aqueous medium, the method comprising: The tripeptide is dissolved in water, wherein the tripeptide is selected from the group consisting of: KYW, KWY, YKW, YWK, WKY, WYK, KYW-NH2, KWY-NH2, YKW-NH2, YWK-NH2, WKY-NH2, WYK-NH2, and combinations thereof; The payload is dissolved in water, and the payload is an organic molecule; Wait for the water to evaporate, thereby allowing the tripeptide to form a porous material when exposed to the payload, thereby encapsulating the payload in the porous material to form an encapsulated payload; as well as Add the encapsulated payload to the aqueous medium.

14. The method of claim 13, wherein the payload is selected from the group consisting of proteins, dyes, fluorescent dyes, aromatic molecules, vitamins, nucleic acids, and drugs.

15. The method of claim 14, wherein the payload is a protein.

16. The method of claim 14, wherein the payload is a nucleic acid.

17. The method of claim 13, wherein the tripeptide is selected from the group consisting of: WKY, WYK, KYW, WKY-NH2, WYK-NH2 and KYW-NH2.

18. The method of claim 13, wherein the tripeptide is selected from the group consisting of WKY, WYK, WKY-NH2 and WYK-NH2.

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

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

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