Aqueous dispensing capsules

Abstract

Aqueous partitioning peptide capsules for delivery of active agents are described. The capsules comprise a membrane having an outer surface and defining a liquid-receiving interior space, configured to encapsulate a water-soluble active agent therein. The capsule membrane is composed of a plurality of linear peptides, each peptide comprising a hydrophobic core of 4 to 12 hydrophobic amino acids flanked by N-terminal and C-terminal hydrophilic segments, each containing 3 to 4 hydrophilic amino acids. Methods for making the peptide capsules and using them to deliver active agents encapsulated therein or bound thereto to various organisms are described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 485,339, filed February 16, 2023, and U.S. Provisional Patent Application No. 63 / 406,388, filed September 14, 2022, each entitled AQUEOUS PARTITIONING CAPSULES, each of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The following application contains an XML file conforming to Standard ST.26 named "SequenceListing57230.xml" created on September 14, 2023, with a size of 54,294 bytes, the contents of which are incorporated by reference herein:

[0003] The present invention relates to self-assembling peptide capsules that entrap aqueous solvents and associated solutes inside for the encapsulation and delivery of a wide variety of active agents. [Background technology]

[0004] Nanotechnology applied to therapeutics, known as nanomedicine, offers many possibilities for healthcare. One major aspect of nanomedicine is drug delivery. Nanotechnology has improved drug delivery systems by increasing the stability of drugs or genetic material by extending their half-life, increasing the solubility of hydrophobic drugs, and helping to control the release of drugs or genetic material.

[0005] Self-assembled nanocarriers are promising delivery vehicles because they overcome certain obstacles, such as poor absorption, observed with other drug delivery species. Compared to liposomal drug delivery systems, polymeric delivery systems have previously been shown to enhance the stability of delivered drugs and aid in the creation of controlled-release systems. Self-assembly of biomolecules is a common phenomenon in nature, such as lipid membranes and multiprotein complexes required for cellular function. The driving force behind self-assembly typically involves interchain hydrogen bonding or hydrophobic interactions. Hydrogen bonding is observed in assemblies composed of peptides that adopt a beta-sheet secondary structure. In the case of hydrophobic interactions, van der Waals interactions dominate. Experimentally, these can be assessed by using hydrophobic solvents that compete for these interactions and lead to capsule degradation. There remains a need for improved encapsulation and delivery technologies. Summary of the Invention

[0006] This paper discloses an improvement in the use of linear peptides that assemble into stable hollow spheres or particles (also known as capsules) to encapsulate water-soluble active ingredients in their aqueous interiors. The outer surface of these capsules is hydrophilic and preferably positively charged, allowing them to disperse in aqueous solutions and promoting uptake by animal and plant tissues, thereby facilitating the delivery of the payload (i.e., active ingredient) into the cells. The active ingredient or targeting moiety can optionally be conjugated to the outer surface of the capsule.

[0007] In one or more embodiments, the present disclosure provides linear peptides comprising a hydrophobic core segment of 4 to 12 hydrophobic amino acids flanked by N-terminal and C-terminal hydrophilic segments each comprising 3 to 4 hydrophilic amino acids. Accordingly, the present disclosure relates to aqueous compositions comprising a plurality of these linear peptides, as well as dry, shelf-stable compositions comprising a plurality of peptides. When dispersed in a low pH buffer system (preferably an aqueous system), these peptides self-assemble into capsules with a capsule membrane having a hydrophilic, preferably positively charged, outer surface and an inner surface defining a liquid-receiving interior space into which an active agent can be dispensed.

[0008] The present disclosure also relates to a method for forming peptide capsules. This method comprises dispersing or dissolving a plurality of linear peptides in a low pH buffer system, optionally containing an active agent to be encapsulated, to form a heterogeneous dispersion or solution of amphipathic peptides (and the active agent). The pH of the buffer system is preferably less than 5, more preferably less than 4, and even more preferably about 2 to about 3.5. An exemplary buffer system includes an aqueous system containing 10 mM glycine-HCl. The mixture is preferably incubated at room temperature under low pH conditions for a period of time (e.g., about 1 to about 20 minutes) during which the peptides self-assemble into capsules. The pH of the mixture is then raised to a neutral pH (about 7) using a neutral or alkaline buffer. It will be appreciated that an appropriate acidic or basic buffer (e.g., 15 mM imidazole-HCl) can be used to adjust the pH to achieve the desired pH value.

[0009] The present disclosure also provides a pharmaceutically acceptable composition comprising a plurality of peptide capsules as described herein and an active agent encapsulated in the capsules. Also provided is a method for targeted delivery of an active agent to a region of a patient. The method comprises administering to a patient peptide capsules as described herein encapsulating an active agent. Advantageously, these capsules can further comprise a targeting moiety attached to the outer surface of the capsule membrane for automatic selective uptake by the target tissue or region of interest. Advantageously, these capsules can be used to encapsulate next-generation therapeutic agents, such as nucleic acids, for direct intracellular delivery of therapeutic payloads.

[0010] The patent or application file contains at least one color drawing. Copies of any color drawing(s) in this patent or patent application publication will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows (A) a cartoon diagram of APC and TEM images taken of APC from Ac-KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) at (B) pH 12, (C) pH 7, and (D) pH 2. [Figure 2] FIG. 2 is a graph of circular dichroism (CD) data for APC at various pH values ​​and in TFE. [Figure 3] FIG. 3 shows a graph of an NTA analysis of APC formed at pH 2 and raised to pH 7 after 5 minutes using imidazole buffer. [Figure 4] FIG. 4 shows a graph of an NTA analysis of APC formed at pH 3 and raised to pH 7 after 5 minutes using imidazole buffer. [Figure 5] FIG. 5 shows a graph of an NTA analysis of APCs with Poly IC attached to the outside of the APCs when formed at pH 2 and raised to pH 7 after 5 minutes using imidazole buffer. [Figure 6]FIG. 6 shows a graph of NTA analysis of APC in encapsulated PolyIC when formed at pH 2 and raised to pH 7 after pH 2 and 5 minutes using imidazole buffer. [Figure 7] Figure 7 shows a confocal microscope image of an APC encapsulating the fluorescent dye rhodamine 6G. The APC is approximately 2 microns in diameter. [Figure 8] FIG. 8 shows graphs of the CD spectra of Ac-KKKFLIVIGSIIKKK-CO-NH2 (SEQ ID NO: 7) APC when formed at pH 3.5 (A) and measured after adjusting the pH to 7.0 (B). [Figure 9] Figure 9 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP plasmid by transfection with APCs at a 1:20 ratio of genetic material to peptide attached to the outside of the APC. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:20 ratio of genetic material to peptide encapsulated inside the APC. (C) These APCs were also characterized by DLS and zeta measurements (Table 12). [Figure 10] Figure 10 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP plasmid by transfection with APCs at a 1:10 ratio of genetic material to peptide attached to the outside of the APC. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:10 ratio of genetic material to peptide encapsulated inside the APC. (C) These APCs were also characterized by DLS and zeta measurements (Table 12). [Figure 11]Figure 11 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP plasmid by transfection with APCs at a 1:5 ratio of genetic material to peptide attached to the outside of the APC. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:5 ratio of genetic material to peptide encapsulated inside the APC. (C) These APCs were also characterized by DLS and zeta measurements (Table 12). [Figure 12] Figure 12 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:20 ratio of genetic material to peptide attached to the outside of the APC. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:20 ratio of genetic material to peptide encapsulated inside the APC. (C) These APCs were also characterized by DLS and zeta measurements (Table 13). [Figure 13] Figure 13 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:10 ratio of genetic material to peptide attached to the outside of the APC. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:10 ratio of genetic material to peptide encapsulated inside the APC. (C) These APCs were also characterized by DLS and zeta measurements (Table 13). [Figure 14] Figure 14 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:5 ratio of genetic material to peptide attached to the outside of the APC. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs at a 1:5 ratio of genetic material encapsulated inside the APC. (C) These APCs were also characterized by DLS and zeta measurements (Table 13). [Figure 15] Figure 15 shows the results of a RiboGreen assay of APCs formed at pH 2, incubated for 5 minutes, and then the pH was increased to 7 using imidazole buffer. The APC conditions included encapsulated genetic material and attached genetic material to the outside of the capsule, at various ratios of genetic material to peptide. [Figure 16] Figure 16 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 2 and increased to pH 7 after 1 minute. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 2 and increased to pH 7 after 5 minutes. (C) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 2 and increased to pH 7 after 20 minutes. (D) These APCs were also characterized by DLS and zeta measurements (Table 14). [Figure 17] Figure 17 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 3 and increased to pH 7 after 1 minute. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 3 and increased to pH 7 after 5 minutes. (C) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 3 and increased to pH 7 after 20 minutes. (D) These APCs were also characterized by DLS and zeta measurements (Table 14). [Figure 18]Figure 18 shows confocal microscopy images of untreated HEK cells. (A) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 3.5 and increased to pH 7 after 1 minute. (B) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 3.5 and increased to pH 7 after 5 minutes. (C) Confocal microscopy images of in vitro uptake of GFP mRNA by transfection with APCs made at pH 3.5 and increased to pH 7 after 20 minutes. (D) These APCs were also characterized by DLS and zeta measurements (Table 14). [Figure 19] FIG. 19 shows a graph of CD when APC prepared at pH 2.0 to 7.0 was dissolved in increasing percentages of TFE. DETAILED DESCRIPTION OF THE INVENTION

[0012] Described herein are linear peptides consisting of a short hydrophobic core segment flanked by an N-terminal cationic amino acid segment and a C-terminal cationic amino acid segment that form water-filled nanocapsules at low pH. These capsules, termed aqueous partitioning capsules (APCs), encapsulate water-soluble active ingredients and deliver them to cells, where they can release their contents.

[0013] The present invention broadly relates to a composition comprising a plurality of APCs suspended in an aqueous carrier. Each APC comprises a peptide capsule having an outer surface and a membrane defining a liquid-receiving interior space, within which a water-soluble active agent can be encapsulated. APCs do not form secondary structures and do not fuse unless they change size. APCs are capable of both delivering and releasing payloads into cells. Advantageously, APCs can cross mucus layers and cell membranes, are rapidly internalized by cells whose peptide membranes have been degraded by normal intracellular processes, and are relatively stable in solution unless organic solvents are added to the solvent system.

[0014] The peptide sequences used to form capsules are linear and contain no branching points, and comprise (or consist essentially of, or consist of) a hydrophobic core segment flanked by N-terminal and C-terminal cationic, hydrophilic segments. Linear peptides generally have a total length of 20 amino acid residues or less, preferably about 6 to about 20 residues, and more preferably about 8 to about 15 residues. These peptides preferably have a molecular weight ranging from about 550 Da to about 2300 Da, more preferably about 675 Da to about 2050 Da, and even more preferably about 800 Da to about 1800 Da. The "molecular weight" of these peptides is the average weight calculated based on the total MW of the bound amino acids actually present divided by the number of residues.

[0015] The hydrophobic core segment can comprise (consist essentially of, or consist of) 4 to 12 amino acid residues. The amino acids used in the hydrophobic core are preferably nonpolar amino acids selected from hydrophobic or very hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, alanine, tyrosine, and methionine. In one or more embodiments, the hydrophobic core can include up to two neutral amino acid residues selected from glycine, serine, cysteine, glutamine, and / or threonine, so long as the overall hydrophobicity of the hydrophobic segment is higher than that of the hydrophilic segment, preferably with an overall hydrophobicity of greater than 1 on the Kyte-Doolittle scale (1982). Particularly preferred hydrophobic amino acids for use in the hydrophobic segment include phenylalanine, leucine, isoleucine, and valine, with leucine being less preferred among the preferred hydrophobic amino acids. When present, the neutral amino acid residues are preferably selected from glycine and / or serine. In one or more embodiments, the hydrophobic segment comprises the sequence XLIVI (SEQ ID NO: 1), XLIVIGSII (SEQ ID NO: 2), XFFIVIL (SEQ ID NO: 3), or XLIVIGSIIVIL (SEQ ID NO: 4), where X is F or V, and the amino acid residues can be in that order or in any order (scrambled, e.g., see SEQ ID NOs: 8-46). In one or more embodiments, the hydrophobic segment comprises the sequence X(LIVI) (SEQ ID NO: 1), X(LIVI)GSII (SEQ ID NO: 2), XFF(IVI)L (SEQ ID NO: 3), or X(LIVI)GSIIVIL (SEQ ID NO: 4), where X is F or V, and the residues in parentheses can be in that order or in any order (scrambled). In one or more embodiments, the residues in parentheses are substituted with all I residues or all V residues. In one or more embodiments, any one residue of the sequences FLIVI (SEQ ID NO: 1), FLIVIGSII (SEQ ID NO: 2), VFFIVIL (SEQ ID NO: 3), or FLIVIGSIIVIL (SEQ ID NO: 4), except for the N-terminal phenylalanine or valine, can be substituted with I or V.In one or more embodiments, the GSII (SEQ ID NO: 5) segment can include one or more substitutions to reduce the hydrophobicity of the segment, such as replacing one or both I residues with a neutral amino acid residue.

[0016] The N-terminal and C-terminal hydrophilic segments can comprise (consist essentially of, or consist of) three to four hydrophilic (polar) amino acid residues (respectively), preferably lysine, but may also include arginine, histidine, aspartic acid, or glutamic acid, which also have charged side chains. A particularly preferred hydrophilic segment consists of three lysine residues. The N-terminal and C-terminal hydrophilic segments may each have the same or different peptide sequences. The N-terminal and C-terminal hydrophilic segments are preferably more hydrophilic than the hydrophobic segments.

[0017] The linear peptide sequence preferably further comprises an N-terminal acetylation and a C-terminal amidation, which removes the charge at both ends of the peptide and allows the peptide to assemble in either orientation (N→C or C→N). Particularly preferred peptide sequences include Ac-KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) and Ac-KKKFLIVIGSIIKKK-CONH2 (SEQ ID NO: 7).

[0018] Surface modification of the resulting APCs can also be achieved by pre-conjugating functional groups and / or various moieties to amino acid residues at the N- or C-terminus of the peptide, which, once formed, presents modified features on the exterior of the APC surface, enabling cell targeting. For example, peptides can be iodinated for targeting. The term "functional moiety" is used herein to encompass functional groups, targeting moieties, and active agents that can be attached to the exterior surface of APCs. Exemplary functional moieties that can be attached include fluorophores, dyes, tissue-targeting moieties and ligands, antibodies, cysteine, cysteamine, biotin, biocytin, nucleic acids, polyethylene glycol (PEG), organometallic compounds (e.g., methylmercury), radiolabels, conjugation chemistry, -COOH, -NH3, -SH, and the like. Multiple such moieties can also be attached sequentially in a chain from the N- or C-terminus, using an aliphatic spacer to separate the different moieties. Thus, the present invention provides the opportunity to create multifunctional APCs.

[0019] Figure 1(A) is an illustration of an APC according to an embodiment of the present invention. In the figure, a water-soluble active agent and aqueous suspension are encapsulated or surrounded by a layer of peptides. As shown in the enlarged view, the wavy lines represent linear peptides arranged to form a peptide membrane or layer, with each cationic N-terminal and C-terminal hydrophilic segment facing the aqueous exterior environment of the suspension, and the internal water-soluble active agent and aqueous suspension are trapped within individual capsules by the peptides. The hydrophobic core residues of each peptide form the core of the membrane and do not directly interact with the solution once the APC is formed. Importantly, the peptides themselves are not conjugated or bonded to the active agent or water-soluble active agent within the capsule, nor are the peptides covalently bonded to each other.

[0020] Peptides can be synthesized using standard procedures. For example, solid-phase peptide synthesis can be performed using a commercially available peptide synthesizer (e.g., a PS3 peptide synthesizer (Protein Technologies Inc.; Tucson, AZ) or a CS Bio CS136X peptide synthesizer (CS Bio; Silicon Valley, CA)) according to the manufacturer's protocol. In one or more embodiments, peptides can be synthesized using Fmoc amino acids, preferably under nitrogen gas. Upon completion of synthesis, the resin can be dried under vacuum, and the peptide can be cleaved using trifluoroacetic acid (TFA), thioanisole, and / or 2% 1,2-ethanedithiol. The cleaved peptide can be filtered to remove excess liquid and then precipitated with diethyl ether, followed by one or more washes to further increase yield. The peptide can then be suspended in deionized water and lyophilized. The completely dried, synthesized peptide can be stored as a solid at room temperature until use.

[0021] When these peptides are incubated in an aqueous, low-pH buffer system (i.e., pH 3.5 or less), the peptides self-assemble into capsules that entrap other solutes dissolved in the solution. Thus, a method for forming APCs includes dispersing a plurality of linear peptides together with one or more active agents in a buffer system. Exemplary buffer systems include any buffer with a pKa of about 2.0. For example, glycine-HCl at a concentration of 10 mM is an exemplary, particularly preferred low-pH buffer. For example, this buffer system can be prepared by mixing solid glycine-HCl with distilled, deionized water to the desired concentration. If necessary, dilute HCl can be added to lower the pH of the buffer system to the desired range (i.e., pH 3.5 or less). Preferably, the peptides and active agents are incubated in the buffer system under low pH conditions (i.e., pH 3.5 or less, preferably pH 2.5 or less) for less than 20 minutes, preferably less than 10 minutes, preferably less than 5 minutes, and more preferably about 1 minute or less. In one or more embodiments, the incubation is performed under ambient conditions (i.e., a temperature of about 22°C and atmospheric pressure). In one or more embodiments, after the components are mixed, incubation is preferably carried out without stirring or shaking the mixture.

[0022] Advantageously, capsules form quickly, minimizing the time that active agents, such as nucleic acids, are exposed to low pH conditions. APC formation can be observed by the mixture changing from a peptide suspension to capsules, which can be monitored using dynamic light scattering. The pH of the mixture is then raised to neutral (approximately pH 7) using an appropriate buffer. Exemplary buffers that can be used to raise the pH include buffers with a pKa of 1.7. Because the refractive index of the buffer affects scattering intensity and the viscosity of the buffer affects the particle diffusion coefficient, the selected buffer preferably also has no optical properties or known properties (refractive index and viscosity) that can be optimized for dynamic light scattering (DLS) analysis. Important optical properties of the buffer can interfere with the interpretation of DLS data. For example, if the buffer absorbs light, it can reduce the amount of scattered light detected. If the buffer scatters light, it can introduce noise into the data. Therefore, any suitable buffer with an appropriate pKa can be used as long as its properties are calibrated for DLS analysis.

[0023] 15mM imidazole buffer is an exemplary and particularly preferred neutralization buffer.If necessary, the prepared APC can be washed with neutral buffer to remove excess molecules used for surface conjugation, such as dye or oligonucleotide.Alternatively, APC can simply be stored in the same neutralization buffer without the need for filtration, washing or recovery from suspension.

[0024] The resulting capsules are shelf-stable in a neutral buffer system (about pH 7) at room temperature (about 22°C), meaning they remain as individual capsules for extended periods (preferably at least 3 months, more preferably at least 6 months, and even more preferably at least 12 months) without agglomeration, coalescence, aggregation, or disintegration. The capsules generally have a maximum surface-to-surface dimension (e.g., the diameter of a substantially spherical capsule) of less than 500 nm, preferably about 50 to about 500 nm, and preferably about 100 to about 300 nm. For ease of reference, the terms "diameter" and "particle size" are used interchangeably herein to refer to the maximum surface-to-surface dimension of each capsule. Furthermore, because the methods of the present invention result in a suspension of multiple capsules, "particle size" as referred to herein may refer to the average (arithmetic mean) diameter of the entire population of capsules in the suspension. An exemplary neutral buffer system that can be used for shelf-stable storage is 15 mM imidazole buffer. In one or more embodiments, the storage buffer further comprises a buffer that is substantially free of histidine or any sulfate or phosphate conjugate acid to improve stability over time. In one or more embodiments, the storage buffer can contain up to 20% DMSO for cryopreservation. Advantageously, the APC is biodegradable, meaning that the APC remains intact and protects the nucleic acid from degradation, but itself eventually degrades over time in an environmentally friendly manner.

[0025] APCs can be used to deliver a wide variety of active agents, including water-soluble active agents that can be encapsulated in APCs and other drugs that can be conjugated to the outside of APCs.For example, water-filled capsules are suitable for delivering nucleic acids (DNA, plasmid DNA, RNA, mRNA, siRNA, microRNA, dsRNA, etc.) for cell delivery, as well as enzymes, peptides, hydrophilic small molecule compounds and other active agents such as drugs, sugars, toxins, enhancers, and detectable labels such as dyes or fluorescent markers.These capsules are also suitable for delivering various substances covalently or electrostatically attached to their outer surface, such as antibodies, antigens, receptor ligands, vitamins, and other targeting moieties.

[0026] These capsules can be used to deliver water-soluble active ingredients to plants, non-human animals (including insects, fish, birds, mammals, reptiles, etc.), and humans in vitro, ex vivo, and in vivo. Accordingly, methods for delivering active agents to plants, animals, or humans are also contemplated herein. These methods comprise administering a plurality of peptide capsules containing the active ingredient to the plant, non-human animal, or human. This can involve directly applying or administering the peptide capsules or providing the peptide capsules in the vicinity of the target plant, non-human animal, or human. For example, the peptide capsules can be applied directly to the leaves or root system of the plant and / or to the soil surrounding the roots. Similarly, peptide capsules can be administered directly to non-human animals or humans topically, orally, or by injection, or can be introduced indirectly, for example, into aquaculture / aquatic systems where animals live (fish, crustaceans, etc.), or into locations where non-human animals may come into contact (e.g., near beehives to treat bees or bees as pests). The peptide capsules can be incorporated into suitable pharmaceutical, horticultural, or veterinary compositions containing suitable carriers, diluents, excipients, or vehicles for administration. Exemplary carriers and vehicles include, but are not limited to, sugars, polysaccharides, and glycerol.

[0027] For human or animal use, a suitable carrier is pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" means that it can be administered to a subject without undue toxicity, irritation, or allergic reaction, and is not biologically or otherwise undesirable in that it does not cause unacceptable biological effects or interact in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers are selected to minimize degradation of the compound or other agent and to minimize adverse side effects in the subject. Pharmaceutically acceptable components include those that are acceptable for veterinary use as well as for human pharmaceutical use, and vary depending on the route of administration. For example, compositions suitable for administration by injection are typically solutions in sterile isotonic aqueous buffers such as phosphate-buffered saline (PBS). Exemplary carriers include aqueous solutions of monosaccharides and disaccharides, etc.

[0028] The composition may include a therapeutically effective amount of peptide capsules (containing an active ingredient) dispersed in a carrier. As used herein, a "therapeutically effective" amount refers to an amount that elicits the biological or medical response in a tissue, system, or subject that a researcher or clinician is seeking, such as an amount that elicits some desired therapeutic or preventative effect against a disease or condition. In the case of delivery of an insecticidal or herbicidal active, it will be understood that the desired effect is growth inhibition, injury, and / or death of the target. Those skilled in the art will recognize that an amount is considered therapeutically "effective" even if the disease, condition, or pest is not completely eradicated or avoided, but the disease or its symptoms and / or effects are only partially improved or alleviated in the subject.

[0029] The compositions may contain other components, such as adjuvants, other active agents, preservatives, buffers (e.g., histidine), salts, and other pharmaceutically acceptable ingredients. The term "adjuvant" is used herein to refer to a substance added to or co-formulated with a therapeutic composition to enhance, elicit, and / or modulate the innate, humoral, and / or cell-mediated immune response to an active ingredient. These compositions are stable for 1-2 weeks once APCs are formed. However, advantageously, peptides, particularly in lyophilized form, are highly stable and can be stored at room temperature for at least about 90 days. These compositions can be provided as kits for on-site preparation of the appropriate APC formulation prior to administration.

[0030] In some embodiments, the peptide, APC, or composition can be provided in a unit dosage form in a suitable container. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for use in plants, non-human animals, or humans. Each unit dosage form can contain a predetermined amount of peptide, preformed APC, or active agent in a suitable carrier calculated to produce the desired effect. In other embodiments, the peptide can be provided separately from the active ingredient, for example, in its own vial, ampoule, sachet, or other suitable container, for mixing with a vehicle or carrier on-site to form an APC before administration to the target plant, non-human animal, or human. Kits containing peptides, preformed APCs, and / or active agents are also disclosed herein. The kits further include instructions for preparing the APC (if necessary) and administering the composition to a subject.

[0031] It will be understood that the therapeutic and prophylactic methods described herein, including targeting pathogens and pests (e.g., fungi, insects, or bacteria) in human and non-human animal systems or otherwise delivering nucleic acids or other active agents to human and non-human animal tissues, are applicable to any suitable non-human animals, including, but not limited to, dogs, cats, and other pets, as well as rodents, primates, horses, cows, pigs, fish, birds, etc. This platform technology is also useful in plants, such as for targeting pathogens and pests (e.g., fungi, insects, or bacteria) in plant systems or otherwise delivering nucleic acids or other active agents to plant tissues. The methods are also applicable to clinical research and / or testing.

[0032] Further advantages of various embodiments of the present invention will be apparent to those skilled in the art upon reviewing the disclosure herein and the examples below. It will be understood that the various embodiments described herein are not necessarily mutually exclusive, unless otherwise specified herein. For example, features described or depicted in one embodiment may, but are not necessarily, included in other embodiments. Thus, the present invention encompasses various combinations and / or integrations of the specific embodiments described herein.

[0033] As used herein, the term "and / or," when used in a list of two or more items, means that any one of the listed items may be employed alone, or any combination of two or more of the listed items may be employed. For example, if a composition is described as including or excluding components A, B, and / or C, the composition may include or exclude A alone; B alone; C alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B, and C.

[0034] Also, numerical ranges are used herein to quantify certain parameters related to various embodiments of the present invention. When numerical ranges are provided, it should be understood that such ranges are to be construed as providing literal support for claim limitations that recite only the lower value of the range as well as for claim limitations that recite only the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for claims recited as "greater than about 10" (without an upper limit) and claims recited as "less than about 100" (without a lower limit). [Example]

[0035] The following examples illustrate methods according to the present invention, however, it should be understood that these examples are provided for illustrative purposes and do not limit the overall scope of the invention.

[0036] overview The aqueous partition capsule (APC)-forming peptides have the primary sequences Ac-KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) and Ac-KKKFLIVIGSIIKKK-CONH2 (SEQ ID NO: 7). Transmission electron microscopy analysis of assemblies formed by Ac-KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) at pH 2 revealed spherical images. Further study of the pH 2 assemblies showed that a low pH is essential for self-assembly, but they remained stable when shifted to neutral pH. APCs efficiently delivered GFP-encoding mRNA and a plasmid into human embryonic kidney (HEK) cells. The stability of APCs encapsulating GFP mRNA and a GFP plasmid was examined over time. A RiboGreen assay was used to confirm whether the genetic material was present on the outside or inside of the APC. Preliminary studies of the APC encapsulation force, temporal stability, and delivery of GFP mRNA and a plasmid into HEK cells are presented.

[0037] When peptides are dispersed in water at low pH (approximately 2-3.5), they form nano-sized spherical structures. Figure 1 shows juxtaposition images at pH 7 and pH 12, showing aggregates and nanofibrils, respectively. One skilled in the art would not expect or predict the formation of water-filled capsules at low pH, because lowering the pH from neutral (approximately 7) to low pH does not change the overall net charge of the molecule. The exact mechanism of this low-pH assembly remains to be explored. Here, we report the unexpected ability of APCs to form under controlled conditions to encapsulate various types of molecules, including dyes, mRNA, and dsDNA. At low pH, nucleic acids lose their negative charge due to protonation of the backbone phosphates. The charge does not return to the nucleic acid until the capsules are placed in a neutral pH environment. Because the primary sequence of APC contains numerous lysine residues that are positively charged at both acidic and neutral pH, the attachment of APCs to the outside (formed at low pH and then increased to neutral pH) was investigated in addition to the encapsulation. It is believed that the negatively charged genetic material electrostatically attaches to these positively charged lysine residues.

[0038] material and method Solid-phase peptide synthesis—The peptides Ac-KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) and Ac-KKKFLIVIGSIIKKK-CONH2 (SEQ ID NO: 7) were synthesized using solid-phase peptide synthesis on a PS3 peptide synthesizer (Protein Technologies Inc.; Tucson, AZ). Peptide synthesis was performed on a 0.1 mmol scale using Fmoc (fluorenylmethyloxycarbonyl). Fmoc amino acids were obtained from AnaSpec, Inc. (San Jose, CA). Synthesis was performed using an automated PS3 peptide synthesizer (Company and City). The peptides were cleaved using 92% trifluoroacetic acid (TFA), 5% thioanisole, and 2% 1,2-ethanedithiol at room temperature for 90 minutes to yield the TFA salt form of the peptide. After cleavage, the liquid was removed and poured into ice-cold diethyl ether. The peptides were precipitated by three additional consecutive washes with diethyl ether. Both peptides were lyophilized for storage and then suspended in deionized water for the following studies.

[0039] Preparation of APC: The peptide was dissolved in 2,2,2-trifluoroethanol (TFE). The peptide concentration was determined by measuring the absorbance of phenylalanine at 258 nm using a CARY 50 Bio UV-Visible Spectrometer (Agilent Technologies, Santa Clara, CA). A final concentration of 1 mM APC was prepared by adding the TFE-dissolved peptide to an Eppendorf tube. The solvent was then removed using a vacuum and a rotor. After drying, the APC peptide was rehydrated at various pH values ​​ranging from 2 to 4. The APC was incubated at room temperature for various times, and then the pH was increased to 7. The size of the peptide nanoparticles (capsules) was measured.

[0040] Dynamic Light Scattering and Zeta Potential Characterization—APC capsules were prepared at a concentration of 1 mM as previously described (APC Preparation and mRNA-APC or dsDNA-APC Nanoparticle Preparation). Particle size and zeta potential for each sample were analyzed using a Zetasizer Nano ZS (Malvern Instruments Ltd, Westboro, MA).

[0041] NTA studies—Nanoparticle tracking analysis—were performed using a NanoSight LM 14 (Malvern Panalytical, UK). A single chamber was connected to a 405 nm laser. The camera type used was a Hamamatsu Photonics CMOS camera model #C11440-50B. Samples were injected into the single chamber using a sterile syringe (BD Discardit II, New Jersey, USA). Video of the sample was recorded at 25.0 fps for 60 seconds. The temperature was maintained constant at 25.0 °C. APC capsules were prepared at a concentration of 1 mg / mL at both pH 2 and pH 3 as previously described (APC preparation). Samples containing PolyIC and dsDNA were prepared at a genetic material to peptide ratio of 1:10 as previously described (mRNA-APC or dsDNA-APC nanoparticle preparation). The software used to analyze the captured images to calculate the number and size of APC nanoparticles in solution was NanoSight NTA 3.3.

[0042] Circular dichroism (CD) spectral data were collected using a Jasco-815 CD spectrophotometer (Jasco Analytical Instruments, Easton, MD). The cuvette type used for data collection was a 1 mm pathlength quartz cuvette. The scan rate was 50 nm / min, and the scanned wavelength range was 260 nm to 190 nm. A 1 nm step interval was used during scanning. A total of five scans were recorded for each sample and averaged. Ellipticity was measured in millidegrees. Data were corrected based on the solvent used; these CD measurements were performed using either TFE, water, or imidazole buffer. A Savitsky-Golay filter was used to smooth the spectra. The analysis software was provided by the CD spectrophotometer manufacturer.

[0043] Encapsulation of APC dye - Two samples were prepared as described above (APC preparation) at 1 mM APC, pH 2 and pH 2 increased to 7. 5 μL of rhodamine 6G stock solution was added to 1.0 mL of APC to give a final rhodamine 6G concentration of 100 μM. The samples were washed three times with water and spun at 3000 RCF for 3 minutes at 25°C in a Labnet Prism® benchtop centrifuge (Labnet, Edison, NJ). The flow-through after the third run was clear. The fourth run was spun in 200 mM sodium trifluoroacetate (Na * 200 μL of TFA was used at 3000 RCF for 3 minutes at 25° C. A final fifth spin was performed at 3000 RCF for 1 minute to quickly remove any remaining unencapsulated Rhodamine 6G dye.

[0044] Samples were analyzed using a CARY Eclipse fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA). Samples of APC encapsulating rhodamine 6G dye were placed on glass slides and allowed to dry. Slides were analyzed using a confocal LSM700 laser scanning microscope (Carl Ziess, Göttingen, Germany). Images captured by the confocal microscope were analyzed using ZEN 3.5 (blue edition).

[0045] Lifespan studies - APC capsules were prepared as previously described (APC preparation and mRNA-APC or dsDNA-APC nanoparticle preparation) and stored at room temperature (22 °C) or 4 °C. The particle size and zeta potential of each sample were analyzed over time using a Zetasizer Nano ZS (dynamic light scattering and zeta potential characterization).

[0046] Preparation of mRNA-APC nanoparticles or dsDNA-APC nanoparticles: Various ratios of genetic material to APC were created by first drying the APC peptide using a vacuum and rotor. The desired amount of APC peptide was removed from a 1 mM APC stock solution in TFE and placed in a microcentrifuge tube. The microcentrifuge tube was placed in a rotor with vacuum to dry the APC peptide. To encapsulate the genetic material, 200 ng of genetic material was added to 20 μL of pH 2 glycine-hydrochloric acid (HCl) buffer. The APC peptide was rehydrated with 20 μL of pH 2 buffer containing the genetic material to form the APC. To attach the genetic material to the exterior of the APC, the APC peptide was rehydrated with 20 μL of pH 2 glycine-HCl buffer. The APC peptide solution was incubated in the pH 2 buffer at room temperature for 5 minutes, after which 200 ng of genetic material was added.

[0047] Transfection of APC-GFP Plasmid - Human embryonic kidney (HEK) cells were grown in a 37°C, 5% CO2 incubator. HEK cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g / L glucose and penicillin-streptomycin and fetal bovine serum (FBS). To prepare for transfection, cells were grown in 8-well cell trays.

[0048] We tested both the attachment of APCs to the exterior and their encapsulation within the APCs, as well as various ratios of APC to plasmid during transfection. The conditions tested for this plasmid transfection included cells only, Lipofectamine (as a positive control), 1:5 exterior, 1:5 interior, 1:10 exterior, 1:10 interior, 1:20 exterior, and 1:20 interior. Each ratio contained 200 ng of genetic material. The amount of peptide was 1 ug for the 1:5 ratio, 2 ug for the 1:10 ratio, and 4 ug for the 1:20 ratio. Each amount of peptide was dried using a rotor and vacuum. After drying, the exterior conditions were rehydrated immediately with 20 μL of pH2 solution. The tubes were incubated at room temperature for 5 minutes, after which 200 ng of GFP plasmid was added. For the interior conditions, 200 ng of GFP plasmid was added to 20 μL of pH2 buffer for each condition. The APCs were rehydrated with the GFP plasmid and pH2 buffer. All tubes were incubated at room temperature for 5 minutes, after which the pH was raised to 7 with 80 uL of imidazole buffer.

[0049] The HEK cell medium was changed immediately before transfection. The conditions were added to separate wells of the cell tray. The transfection was incubated for 44 hours. Transfection was terminated using perfluoroalkoxyalkane (PFA). After transfection, the medium was replaced with phosphate-buffered saline (PBS). To permeabilize the cell membrane, Triton X was added to the wells and allowed to stand for approximately 1 hour. The cells were washed with PBS. After the cell tray was completely dried, SlowFade™ Diamond Antifade Mountant containing DAPI was added. A cover glass was placed on the cell tray and sealed with clear nail polish. The cell tray was covered with aluminum foil and stored at room temperature before analysis using a confocal microscope. Images taken with the confocal microscope were analyzed using ZEN3.5 (blue plate).

[0050] Transfection of APC GFP mRNA - Human embryonic kidney (HEK) cells were grown in a 37°C, 5% CO2 incubator. HEK cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g / L glucose and penicillin-streptomycin and fetal bovine serum (FBS). To prepare for transfection, cells were grown in 8-well cell trays.

[0051] As described above (APC GFP plasmid transfection), APCs were prepared at ratios of 1:5, 1:10, and 1:20. At each ratio, APCs encapsulating GFP mRNA and APCs with GFP mRNA attached to the outside of the capsule were prepared.

[0052] Immediately after drying, the outer conditions were rehydrated with 20uL of pH2 solution. The tubes were incubated at room temperature for 5 minutes, after which 200ng of GFP mRNA was added. For the inner conditions, 200ng of GFP plasmid was added to 20uL of pH2 buffer for each condition. The APCs were rehydrated using GFP mRNA and pH2 buffer. All tubes were incubated at room temperature for 5 minutes, after which the pH was raised to 7 with 80uL of imidazole buffer.

[0053] The HEK cell medium was changed immediately before transfection. The conditions were added to separate wells of the cell tray. The transfection was incubated for 44 hours. Transfection was terminated using perfluoroalkoxyalkane (PFA). After transfection, the medium was replaced with phosphate-buffered saline (PBS). To permeabilize the cell membrane, Triton X was added to the wells and allowed to stand for approximately 1 hour. The cells were washed with PBS. After the cell tray was completely dried, SlowFade™ Diamond Antifade Mountant containing DAPI was added. A cover glass was placed on the cell tray and sealed with clear nail polish. The cell tray was covered with aluminum foil and stored at room temperature before analysis using a confocal microscope. Images taken with the confocal microscope were analyzed using ZEN3.5 (blue plate).

[0054] RiboGreen assay—We used the RiboGreen assay because RiboGreen reagent is a fluorescent dye capable of detecting genetic material. RiboGreen reagent detects genetic material attached to the outside of APC capsules but not genetic material encapsulated within them. The genetic material-to-peptide ratios used in this assay were 1:1, 5:1, and 10:1. APC nanoparticles were prepared at these ratios as previously described (APC preparation and mRNA-APC nanoparticles or dsDNA-APC nanoparticles preparation). The assay was set up in a 96-well plate. 0.5 μL of RiboGreen reagent in 100 μL of TE buffer was added to each well containing APC nanoparticles. After adding the reagent, fluorescence was measured using a plate reader.

[0055] APC GFP mRNA transfection efficacy test - HEK cells were prepared as described above (APC GFP mRNA transfection). APC was prepared at a 1:10 ratio with GFP mRNA encapsulated. Each sample was rehydrated with 200 ng of GFP mRNA and 20 μL of pH 2, pH 3, or pH 3.5 solution. Each sample was incubated at room temperature for 1, 5, or 20 minutes, after which the pH was raised to 7 with 80 μL of imidazole buffer. The samples were added to HEK cells and incubated for 44 hours, after which the transfection was terminated with PFA. Cells were prepared for confocal microscopy as described above (APC GFP mRNA transfection).

[0056] APC characterization Our studies first focused on a shorter peptide sequence (SEQ ID NO: 6, KKKFLIVIKKK-CONH2). DLS was used to analyze the size of KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) APCs formed and / or transferred to three pH values ​​(pH 2, 7, and 12). The APCs were smallest at pH 2, with a mean diameter of 239.1 nm across three experiments, with a standard deviation of 28.4 nm (Table 1).

[0057] [Table 1]

[0058] APC was largest at pH 12, measuring 3406 nm, with a standard deviation of 1832 nm across three experiments (Table 1). At pH 7, the size of APC was 664.3 nm. However, when APC was first prepared at pH 2 and then increased to pH 7, the size was measured at 340.5 nm, a size similar to that of the pH 2 aggregates. These results indicate that APC can be prepared initially at pH 2 and then increased to pH 7, maintaining a similar size and net positive charge. This is consistent with the CD measurements, which indicate that the random coil secondary structure formed at low pH remains unchanged when capsules are formed at low pH and then increased in pH. The zeta potential at each pH value remained positive. The positive zeta potential indicates that the positive surface charge may be due to the exposure of positively charged lysine residues on the surface of the APC capsules.

[0059] Initial experiments to characterize APC focused on DLS, zeta potential measurements, and CD. Dynamic light scattering was used to measure hydrodynamic diameter, polydispersity, and surface charge (zeta potential). Circular dichroism was used to determine the secondary structure of APC at various pH values. At pH 2, APC was shown to form a random coil secondary structure (Figure 1). At pH 7 and 12, APC was shown to form a β-sheet structure. In TFE, the APC peptide was shown to form an α-helix. Notably, when the peptide was assembled at pH 2, increasing the pH to 7 did not induce a β-sheet secondary structure, but instead remained in a random coil secondary structure (Figure 1). This finding suggests that once self-assembly at low pH occurs, the secondary structure is fixed and resistant to change even when shifted to higher pH values.

[0060] Results were recorded for 1, 3, 4, and 5 minutes of KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) APC formation (Table 2). Even at the earliest time point of 1 minute, self-assembly appears complete. Polydispersity values ​​appear excellent, with capsule sizes just under 100 nm. The sizes for the 4 and 5 minute incubation times are artifacts due to small sample sizes. The zeta potentials for each sample were all shown to be positive, again indicating a positive surface charge due to surface-exposed lysine residues.

[0061] [Table 2]

[0062] In the next study, we used a wider pH range, from 2.00 to 4.56, and followed APC formation over two time periods (Tables 3 and 4). Up to pH 3.5, the two incubation times yielded APCs of similar size. Above pH 3.5, the hydrodynamic size began to increase, a characteristic of material beginning to aggregate. In the 5-minute incubation sample, the capsule size remained around 100 nm up to pH 3.51, at which point it dramatically decreased in size to 13 nm, indicating aggregation (Table 3). The zeta potential remained positive at all pH values, reaching a maximum of 14.3 mV at pH 2 and a minimum of 5.9 mV at pH 3.51. In the 20-minute incubation sample, the capsule size remained around 100 nm up to pH 3.51, at which point it dramatically decreased in size to 23 nm, indicating aggregation (Table 4). This size decrease suggests that aggregation may have begun at this pH. The zeta potential remained positive at all pH values, reaching a maximum of 14.4 mV at pH 2 and a minimum of 5.5 mV at pH 3.90. Overall, at each incubation time, APC appeared to show signs of aggregation at pH values ​​greater than 3.51. The zeta potential of APC was most positive at pH 2 and decreased as the pH increased. However, the zeta potential remained positive even at pH 4.56, indicating that the lysine residues on the exterior of the capsule were protonated.

[0063] [Table 3]

[0064] [Table 4]

[0065] Nanoparticle Tracking Analysis To examine the size and distribution of APC capsules in more detail, nanoparticle tracking analysis (NTA) was performed to show the size distribution of APCs formed at pH 2 and then raised to pH 7 after 5 minutes (Figure 2). APCs formed at pH 3 were also analyzed, yielding similar results to those formed at pH 2 (Figure 3). Analysis of these APCs showed that most particles varied in diameter between 50 and 200 nm, with very few exceeding 300 nm. The mean particle size of APCs formed at pH 2 was 139.1 nm with a standard error of 8.9 nm. The mean particle size of APCs formed at pH 3 was 240.1 nm with a standard error of 104.0 nm. These results are consistent with those obtained by DLS of the samples (Tables 1 and 3). Both NTA and DLS indicated that the size of APCs formed at pH 2 and raised to pH 7 was approximately 100-200 nm. Similarly, APCs formed at pH 3 showed similar results by both NTA and DLS, with sizes in the range of 200–300 nm.

[0066] APCs with genetic material encapsulated inside and attached to the outside of the capsules were also analyzed using NTA (Figures 4 and 5). NTA analysis of APCs encapsulated with PolyIC showed that the size ranged primarily between 200 and 400 nm, with very few particles larger or smaller (Figure 4). The average size of APCs encapsulated with PolyIC was 270.7 nm, with a standard error of 105.9 nm. NTA analysis of APCs with PolyIC attached to the outside of the particles showed that the size ranged between 100 and 300 nm, with very few particles larger or smaller (Figure 5). The average size of APCs with PolyIC attached to the outside was 246.3 nm, with a standard error of 23.1 nm.

[0067] Fluorescence spectroscopy and confocal microscopy confirmed that rhodamine-6G dye was successfully encapsulated using APCs prepared in dilute HCl at pH 2.0. Confocal microscopy images show several APCs containing rhodamine-6G dye (Figure 7).

[0068] The low number of capsules is due to the detection limit of confocal microscopy (1 micron), so smaller aggregates cannot be observed. Light diffraction studies below show that most of the capsules are much smaller. This result indicates that these peptides form water-filled capsules that can trap solutes.

[0069] Stability testing The stability of APC capsules over time was analyzed using DLS and zeta potential measurements to track changes in size and surface charge. Samples were stored at room temperature (22°C) or in a refrigerator at 4°C. Various samples, including APCs encapsulating GFP plasmid and APCs encapsulating GFP mRNA, were analyzed over time for 12 to 24 days. Daily samples were analyzed to determine size, polydispersity, and surface charge.

[0070] APC capsules containing GFP plasmid were stored at room temperature and analyzed over a two-week period. The initial size of the capsules was 389 nm (Table 5). The characteristics were tracked over a two-week period while the capsules were stored at room temperature. Each sample was read three times, and the results are the average values. Standard deviations for size and hydrodynamic diameter are included in the table.

[0071] The final capsule size measurement on day 13 was 411 nm, indicating that the initial and final measurements of capsule size remained relatively similar. However, the surface charge measurements were substantially different, with an initial measurement of 6.6 mV and a final measurement of 0.5 mV (Table 6). The properties were tracked over a period of one month and stored at room temperature. Each sample was read twice, and the results are the average of those readings. Standard deviations for size and hydrodynamic diameter are included in the tables.

[0072] [Table 5]

[0073] [Table 6]

[0074] The stability of APC capsules encapsulating GFP mRNA was investigated over a 24-day period at both room temperature (20°C) and 4°C (Tables 7 and 8). Properties were tracked for one month and then stored at 4°C. In Table 7, each sample was read twice, and the results are the average values, with standard deviations included for size and hydrodynamic diameter. In Table 8, each sample was read three times, and the results are the average values, with standard errors included for size and hydrodynamic diameter.

[0075] [Table 7]

[0076] [Table 8]

[0077] The size of the APC capsules started at approximately 108 nm for samples stored at room temperature. After 24 days of standing at room temperature, the size of the APC capsules reached 150 nm. Overall, the size of the APC capsules did not change substantially over time, but the surface charge changed dramatically over time. The initial measurement showed a surface charge of 19.7 mV, while the final measurement showed a surface charge of 1.9 mV. The surface charge of the samples at 4°C also fluctuated, starting at 15.9 mV and reaching a final measurement of -0.3 mV. These results indicate that APC may be stable at both 4°C and room temperature (20°C).

[0078] The decrease in surface charge over time, along with an increase in the polydispersity and hydrodynamic diameter of the aggregates, suggested aggregation. Because cellular uptake depends on both surface charge and diameter, these aggregates likely result in inefficient delivery and lack of intracellular cargo.

[0079] Stability studies of the Ac-KKKFLIVIKKK-CONH2 (SEQ ID NO: 6) sequence revealed that it was stable for only a few days when prepared and stored at room temperature or 4°C. This instability limits its application to samples prepared immediately before use. In an attempt to improve the stability of APCs, the longer Ac-KKKFLIVIGSIIKKK-CONH2 (SEQ ID NO: 7) sequence was tested. It was reasoned that a longer hydrophobic sequence would enhance stability. In Table 9, this longer sequence formed a stable APC when formed at pH 3.5. Assembly at pH 2.0 yielded mixed results, requiring a longer inoculation time such that the RNA was disrupted before complete encapsulation. At this pH, assembly occurred rapidly, within 1 minute.

[0080] [Table 9]

[0081] Assembly at pH 3.5 prevents autolysis of mRNAs21 during the encapsulation process. An incubation time of 1 min produced the smallest capsules. In all subsequent experiments, the incubation time before pH neutralization was 1 min.

[0082] The stability of capsules prepared with the larger Ac-KKKFLIVIGSIIKKK-CONH2 (SEQ ID NO: 7) sequence was examined at both room temperature and 4°C. Table 10 shows the stability at room temperature. Based on the observed polydispersity values, these capsules are stable for approximately 7 days. Although the size did not increase significantly, the hydrodynamic diameter began to increase after the 8th day.

[0083] [Table 10]

[0084] Each sample was read in triplicate using dynamic light scattering to determine size, hydrodynamic diameter, and polydispersity. Results are the average of these values, and the standard error of the mean for size and hydrodynamic diameter is included in the tables.

[0085] The stability of larger peptide capsules was also tested at 4°C (Table 11). Lowering the temperature extended the stability of these preparations by one week. The mean size was around 200 nm, and the polydispersity index remained in the mid-20s. The hydrodynamic range, with no significant standard error of the mean, was between 400 and 500 nm. Keeping these aggregates at a low temperature significantly improved their viability. Each sample was read three times using dynamic light scattering to determine size, hydrodynamic diameter, and polydispersity. Results are averages, and the standard error of the mean for size and hydrodynamic diameter is included in the table.

[0086] [Table 11]

[0087] To investigate factors that may contribute to the observed increased stability, circular dichroism studies were performed (Figure 8). Spectra recorded at both the assembly pH and the subsequent neutralization pH confirm a nearly identical random coil secondary structure. This result indicates that the increased stability of this preparation is due to the presence of additional hydrophobic residues in the sequence, which facilitate additional van der Waals interactions.

[0088] Transfection of GFP plasmid Confocal images in Figure 9 show the results of transfection of GFP plasmid with APC in the 1:20 outer (B) and 1:20 inner (C) conditions, as well as cells alone (A). These confocal images were taken with a confocal LSM700 laser scanning microscope and analyzed using Zen Blue photoediting. The DAPI-stained HEK cells alone condition showed no GFP autofluorescence (A). HEK cells treated with the 1:20 outer (B) and 1:20 inner (C) APC conditions both showed GFP expression (Figure 9). HEK cells in both the 1:20 outer (B) and 1:20 inner (C) conditions showed GFP expression (Figure 9). While both conditions showed GFP expression, HEK cell confluence was significantly higher in the 1:20 outer (B) condition than in the 1:20 inner (C) condition. Nevertheless, GFP expression in both conditions indicates that the APC capsules were able to enter HEK cells, deliver the GFP plasmid, and result in GFP expression.

[0089] The confocal images in Figure 10 show the results of GFP plasmid transfection in the 1:10 outer APC condition (B), the 1:10 inner APC condition (C), and cells alone (A). In the HEK cell-only condition, blue DAPI staining is evident, but no GFP fluorescence is observed. In the 1:10 outer APC condition (B), DAPI staining and GFP expression are evident (Figure 10). In the 1:10 inner APC condition (C), DAPI staining and GFP expression are evident (Figure 10). Compared to the 1:20 condition shown in Figure 9, the 1:10 condition in Figure 10 shows much higher GFP fluorescence expression in HEK cells. The HEK cell confluence in both the 1:10 outer and 1:10 inner conditions is high and relatively similar.

[0090] Both the 1:10 outer and inner conditions expressed GFP, indicating that HEK cells were able to express the GFP plasmid delivered via the APC capsules under these conditions. Both encapsulation of the GFP plasmid and attachment of the GFP plasmid to the outside of the capsules demonstrated the ability to deliver and express genetic material in HEK cells.

[0091] The confocal images in Figure 11 show the results of GFP plasmid transfection in the 1:5 outer (B) and 1:5 inner (C) APC conditions, as well as cells alone (A). The left column shows the results for the HEK cell-only condition stained with DAPI. In the 1:5 outer APC condition (B), cells are stained with DAPI and express GFP fluorescence. In the 1:5 inner condition (A), cells are stained with DAPI and express GFP fluorescence.

[0092] Transfection of GFP mRNA Confocal images in Figure 12 show the results of GFP mRNA transfection for the 1:20 outer APC condition (B) and the 1:20 inner APC condition (C), as well as cells alone (A). Similar to the results for the plasmid transfection, these images were taken with a confocal LSM700 laser scanning microscope and analyzed using Zen Blue photo editing software. In the HEK cell-only condition (A), stained with DAPI, no GFP fluorescence is expressed (Figure 12). In the 1:20 outer APC condition (B), GFP expression is visible, stained with DAPI (Figure 12). In the 1:20 outer condition (C), GFP expression is visible, stained with DAPI (Figure 12).

[0093] Confocal images in Figure 13 show the results of GFP mRNA transfection in the 1:10 outer APC condition (B) and the 1:10 inner APC condition (C), as well as HEK cells alone (A). In the HEK cell alone condition (A), cells are stained with DAPI and do not express GFP fluorescence (Figure 13). In the 1:10 outer condition (B), cells are stained with DAPI and some cells express GFP (Figure 13). In the 1:10 inner APC condition (C), cells are stained with DAPI and express GFP (Figure 13). Cells in the 1:10 inner condition are much more confluent than in the other two conditions, and therefore, a smaller percentage of cells express GFP fluorescence in the confocal images.

[0094] Confocal images in Figure 14 show the results of GFP mRNA transfection for the 1:10 outer APC condition (B), the 1:10 inner APC condition (C), and cells alone (A). In the HEK cell-only condition (A), cells were stained with DAPI and showed no GFP fluorescence (Figure 14). In the 1:10 outer condition (B), cells were stained with DAPI and some cells were seen to express GFP (Figure 14). In the 1:10 inner condition (C), cells were stained with DAPI and showed GFP expression (Figure 14). Cells in the 1:10 inner condition (C) were much more confluent than the other two conditions, and therefore, a smaller percentage of cells expressed GFP fluorescence in the confocal images. These results indicate that both the GFP mRNA encapsulated in the APC and the GFP mRNA attached to the outer surface were able to be taken up and expressed by HEK cells (Figures 12, 13, and 14).

[0095] RiboGreen assay The RiboGreen assay revealed that the difference between the encapsulated genetic material and the genetic material attached to the outside of the APC capsules was greatest at an equal ratio of 1:1. At this ratio, a significant difference was observed between the fluorescence measurements of the 1:1 outer APC and 1:1 inner APC (Figure 15). Because the RiboGreen reagent fluoresces strongly when exposed to genetic material, stronger fluorescence measurements for the 1:1 outer APC capsules indicate more genetic material was detected in the sample. For the 1:1 inner APC capsules, less genetic material was detected by the RiboGreen reagent, so lower fluorescence measurements indicate the potential for encapsulated genetic material. The results from this 1:1 condition suggest efficient internal packaging of dsRNA. Only minor differences were observed at genetic material-to-peptide ratios of 5:1 and 10:1. At these ratios, the outer condition had slightly higher fluorescence measurements than the inner condition. It is possible that some dsRNA remains on the surface after self-assembly, explaining why fluorescence is detected in each inner condition.

[0096] APC GFP mRNA transfection efficacy results Based on the confocal microscopy images shown in Figure 16, the optimal conditions for mRNA transfection using APC capsules are to prepare APCs at pH 2, wait 1 minute, and then raise the pH to 7 using imidazole buffer (B). These images show the highest GFP expression in HEK cells (B). The leftmost image in Figure 16 shows HEK cells stained with DAPI only; HEK cells not treated with APCs show no GFP expression (A). These images show the highest GFP expression in HEK cells. When APCs are prepared at pH 2, incubated for 5 minutes, and then the pH is raised to 7, no GFP expression is observed in HEK cells (C). HEK cells treated with GFP mRNA-containing APCs prepared at pH 2, incubated for 20 minutes, and then raised to pH 7 show GFP expression (D).

[0097] The 1-minute incubation time resulted in the highest GFP expression and the greatest confluence of HEK cells. These same incubation times were repeated using APCs at pH 3 and pH 3.5, but the results showed almost no GFP expression (Figures 17 and 18). Of these conditions, pH 3.5 and a 20-minute incubation showed the highest GFP expression. Overall, these results demonstrate that APCs can deliver and express GFP mRNA in HEK cells.

[0098] conclusion Using circular dichroism to assess the secondary structure of APC at various pH values, we found that APC capsules formed at pH 2 formed random coils. When initially formed at pH 2 and then increased to pH 7, APC maintained its random coil secondary structure. This finding suggests that once assembled at low pH, the secondary structure becomes fixed and less susceptible to change when shifted to higher pH values. Examining the effect of exposing APC to increasing concentrations of TFE, we found that APC is held together by hydrophobic interactions, which can be disrupted at TFE concentrations above 30% (Figure 19). Below this concentration, the secondary structure is random coil; however, increasing the TFE concentration to 30% switches the secondary structure to an α-helix. Most α-helices are monomeric, meaning that APC prepared at pH 2 disintegrated at these higher TFE concentrations. If APC can be opened using TFE, quantification of encapsulated solutes may be possible in future experiments. These self-assembled capsules are approximately 100-200 nm in size, as confirmed by both DLS and NTA analysis. The surface charge of the APC capsules is positive due to the hydrophilic lysine residues lining the exterior of the capsules, as shown in Figure 1A.

[0099] The cationic surface allows anionic molecules, such as DNA and RNA, to electrostatically attach to the exterior of APC capsules. APC capsules can also encapsulate anionic molecules. These capsules are readily internalized by cells, where they open and release the encapsulated solute. Surface-bound substances are also released into cells, indicating uptake of the encapsulated genetic material. In vitro cellular activity of APC capsules containing genetic material was confirmed by transfecting HEK cells with a GFP plasmid and GFP mRNA. These results suggest that it is possible to deliver two different types of molecules, one inside and one outside. An example of this would be the Crispr Cas9 enzyme inside and an RNA guide outside. Delivery of both into the same cell could facilitate gene editing.

[0100] Supplementary Data [Table 12]

[0101] [Table 13]

[0102] [Table 14]

[0103] After encapsulating the dye, the APCs were analyzed by DLS before and after drying and rehydration. The size, polydispersity, and zeta potential remained similar before and after drying and rehydration, all around 20 mV (Table 15). The size remained around 500 nm, and the zeta potential remained around 20 mV (Table 15).

[0104] [Table 15]

[0105] [Table 16]

[0106] [Table 17]

[0107] [Table 18]

[0108] As shown in Figure 1 for short peptide sequences, similar nanofibril assembly is observed for longer peptide sequences when "assembled" at neutral pH (about 7) or high pH (about 12).

[0109] APC Peptide Assembly Protocol 1. After synthesis and cleavage (and storage, if applicable), dissolve the dry peptide in 2,2,2-trifluoroethanol >99% (TFE). 2. The concentration of the peptide solution is determined by measuring the absorbance of phenylalanine at a wavelength of 257.5 nm with a CARY 50 Bio UV-visible spectrometer (Agilent Technologies, Santa Clara, Calif.). 3. The concentration of the peptide in the solution is then calculated using Beer's law. A=εcl where A = absorbance, ε = molar extinction coefficient, c = concentration, and l = path length. The absorbance was measured using a 0.3 cm long quartz cuvette, so the optical path length was 0.3 cm. The molar extinction coefficient of phenylalanine at 257.5 nm is 195 M -1 cm -1 was used in the calculation. The final concentration of peptide is typically prepared at 1.0-2.0 mg peptide per mL of TFE in an Eppendorf tube unless another concentration or amount is specified. 4. The solvent is then evaporated using a Speed-Vac vacuum system for 20 minutes. 5. Once the peptide drying step is complete, rehydrate the peptide with 20 μL of low pH buffer: 10 mM glycine-HCl buffer pH 2.0 (or 3.5). 6. Incubate the rehydrated peptide in low pH buffer at room temperature for 1-20 minutes. 7. Next, increase the pH of the solution to a neutral pH of 7.0 by adding 20 μL of 10 mM imidazole buffer at pH 7.0 along with HCl. The imidazole buffer is prepared by dissolving solid imidazole in water and then adjusting the pH to 7.0 by adding concentrated HCl dropwise. This solution is added to the newly formed APC at a low pH to adjust it to a neutral pH.

Claims

1. A peptide capsule configured to encapsulate a water-soluble active agent therein, the peptide capsule comprising a membrane having an outer surface and defining a liquid-receiving interior space, the membrane being comprised of a plurality of linear peptides, each peptide comprising a hydrophobic core of 4 to 12 hydrophobic amino acids flanked by N-terminal and C-terminal hydrophilic segments each comprising 3 to 4 hydrophilic amino acids.

2. 2. The peptide capsule of claim 1, wherein the capsule has a particle size of about 50 to about 500 nm.

3. The peptide capsule of claim 1 , wherein the outer surface has a positive surface charge.

4. The peptide capsule of claim 3 , wherein the outer surface has a zeta potential of at least about +15 mV.

5. 2. The peptide capsule of claim 1, wherein the active agent is selected from the group consisting of a nucleic acid, a fluorescent dye, and a water-soluble active ingredient (e.g., a therapeutic compound, a prophylactic compound, or a toxin / toxic compound).

6. The peptide capsule of claim 1, wherein the peptide is 20 amino acid residues or less in length.

7. 1. A method of forming a peptide capsule for encapsulating a water-soluble active agent, comprising: dispersing or dissolving a plurality of linear peptides in a low pH buffer system having a pH less than 5, optionally including an encapsulated active agent, to form a dispersion of peptides and active agent (if present), wherein said peptides each comprise a hydrophobic core of 4 to 12 hydrophobic amino acids flanked by N-terminal and C-terminal hydrophilic segments comprising 3 to 4 hydrophilic amino acids; incubating the dispersion for less than 20 minutes, wherein the peptides self-assemble into peptide capsules having an exterior surface and comprising a membrane defining a liquid-receiving interior space, the membrane consisting of a plurality of the peptides, and the active agent, if present, is entrapped in the liquid-receiving interior space of the capsule; and Raising the pH of the mixture to about 7 The method comprising:

8. 8. The method of claim 7, wherein the pH of the low pH buffer system is less than 4.

9. 8. The method of claim 7, wherein the low pH buffer system has a pH of about 2 to about 3.

5.

10. 8. The method of claim 7, wherein the low pH buffer system comprises glycine HCl or a mixture thereof in water.

11. 8. The method of claim 7, wherein the pH of the mixture is raised to about 7 using a neutral or alkaline buffer, preferably comprising imidazole-HCl, in case of alkaline conditions, NaOH or mixtures thereof in water.

12. 8. The method of claim 7, wherein the capsules form during the incubating step within 5 minutes of forming the dispersion.

13. 8. The method of claim 7, further comprising drying the capsule to a dry powder or a lyophilized powder.

14. 14. The method of claim 13, further comprising rehydrating the dry or lyophilized powder by mixing and suspending it in a neutral buffer.

15. 1. A method for delivering a water-soluble active agent to an organism, comprising administering or applying to said organism a composition comprising a plurality of peptide capsules, each peptide capsule having an outer surface and a membrane defining a liquid-receiving interior space, wherein the water-soluble active agent is encapsulated therein, and wherein the membrane is comprised of a plurality of linear peptides, each peptide comprising a hydrophobic core of 4 to 12 hydrophobic amino acids flanked by N-terminal and C-terminal hydrophilic segments, each comprising 3 to 4 hydrophilic amino acids.

16. 16. The method of claim 15, wherein the organism is a plant and the administering or applying comprises applying the composition to the leaves or roots of the plant, or applying the composition to the soil or growth medium.

17. 16. The method of claim 15, wherein the organism is a human or non-human animal, and the administering or applying comprises topically applying the composition to the organism, administering the composition directly to the organism orally or by injection, or introducing the composition into an area where the organism will come into contact with the composition.

18. The method of claim 15, wherein the capsule is taken up intracellularly by the organism after the administration or application.

19. 20. The method of claim 18, wherein the active agent is delivered intracellularly.

20. 19. The method of claim 18, wherein the peptide capsule membrane is degraded by endogenous intracellular mechanisms of the organism to release the active agent.

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