Confining amphiphilic peptide colloids

Abstract

Peptide-based colloidal particles capable of encapsulating and stabilizing non-polar excipients, including lipids that are solid at room temperature, and hydrophobic and / or poorly water-soluble active agents for storage and delivery in aqueous media, methods of making and using said peptide-based colloidal particles, methods of customizing and sizing such colloids, and peptide-based colloidal particles as delivery vehicles for a variety of hydrophobic and / or poorly water-soluble active agents.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 335,847, filed April 28, 2022, entitled “CORRALLING AMPHIPATHIC PEPTIDE COLLOIDS,” the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing The following application contains a sequence listing that has been submitted electronically as a standard ST.26 compliant XML file entitled "SequenceListing57229.xml", created on April 27, 2023 as 40,960 bytes. The contents of the XML file are incorporated herein by reference. [Technical field]

[0003] FIELD OF THEINVENTION The present invention relates to formulations and methods for stabilizing non-polar compounds and excipients for delivery in aqueous systems. [Background technology]

[0004] Description of the Prior Art There has been a continuous search for a method and composition that can effectively and efficiently deliver lipid or oil, as well as hydrophobic active agent, into aqueous media.The conventional approach to prepare formulations containing hydrophobic active agents has many shortcomings, including large structures that aggregate and eventually cause complete phase separation.Due to their physical instability and lack of homogeneity, these formulations also suffer from poor and variable cell absorption.There is still a need for improved techniques to formulate and deliver hydrophobic or poorly water-soluble active agents. Summary of the Invention

[0005] The present disclosure generally relates to a composition of colloid particles, each of which comprises a peptide layer that encapsulates droplets of non-polar excipients (e.g., lipids, oils, greases, or non-polar solvents, etc.), optionally with one or more hydrophobic and / or poorly water-soluble active agents dispersed or distributed therein. Described herein are methods for controlled sizing and resizing of the colloid particles, as well as methods for freeze-drying and rehydrating the colloid particles. Described herein are new peptide sequences for preparing the colloid particles, as well as new techniques for encapsulating and stabilizing lipids, oils, and fats that are solid at room temperature (e.g., such lipids that are in a solid state at room temperature and body temperature, usually long-chain triglycerides or partial glycerides, etc.) in the colloid particles.

[0006] Also described herein are methods for delivering a hydrophobic and / or poorly soluble active agent to a subject in need thereof, comprising administering to the subject a composition according to various embodiments described herein.

[0007] The present application also relates to a method for delivering a hydrophobic and / or poorly soluble active agent to a plant, the method comprising applying a composition according to various embodiments described herein to at least a portion of the plant and / or to the soil in which the plant is or will be planted. In some embodiments, the active agent is applied to the plant and / or to the soil in which the plant is or will be planted, for the purpose of delivering the active agent to a pest.

[0008] Also described herein is a method for delivering a hydrophobic and / or poorly soluble active agent, particularly an insecticide, to an insect, the method comprising contacting the insect with a composition according to various embodiments described herein. [Brief description of the drawings]

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1] FIG. 1 is a schematic illustration of a colloidal particle according to one embodiment of the present invention, including a close-up of a peptide monolayer coating and stabilizing a non-polar excipient droplet. [Diagram 2] FIG. 2 shows the circular dichroism (CD) spectra of the CAPC peptide encapsulating different solvents or oils. [Diagram 3] Figure 3 shows the CD spectra of different CAPC peptide sequences used to encapsulate soybean oil. The C- and N-terminal protecting groups are not included in the legend. [Figure 4] FIG. 4 shows mass spectra from tryptic digestion of free and aggregated CAPC peptides: A. intact control CAPC, B. CAPC after 1 hour, and C. free aggregated CAPC peptide digested for 1 hour. [Figure 5A] Figure 5A shows a graph of the NTA analysis of the colloidal particle mixture immediately after sonication. The traces to the right of the distribution curve are the individual data sets generated for the readings recorded by the instrument. [Figure 5B] Figure 5B shows a graph of the NTA analysis of the sized colloidal particle mixture. The traces to the right of the distribution curve are the individual data sets generated for the readings recorded by the instrument. [Figure 6] FIG. 6 is a bar graph of particle size and temperature stability of CAPC colloidal particles containing coconut oil prepared at 37° C. (n=3). [Figure 7] FIG. 7 is a graph of the differential scanning calorimetry analysis: peptide prepared without oil (h5L, smaller dashes), peptide prepared with coconut oil (CCO, larger dashes), and CAPC colloidal particles prepared using the same peptide sequence together with coconut oil (CAPC, solid line). [Figure 8]FIG. 8 shows confocal microscopy images of A. green CF-labeled peptide in water (reference bar, 10 microns), B. soybean oil containing Nile Red (reference bar, 20 microns), and C. CAPC colloidal particles forming a green monolayer containing soybean oil with Nile Red (reference bar, 5 microns). [Figure 9] FIG. 9 is a transmission electron microscope (TEM) image of resized methylmercury-labeled CAPC. [Figure 10] Figure 10 shows confocal microscopy images of dried / rehydrated CF-labeled CAPC encapsulating soybean oil containing Nile Red: A. Image at wavelength showing only the CF-labeled peptide, B. Image at wavelength detecting soybean oil containing Nile Red, and C. Image of the merged image. Reference bar is set at 2 microns. [Figure 11] Figure 11 shows imaging of in vitro cellular uptake by CHO cells of CF-CAPC colloidal particles containing soybean oil with Nile Red. The bottom left image is a bright field image, the bottom center image is a merged photograph showing all of the different fluorescent components. The bottom right image is a reference blank. [Figure 12] FIG. 12 is a graph showing the effect of sonication time on colloidal particle size and distribution density. [Figure 13] FIG. 13 is a graph showing the results of oral administration of CBD nanoformulation CAPC (NANO) or MCT oil (MCT) as a control to dogs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description The present disclosure relates to peptide-based colloidal particles that can encapsulate and stabilize non-polar compounds for storage and delivery in aqueous media.For example, the peptides can be used to encapsulate and stabilize droplets or particles of lipids, oils, greases, fats and non-polar solvents in a hydrophobic core surrounded by a peptide monolayer.The hydrophobic core advantageously sequesters non-polar excipients (such as lipids, oils or non-polar solvents) into discrete colloidal particles that can remain stably suspended or dispersed in aqueous media, optionally with one or more hydrophobic and / or poorly soluble active agents dispersed or distributed therein.

[0011] More specifically, we disclose improvements in the use of linear peptides that can encapsulate or form a coating around droplets or particles of lipid oils and other hydrophobic or poorly (water) soluble active ingredients, allowing them to be dispersed as stable colloidal particles suspended in water. The cationic outer surface of these particles is hydrophilic, allowing them to be dispersed in aqueous solutions and also promoting uptake by animal and plant cells and tissues, thereby facilitating delivery of lipid soluble active ingredients to the interior of cells. Other carriers, adjuvants, synergists, dispersants or solutions may also be included within / with the particles. These particles also shield the active agent from the external environment that may prematurely inactivate the active agent. As drug delivery vehicles, these novel colloidal particles can also be used to alter the biological half-life of the active ingredient.

[0012] The present invention generally relates to a composition comprising a plurality of colloidal particles suspended in an aqueous carrier. Each of these colloidal particles comprises a peptide layer or coating having a cationic, hydrophilic outer surface in which a non-polar excipient (e.g., lipid, oil, or non-polar solvent, etc.) is sequestered, optionally together with one or more hydrophobic and / or poorly soluble active agents dispersed or distributed therein. The particles are characterized by a cationic, hydrophilic outer surface formed from hydrophilic segments at the C-terminus of the peptides that are oriented outwardly towards the external environment in each particle. The inwardly facing surface of the peptide layer is hydrophobic, formed from hydrophobic segments at the N-terminus of the peptides that are oriented towards the internal hydrophobic core of the particle. Preferably, the peptide layer or coating is homogeneous, meaning that the peptide layer or coating is composed of a plurality of peptides of the same type (i.e., peptides having the same amino acid sequence).

[0013] The peptide sequences used to prepare the peptide coating or layer are amphiphilic and linear without branch points and comprise (or consist essentially of) an N-terminal hydrophobic segment (first end) and a C-terminal hydrophilic segment (second end) or consist of an N-terminal hydrophobic segment (first end) and a C-terminal hydrophilic segment (second end). The peptides preferably have a molecular weight in the range of about 550 Da to about 2300 Da, more preferably in the range of about 675 Da to about 2050 Da, and even more preferably in the range of about 800 Da to about 1800 Da. The "molecular weight" of these peptides is the average weight calculated based on the total molecular weight (MW) of the actual bound amino acids present divided by the number of residues. Linear peptides have lengths ranging up to 20 amino acid residues in total, preferably from about 5 to about 20 residues, more preferably from about 8 to about 15 residues, and even more preferably from about 8 to about 12 residues. Peptides can be synthesized using conventional Fmoc chemistry.

[0014] The N-terminal hydrophobic head groups are preferably each about 3 to about 11 residues in length, more preferably about 4 to about 10 residues in length, and even more preferably about 5 to about 9 residues in length. The amino acids used for the N-terminal hydrophobic segment are preferably selected from hydrophobic or very hydrophobic residues, such as leucine, isoleucine, valine, phenylalanine, and methionine. In one or more embodiments, the N-terminal hydrophobic segment may contain up to two neutral amino acid residues selected from glycine, serine, and / or threonine. In one or more embodiments, the N-terminal hydrophobic segment does not contain an alanine residue. Particularly preferred hydrophobic amino acids for use in the hydrophobic segment include phenylalanine, leucine, isoleucine, and valine. 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 in these formulas, the amino acid residues can be in this order or in any order (scrambled, see e.g., SEQ ID NOs:14-32). In one or more embodiments, the N-terminal hydrophobic segment comprises the sequence X(LIVI) (SEQ ID NO:1), X(LIVI)GSII (SEQ ID NO:2), XFF(IVI)L (SEQ ID NO:2), or X(LIVI)GSIIVIL (SEQ ID NO:4), where X is F or V, and in these formulas, the residues in brackets can be in this order or in any order (scrambled). In one or more embodiments, the residues in brackets are replaced with all I residues or all V residues. In one or more embodiments, any one of the residues in the sequences XLIVI (SEQ ID NO:1), FLIVIGSII (SEQ ID NO:2), VFFIVIL (SEQ ID NO:3), or FLIVIGSIVIL (SEQ ID NO:4), except for the N-terminal phenylalanine, can be replaced by I or V.

[0015] The C-terminal hydrophilic (polar) tail segment preferably comprises from about 1 to about 7 hydrophilic and cationic amino acid residues (respectively), preferably comprising lysine, but may also comprise histidine, arginine, aspartic acid, or glutamic acid, which also have electrically charged side chains. In one or more embodiments, the hydrophilic tail segment does not comprise any arginine residues (preferably, the entire peptide does not comprise any arginine residues). More preferably, the C-terminal hydrophilic tail consists of lysine residues, more preferably from about 1 to about 6 lysine residues, and even more preferably from about 1 to about 5 lysine residues. A particularly preferred lysine sequence is KKKKK (SEQ ID NO: 5).

[0016] In one or more embodiments, the peptide includes an added cysteine ​​residue at the C-terminus of the peptide, preferably linked at a terminal lysine position, to facilitate further functionalization. In some embodiments, the N-terminus of each hydrophobic segment can be capped with an acetyl group (Ac).

[0017] Exemplary peptides are also described in International Patent Application PCT / US2020 / 023891 (filed March 20, 2020, published as International Publication WO2020 / 198020, which is incorporated herein by reference in its entirety). Now termed Corralling Amphipathic Peptide Colloids (CAPC), these linear peptides do not form bilayers or micelles, but rather, when mixed with hydrophobic compositions, transform lipid solutions into colloids with encapsulated lipid droplets or particles with cationic surfaces that are easily taken up by cells. Hydrophobic active ingredients that are soluble in lipids, oils, and non-polar solvents can be delivered using these colloids. Moreover, these peptides can capture nearly 100% of the active ingredient dissolved in the hydrophobic phase.

[0018] In one or more embodiments, functional groups and / or various moieties can be added to the C-terminal lysine or C-terminal carboxyl groups, or to the free sulfhydryl groups in the case of C-terminal cysteine. In this way, the peptides can be modified with various targeting moieties, which will be located on the outside of the colloid and can be used for targeting, detectable labeling (e.g., fluorescent labeling), and the like. For example, the peptides can be iodized for targeting. The term "functional moiety" is used herein to encompass functional groups, targeting moieties, and active agents that may be added to the outer surface of the particle. Exemplary functional moieties that can be added 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 be similarly added in a chain of consecutive order from the C-terminus, using an aliphatic spacer to separate the different moieties. Thus, the present invention provides an opportunity to create multifunctional colloidal particles. Since the individually modified peptides self-assemble to form a matrix, any number of functional moieties of different stoichiometries can be added to the individual peptide sequences that make up part of the assembled colloidal particle.

[0019] FIG. 1 provides an illustration of a colloid particle according to one embodiment of the present invention. In the figure, a hydrophobic droplet or particle is encapsulated or enveloped by a peptide layer. As shown in the enlarged view, the squiggly lines represent amphipathic peptides. The peptides form a peptide membrane or layer with their cationic hydrophilic residues facing the aqueous exterior environment and their hydrophobic residues extending towards the interior / core of the colloid, interacting with lipid, oil or non-polar solvent molecules such that the peptides assemble to form a monolayer at the oil-water interface, thereby encapsulating the lipid, oil or non-polar solvent within the discrete particle. Importantly, however, the peptides themselves are not conjugated (conjugated) or otherwise bound to the active agent or hydrophobic core material.

[0020] However, advantageously, the peptide layer interacts with the hydrophobic droplets to form a monolayer that stabilizes the encapsulated hydrophobes, so that the colloid particles are stable as discrete colloid particles in aqueous solutions for extended periods of time (preferably at least 3 months, more preferably at least 6 months, and even more preferably at least 12 months) without aggregation, coalescence, or collapse. Advantageously, the colloid particles are stable in suspension and do not coalesce into larger aggregates or larger colloid particles over time. This is referred to herein as the "shelf life" or "storage stability" of the colloids. In particular, this report details the advantageous storage stability of the colloids. For example, the colloids formed exhibit storage stability at ambient conditions when stored in aqueous solutions (e.g., water) for over 400 days.

[0021] Specific improvements to the techniques reported herein include controlled size distribution of colloids, as well as techniques for sizing colloids. For example, as reported herein, the size of colloids can be adjusted by changing the temperature of the reaction solution, e.g., the average size of colloids shrinks at lower temperatures.

[0022] Approaches for freeze-drying or spray-drying of colloids followed by rehydration have also been described, confirming that the hydrophobic entities remain encapsulated in the peptide monolayer and do not break apart during the freeze-drying period, and that the freeze-dried powder can be reconstituted via hydration in aqueous solvents.

[0023] Colloidal particles are prepared by mixing lipids, oils, fats, greases or non-polar solvents (i.e., excipients) with the peptide in a reaction vessel. In one or more embodiments, the active agent is first dispersed or dissolved in a hydrophobic bulk excipient. Preferred lipids, fats and oils are vegetable oils (coconut, soybean, avocado, etc.), mineral oil, migroil oil, paraffin oil, Solutol®, and the like, or combinations thereof. The oil may itself be an active form or may contain an active form. Preferred non-polar or low dielectric solvents (i.e., solvents with low dielectric constants) include any solvent that is essentially immiscible with water. Water has a dielectric constant of about 78.2 at room temperature (about 25° C.). Exemplary solvents include those with a dielectric constant of less than 50, preferably less than 30, at room temperature (about 25° C.). Examples include alkanes (pentane, hexane, heptane and n-decane), cycloalkanes (cyclohexane), diethyl ether, carbon tetrachloride, methylene chloride, aromatics (benzene, toluene and xylene), phthalates (diethyl phthalate), piperonyl butoxide, and the like, or combinations thereof.

[0024] Preferably, the active agent, if present, is first dissolved, suspended or dispersed in the bulk excipient. The peptide is added in an amount sufficient to encapsulate all of the excipients present, mixed with the excipients, and then allowed to stand for at least about 15 minutes, preferably about 15 minutes to about 30 minutes. In one or more embodiments, the peptide is added at a concentration of about 0.5 mM to about 5 mM, preferably about 1 mM to about 3 mM. In one or more embodiments, the weight ratio of peptide to excipient is about 1:50 to about 1:20, preferably about 1:25 to about 1:10. Water (preferably distilled / deionized water) or other aqueous solvent system is then added, preferably in excess, and the resulting emulsion (otherwise immiscible excipients) is mixed or stirred to distribute or suspend evenly in the aqueous solvent system. More preferably, the mixture is mixed or stirred using a vortex mixer or bath sonicator for at least about 5 minutes, preferably about 5 minutes to about 15 minutes. The homogenous or uniformly mixed composition becomes somewhat turbid as colloids form, as the peptides encapsulate the excipient particles or droplets and stabilize them within the aqueous solvent system, so that they become suspended and distributed throughout the aqueous solvent system. Upon centrifugation, the colloids move to the top of the water column, and notably, there is no longer an oil layer visible. As shown in Figure 1, the hydrophobic amino acids in the peptide sequence point towards the interior of the colloid and interact with the bulk excipient droplets that are enveloped by the peptide monolayer.

[0025] Moreover, as described herein, colloids can be advantageously used to encapsulate lipids, fats, oils and oils that are solid at room temperature. For example, peptides can be dispersed in a solution containing lipids, fats, oils or oils under conditions above their melting temperature, resulting in the melted lipids, fats, oils or oils being in a liquid state. Once colloids are formed around the melted lipids, fats, oils or oils, the colloidal suspension can be returned to room temperature, and the encapsulated lipids, fats, oils or oils are solidified with a peptide coating or layer that stabilizes and protects the lipid, fat, oil or oil payload. Examples of such high melting lipids include coconut oil and glycerol, triglycerides (tristearin), partial glycerides (Imwitor), fatty acids (stearic acid, palmitic acid), steroids (cholesterol), and waxes (cetyl palmitate) based on stearic acid.

[0026] In one or more embodiments, the colloidal particles have a maximum surface-to-surface dimension (e.g., diameter of a substantially spherical particle) greater than about 25 nm, preferably from about 100 nm to about 5 microns, more preferably from about 200 nm to about 1,000 nm. For ease of reference, the terms "diameter" or "particle size" are used interchangeably herein to refer to the maximum surface-to-surface dimension of each particle. Moreover, because the methods of the present invention produce suspensions of a plurality of particles, "particle size" as referred to herein may refer to the average (arithmetic mean) diameter of the entire population of particles in the suspension.

[0027] The particles can be resized (or reduced in size) if desired, for example, by extruding the composition through any combination of filters, small diameter conduits (e.g., hollow syringes, etc.) and the like, with the desired size of opening (opening bore) size or pore size. For example, to improve cellular uptake, particles of 200 nm or less in size are generally desired. Colloid particles can be resized, for example, by passing through a combination of syringes and / or filters, which causes larger colloid particles to be vesiculated or pinched into smaller colloid particles. In other words, the larger colloid particles are broken down into smaller particles, so that the suspension before resizing has the same volume, but contains more particles after resizing. As discussed herein, sizing can also be controlled by reducing the temperature to about 4° C. during the manufacturing process to reduce the size of the colloid particles formed. However, it will also be understood that larger sized colloids are useful for electrostatically binding and delivering much larger oligonucleotides, up to 10,000 bases, to their surfaces.

[0028] Advantageously, the colloidal particles have low polydispersity, with a PDI of less than 250%, preferably less than 100%, more preferably less than 50%, even more preferably less than 40%, and even more preferably from about 2% to about 30%. Another important aspect of the design of colloidal particles is the cationic nature of the surface exposed to the solvent. The colloidal particles have a zeta potential of about 1 mV to about 400 mV, preferably from about 20 mV to about 100 mV.

[0029] One concern for the pharmaceutical industry is the perception that many pipeline "investigational new drugs" are not readily soluble in water, thereby reducing their bioavailability. This hurdle is often insurmountable, preventing potentially effective drugs from progressing through clinical trials. The technology described herein addresses this problem by packaging hydrophobic molecules into submicron colloids.

[0030] Advantageously, colloidal particles can be prepared for targeting of specific cell surface receptors through the addition of different molecules or functional groups (such as cholesterol, mannose, TAT peptide, insulin, biotin, nucleotides, or any other suitable known surface targeting molecules, and combinations thereof) to the C-terminal lysine. Colloidal particles with such targeting moieties attached to their outer surface can therefore be localized and selectively taken up by specific cells or tissues of the patient. Thus, colloidal particles can be used for targeted therapy (gene therapy, cancer treatment, etc.) and nano drug delivery by administering colloidal particles with targeting moieties to a patient. The targeting moiety is added to the hydrophilic component of the peptide used to form the colloidal particle: in this case, the hydrophilic component mainly occupies the outer layer of the particle, and thus the targeting moiety is presented on the outer surface of the colloidal particle after formation. This moiety is recognized by the target area or tissue in the patient, and the colloidal particle automatically localizes in that area or tissue. Targeting these structures to specific cell types can reduce the amount of active ingredient required, as well as limit off-target effects.

[0031] Colloidal particles find application in the cellular delivery of poorly (water) soluble compounds / drugs that are currently too hydrophobic to be delivered effectively. As used herein, a "poorly soluble" active agent refers to compounds and substances that have low solubility in aqueous solvent systems (e.g., less than 1 milligram per mL of agent at neutral pH in physiological buffer (37±1° C.)), as opposed to agents that can be fully dispersed or dissolved in aqueous systems. This allows small molecules with poor solubility and poor cell permeability to become promising drug candidates. There are numerous synthetic and vegetable oils that can preferentially solubilize hydrophobic molecules. The aforementioned technology is useful, for example, for preparing insecticides, fungicides, antiparasitic agents, anticancer drugs, and for improving the bioavailability of many lipid-soluble active ingredients. In particular, the technology is particularly suitable as a delivery vehicle for formulating BCS class II (low solubility, high permeability) and BCS class IV (low solubility, low permeability) drugs that have poor solubility and poor bioavailability. The Biopharmaceutic Classification System (BCS) is a scientific approach based on the aqueous solubility and intestinal permeability characteristics of a drug substance(s).

[0032] Colloid particles can be used in a pharma- ceutically acceptable composition for delivering colloid particles to a subject. In one or more embodiments, the composition comprises a therapeutically effective amount of colloid particles dispersed in a pharma- ceutically acceptable carrier. As used herein, a "therapeutically effective" amount refers to an amount of colloid particles that induces a biological or medical response in a tissue, system, animal, or human, as sought by a researcher or clinician, in particular, that induces some desired therapeutic effect. Those skilled in the art will recognize that an amount may be considered therapeutically effective even if the condition is only partially improved, rather than completely eradicated. As used herein, the term "pharma-ceutically acceptable" means that it is not biologically or otherwise undesirable in that it can be administered to a subject, cell, or tissue without undue toxicity, irritation, or allergic response, and does not produce any undesirable biological effects or adversely interact with any of the other parts of the composition in which it is contained. Pharmaceutically acceptable carriers are naturally selected to minimize degradation of colloid particles, functional groups, or active agents, and to minimize adverse side effects in the subject, cell, or tissue, as is well known to those skilled in the art. Pharmaceutically acceptable ingredients include those that are acceptable for veterinary use, as well as for human pharmaceutical use. Exemplary carriers and excipients include aqueous solutions, such as normal (n.) saline (about 0.9% NaCl), phosphate buffered saline (PBS), and / or sterile water (DAW), oil-in-water or water-in-oil emulsions, and the like.

[0033] Also described herein is a method for targeted delivery of an active agent to a region of a patient, the method comprising administering to the patient colloidal particles as described herein, the colloidal particles comprising a targeting moiety on an outer surface thereof, the moiety being recognized by a target region or tissue of the patient, and the colloidal particles automatically localizing to the region or tissue. The colloidal particles can be injected directly into the target tissue or can be administered systemically.

[0034] Colloid particles are taken up by cells via the endocytic pathway and then metabolized to release their payload / contents and / or any surface conjugates. In particular, the cationic surface of colloid particles allows them to be taken up by the cell membrane to form early endosomes. Colloid particles begin to degrade in late endosomes, releasing their contents into the perinuclear cytosol. Furthermore, the new pH of this intracellular environment results in a decrease in electrostatic attraction, releasing the surface payload, if present. In this way, surface-bound nucleic acids and any encapsulated active agents are released from the colloid particles into the cytosol.

[0035] Colloidal particles have been shown to effectively deliver nucleic acids, with time-dependent release of the nucleic acids. Colloidal particles can also be used for surface attachment of proteins, peptides, plasmids, and nucleic acids, including CRISPR-Cas9 components for delivery. Colloidal particles can also be used to encapsulate a wide variety of active agents, including small molecules, fat-soluble vitamins, pheromones, and fatty acids.

[0036] Also described herein is a method for delivering active agents to plants, such as the leaves, stems, roots, or other tissues or cells of plants.The method can be used to deliver various active agents, including for treating and / or preventing pests, diseases, infections, and the like.The method includes applying colloidal particles to at least a part of the plant and / or the soil in which the plant is or will be planted.

[0037] Thus, also contemplated herein is a method for delivering an active agent to a plant, animal or human. The method includes administering a plurality of colloids containing an active ingredient to a plant, animal or human. This can include directly applying or administering the colloid, or providing the colloid in the vicinity of the target. For example, the colloid can be applied directly to the leaf system or root system of the plant, or to the soil around the root. Similarly, the colloid can be administered topically, orally, or directly by injection into the animal, or it can be introduced indirectly, for example, into an aquaculture / water system where the animal is present, or where the animal may come into contact (e.g., near a beehive, etc.). The colloid can be incorporated into a suitable pharmaceutical, horticultural or veterinary composition, including a carrier, diluent, excipient or vehicle suitable for administration.

[0038] Also described herein is a method of delivering an active agent (e.g., an insecticide) to an insect by contacting the insect with colloid particles carrying the active agent. The method can include applying the colloid particles to the leaves, stems, roots, or other tissues or cells of a plant, or otherwise disposing the colloid particles where the insect / pest will come into contact with the colloid particles. In some embodiments, the colloid particles can be ingested by the insect. In some embodiments, the colloid particles can be provided in an insect bait along with an edible insect attractant (sugar, carbohydrate, yeast, fat, oil, protein). The bait can be in the form of a liquid, gel, or solid tablet or granule.

[0039] The technology described herein can be used to deliver a wide variety of active agents, including but not limited to, imaging agents, detectable dyes, fungicides, anticancer agents, insecticides, herbicides, metabolic inhibitors, and the like.

[0040] In some embodiments, the peptide, colloid 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 administration for human, plant or animal use. Each unit dosage form may contain a predetermined amount of the peptide, colloid or composition in a suitable carrier calculated to produce a desired effect. In one or more embodiments, the kit includes a lyophilized colloid, where the preformed colloid is lyophilized or spray dried, together with instructions for reconstituting the lyophilized colloid for use. In one or more embodiments, the kit includes a peptide, colloid or composition together with instructions for preparing the colloid or composition and administering the composition to a subject.

[0041] It is understood that the therapeutic and prophylactic methods described herein can be applied to humans, as well as any suitable animals, including, but not limited to, dogs, cats and other pets, as well as rodents, primates, horses, cows, pigs, etc. These methods can also be applied for clinical research and / or investigation. This platform technology is also useful in plants, for example, to target pathogens in plant systems, or to deliver various active agents to plant tissues or their pests or pathogens in other cases.

[0042] Further advantages of various embodiments of the present invention will be apparent to those skilled in the art when considering the disclosure herein and the examples below.It is understood that the various embodiments described herein are not necessarily mutually exclusive, unless otherwise indicated herein.For example, the features described or depicted in one embodiment may also be included in other embodiments, but are not necessarily included in other embodiments.Therefore, the present invention encompasses various combinations and / or integrations of the specific embodiments described herein.

[0043] As used herein, the word "and / or," when used in a list of two or more items, means any one of the listed items may be used alone or in any combination of two or more of the listed items. For example, if a composition is described as containing or excluding component A, component B, and / or component C, the composition may contain or exclude A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0044] Numerical ranges are also 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 interpreted as providing literal support for the limitations of claims that only recite the lower limit of the range, as well as for the limitations of claims that only recite the upper limit of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for claims that recite "greater than about 10" (without an upper limit) and claims that recite "less than about 100" (without a lower limit). EXAMPLES

[0045] The following examples describe methods according to the present invention, however, it should be understood that these examples are provided by way of illustration and that nothing in the examples should be construed as a limitation on the overall scope of the invention.

[0046] Example 1 Linear amphiphilic oligopeptides stably encapsulate solutes dissolved in low dielectric oils / solvents dispersed in water

[0047] summary This report presents amphipathic peptides that stably encapsulate droplets of oil and low dielectric solvents in water. The amphipathic peptides produce monodisperse, approximately 20-2000 nm colloidal particles that can encapsulate and resize these liquids. They are stable over long periods of time and in the temperature range of 4-90°C. The cationic colloids have a zeta potential of -6.1 mV to +50 mV. The peptides remain unstructured, with the lysyl residues fully exposed to the solvent. The encapsulated coconut oil retains its major phase transitions, indicating that the oil remains in a liquid state. Finally, these inclusions are rapidly taken up by cells in culture, suggesting that these oil-filled colloids may be applicable for the delivery of hydrophobic therapeutic agents.

[0048] material and method Peptide synthesis Test peptides based on -Ac-FLIVI-KKKKK-CO-NH2 (SEQ ID NO:6) were synthesized using standard synthesis. Amino acids (F, I, L, V, A) along with HOAt and HATU were obtained from P3 Biosystems (Loiusville, KY), and amino acid (K) was obtained from AnaSpec Inc. (Fremiont, CA). DMF was obtained from (Thermo-Fisher, Waltham, MA), N-methyl-2-pyrrolidone from (Sigma-Aldrich Corp., St. Louis, MO), piperidine from (American BioAnalytical, Canton, MA), and CLEAR® amide resin from (Peptides International, Louisville, KY).

[0049] The peptide was cleaved in 1 M HCl (Thermo-Fisher, Waltham, MA) in 1,1,1,3,3,3-hexafluoroisopropyl alcohol (HFIPA) (Sigma-Aldrich Corp., ST Louis, MO) for 4 hours at room temperature. After cleavage, the peptide was dried under vacuum. The calculated mass of the peptide was confirmed by mass spectrometry. 5(6)-carboxyfluorescein (5(6)-CF) labeled peptide was prepared by deprotecting the attached Boc-protected lysyl-epsilon-amino group with 20% TFA in dichloromethane (DCM) for 20 minutes. After washing the deprotected peptide with DCM, 5(6)-carboxyfluorescein was added (at a concentration of 1 part to 5 parts lysine) in the presence of HOAT-HATU in DMF for 20 minutes. The reaction was stopped by filtering the dye followed by washing with more DCM. The material was cleaved from the resin and analyzed as described above. MALDI-mass spectral analysis showed that the products from this synthesis by partial coupling of CF were unlabeled peptide and monosubstituted peptide.

[0050] Encapsulation studies- 50 μL of winterized soybean oil (Archer Daniel Midlands, Chicago, IL) was added to 2 mg of dried peptide and vortexed for 2 min. Deionized distilled-reverse osmosis (DDI-RO) water was added to a final volume of 1 mL. The mixture was sonicated (bath sonicator) for 15 min at 37°C. The resulting CAPCs were kept at room temperature for at least 1 h prior to confocal microscopy. Reagent grades for solvent / oil encapsulation studies were benzene (Sigma-Aldrich, St. Louis, MO), cyclohexane (Acros Organics, Geel, Belgium), n-decane (grade 99+%, Sigma-Aldrich, St. Louis), Migloyl® 812N; capric triglyceride (Pharma Grade, IOI Oleo GmbH, Witten, Germany), and mineral oil paraffin (USP, Thermo-Fisher Scientific, Waltham, MA), and wax-free soybean oil (Archer Daniel Midlands, Chicago, IL). The CAPCs were prepared using 50 μL of solvent / oil with 480 μL of DDI-RO water and sonicated for 15 minutes. 2 mg of CAPC was subsequently added and sonicated for 10 minutes. The samples were measured for size, zeta potential, and their circular dichroism (CD) spectra were recorded after 24 hours.

[0051] Particle Size / Zeta Potential - Samples were analyzed at 25°C using a Litesizer™ 500 particle size analyzer (Anton Paar GmbH, Graz, Austria) using an Omega cuvette. Particle size distribution was analyzed with the proprietary Kalliope™ v.2.16 software (Anton Paar GmbH, Graz, Austria) using an intensity weighted model. For zeta potential analysis, the suspensions used above were diluted 5-fold with DDI-RO prior to analysis.

[0052] NTA research-NTA measurements were performed with a NanoSight LM 14 (Malvern Panalytical) using a sample chamber connected to a 405 nm laser and equipped with a Hamamatsu Photonics KK CMOS camera (Model#C11440-50B). CAPC samples were injected into the chamber using a sterile syringe (BD Discardit II, New Jersey, USA) and particle tracking was performed with 5 individual captures at 25 frames / sec over a 60 sec time lapse. All measurements were performed at 25°C and the captured images were analyzed by NanoSight NTA 3.3 software to calculate the concentration and size of nanoparticles in the suspension.

[0053] Circular dichroism The same samples used for the zeta potential measurements, 50–100 μM CAPC solutions, were analyzed using a Jasco J-815 CD spectrophotometer (Jasco Analytical Instruments, Easton, MD). Samples were scanned in a rectangular cuvette with a 1 mm path length from 260 nm to 190 nm at 50 nm / min with 1 nm step intervals.

[0054] Differential scanning calorimetry - Differential scanning calorimetry (DSC) experiments were performed using a VP-DSC calorimeter (MicroCal Inc., Northampton, MA) at a scan rate of 1 K / min. The instrument baseline was obtained by first measuring the heat capacity profile of a water blank and subtracting each measurement from the reference baseline.

[0055] temperature stability -Temperature stability was screened using CAPC consisting of 3 mM peptide containing coconut oil. Particles in suspension were diluted to 2% in DDI-RO for particle size analysis and 0.05% for zeta potential measurement. Samples were analyzed in the range of 5-70 °C using a Litesizer™ 500 particle size analyzer (Anton Paar GmbH, Graz, Austria) using an Omega cuvette.

[0056] Shelf Life Stability A CAPC sample containing a lipid mixture was prepared as described above and characterized for average size, surface charge and polydispersity. A portion of this sample was set aside for stability testing at room temperature in the dark.

[0057] Confocal microscopy -CAPC was prepared with both CF-tagged peptides and soybean oil containing Nile Red (TCI America, Portland, OR). The dye is insoluble in water and fluoresces only in lipids and organic solvents. This dye has been used previously to visualize encapsulated lipids using confocal microscopy. The two dye-containing CAPC suspended in water was mixed with 0.7% low melting point agarose in a 1:1 ratio to immobilize the particles. A single drop of 20 μL was spotted onto a microscope slide and a coverslip was applied immediately after. Single confocal sections were photographed using a 20× air objective and a 63× oil immersion objective. A 488 nm filter was used to visualize the CF peptides and a 594 nm filter was used to image the Nile Red. Preparations were imaged using a Zeiss LSM 700 (Zeiss Inc., Carl-Zeiss-Strasse 22, 73447 Oberkochen, Germany) and images exported as jpeg files for publication were processed using Zeiss Zen software.

[0058] CAPC Sizing- Samples with an average diameter of >600 nm, prepared as described in the previous section, were individually placed in gas-tight syringes and extruded through 19 mm Whatman Nuclepore Track-Etched polycarbonate extrusion membranes with 0.2 micron pores (Avanti Polar Lipids, Inc., Alabaster, AL). In addition, for some experiments, 19 mm Whatman Nuclepore Track-Etched polycarbonate extrusion membranes with 0.1 micron pores were extruded through an Avanti Mini-Extruder (with at least 25 membrane passes per sample). Attempts to directly size through 0.1 micron were unsuccessful, so a two-step process was necessary. The resized samples were used for cellular uptake experiments.

[0059] TEM research - Transmission electron microscopy (TEM) images were obtained using a Hitachi STEM 4800EM (Hitachi High Tech Group, Europark Fichtenhain A12, 47807 Krefeld, Germany) with samples prepared as described above and sized to 0.2 microns. The peptides used in this experiment were labeled with Me-Hg. A drop (20 μL) of each sample was placed on a 200 mesh Formvar coated grid (Lacey TEM grid) for 10 minutes, and excess sample was blotted using filter paper. Samples were dried for 30 minutes in an open glass Petri dish set in a drying oven at 55°C prior to visualization.

[0060] Cellular uptake studies-CHO cells (ATCC10801, University Blvd., Manassas, VA) were cultured in Dulbecco's modified Eagle's medium / Ham's F12 (DMEM / Ham's F12) (Gibco, USA) supplemented with 10% (v / v) fetal bovine serum (FBS) (Gibco, USA), 1% penicillin / streptomycin (Gibco, USA) and maintained in a humidified incubator at 37 °C and 5% CO2. For cellular uptake experiments, cells were plated at 2.5 × 10 per well in a Thermo Scientific Lab-Tek II CC2 (8-well glass microscope slide chamber) (Thermo Fisher Scientific, Waltham, MA, USA). 4 Cells were seeded at 100x and maintained in growth medium until they reached 60-70% confluence. Prior to adding CAPCs, the growth medium was replaced with Opti-MEM Reduced Serum (Thermo Fisher Scientific) and CAPCs with carboxyfluorescein-labeled peptides and Nile red dissolved in oil were mixed into the medium to a final concentration of 5% in a final volume of 200 μL. These CAPCs were sized as described above, incubated with the cells for 4-6 hours at 37°C and 5% CO2, fixed with 4% paraformaldehyde, followed by incubation with 0.2% Tween 20 for cell permeabilization, mounted on slides using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) for nuclear staining, and imaged by confocal microscopy as described previously.

[0061] Results and Discussion Topography Study The positive zeta potential of CAPC in Table 1 indicates that the peptide is oriented with its oligo-lysyl ε-amino group exposed to the solvent.

[0062] [Table 1]

[0063] This orientation of the lysyl residues was confirmed by performing tryptic digestion on preformed CAPCS and on peptide aggregates formed in water as controls (Figure 4). In Figure 4 (S1-A), the mass spectrum of the intact CAPC peptide shows the expected mass of 1284.89 Da. By 1 h (S1-B), segments with one to four remaining lysyl groups were observed. The 1 h control (S1-C, aggregated peptide) was almost completely digested. This result suggests that the oligo-lysines of CAPC have a range of solvent accessibility. Lysyl residues can become partially buried in hydrophobic environments, especially in membranes, with their epsilon amino groups burrowing outward to reach the aqueous phase.

[0064] Biophysical research: The first experiments documenting the complexation of the first CAPC peptide sequence, Ac-FLIVIKKKKK-CO-NH2 (SEQ ID NO: 6), with soybean oil droplets focused on DLS, MALDI-MS and CD measurements. The samples were analyzed for their hydrodynamic size and polydispersity by dynamic light scattering, as well as for their zeta potential. The diameter of the measured particles was derived from the evaluation of the DLS intensity peaks. After the sonication step, the mixture was turbid and no free oil was aggregated at the surface of the samples. The average (mean) diameter was 465 nm and the positive zeta potential was 44.8 mV (Table 1). These samples were subjected to trypsin digestion to evaluate the water exposure of the lysyl residues (Figure 4). Within 1 h, all five lysine residues were proteolytically released: this indicates that the protease had reached the carboxyl terminus of each residue. The conformational state of the Ac-FLIVIKKKKK-CO-NH2 (SEQ ID NO: 6) peptide in CAPCS was assessed by circular dichroism spectroscopy of this mixture. The CD spectrum had a strong minimum at 198 nm and a slight maximum at about 218 nm. This spectrum is consistent with the peptide in a random coil conformation.

[0065] This initial study was carried out using a natural mixture of triacylphospholipids containing mainly 18:1 and 18:2 phospholipids. We subsequently tested various oils and non-polar solvents for encapsulation according to their diameter, polydispersity index and zeta potential (Table 1).

[0066] All solvents and oils tested were encapsulated by peptides producing capsules at 37°C with positive zeta potential. With the exception of cyclohexane, the others produced a single mean diameter population. With the exception of mineral oil (>30%), they showed good polydispersity index. The mean capsule diameter was highly variable, with the lowest being 168 nm for benzene and the highest being 804 nm for n-decane. Most oils, except mineral oil, were linear saturated alkyl chains or curved unsaturated alkyl chains, resulting in colloid diameters in the range of 450-530 nm. Mineral oil contains bulkier unsaturated branched or polycyclic alkyl chains that produced colloids with diameters of approximately 700 nm. The larger diameter of the n-decane colloids does not appear to fit models predicting that the molar volume of the hydrophobic oil or solute determines the size of peptide-encapsulated colloids.

[0067] Circular dichroism was used to determine the secondary structure of the encapsulated peptide for each of the solvents in Table 1 (Figure 2). The CD spectra of all colloids appeared remarkably similar, all with a strong minimum at 198 nm and a slight maximum at approximately 218 nm. These spectra are consistent with the peptides in a random coil conformation. The absence of beta structure implies that the assembled peptides are not interacting with each other via hydrogen bonds, but rather through peptide-peptide hydrophobic contacts and oil / solvent.

[0068] To test whether short hydrophobic sequences within amphipathic peptides can effectively encapsulate soybean oil, six different peptide sequences were prepared (Table 2). The presence of a phenylalanine residue in all sequences allows for concentration determination. One sequence contains a C-terminal cysteine ​​to facilitate attachment of methylmercury (Me-Hg) used in our TEM studies. Secondary structure analysis as well as size and charge determinations were performed.

[0069] All of the non-polar sequences were able to form colloids; however, not all yielded useful sizes and degrees of polydispersity. Of note is the observation that all of the peptides interacting with soybean oil remained unstructured based on their CD spectra (Figure 3). Branched versions of similar peptide sequences with two lysine tails are strong beta-forming sequences capable of forming self-assembling peptide bilayers in water. Linear sequences do not adopt a beta structure when immersed in oil.

[0070] [Table 2]

[0071] Examining their size and charge, they form CAPCs with average diameters of 560 nm and 300 nm, respectively. The average diameters in Table 2 are slightly larger compared to the Ac-FLIVI-KKKKK-CONH2 (SEQ ID NO: 6) soybean oil CAPC shown in Table 1. We note that the diameters vary from batch to batch, ranging from 400 nm to 700 nm. The larger sequences result in smaller CAPCs with lower zeta potentials. The methylmercury-containing sequences behaved similarly to the parent sequence containing the FLIVI (SEQ ID NO: 1) segment. This was expected since the added mercury is present in the portion of the peptide that is exposed to water. The scrambled sequence containing the sequence IVFLI (SEQ ID NO: 27) produced a CAPC very similar to the parent sequence, indicating that the order of amino acids is not important. The two outlier sequences in this study are sequences that contain repeating leucine or alanine residues. The oligoleucine-containing sequence has a similar ΔG to the parent sequence. Isoleucine and valine are structurally different from leucine in that they both have an internal branching methyl group on the beta carbon. The oligoalanine peptides, while still non-polar, are much less hydrophobic, reducing their ability to interact with hydrophobic droplets. Both show fairly high polydispersity values ​​and low zeta potentials. Both of these parameters indicate a tendency towards aggregation. Taken together, these values ​​suggest that these two sequences do indeed form smaller CAPCs that then aggregate to give the diameter values ​​reported in Table 2. The fact that the parent sequences contain beta-substituted amino acids (Ile and Val) may have some effect on the CAPC properties. The smaller CAPC formed by the longer sequence (Ac-FLIVIGSII-KKKKKK-CONH2 (SEQ ID NO: 7)) was unexpected. It seems likely that the addition of the four extra amino acids may limit the volume of oil droplets that form during the sonication step. Due to the additional cost of producing the longer sequence, and the fact that the shorter CAPCs are resized (see below), this sequence was not tested further.

[0072] During the initial CAPC formation, various sizes are formed, ranging from nm to micron size (later shown in the microscopy section). Their flexibility allows them to be resized by extrusion through successive polycarbonate filters with 200 nm pores and then 100 nm pores (Table 3). Considering that cationic 100 nm nanospheres are easily taken up by cells, freshly prepared CAPC were extruded successively through a polycarbonate membrane filter with 200 nm pores 21 times and then through a polycarbonate membrane filter with 100 nm pores 21 times (Table 3). Attempts to pass the initial heterogeneous suspension through the 100 nm filter were unsuccessful due to high resistance.

[0073] [Table 3]

[0074] The hydrodynamic values ​​of CAPC before resizing show a typical heterogeneous size population with good polydispersity and positive zeta potential. After repeated extrusion of the heterogeneous mixture back and forth through a filter with 200 nm pores, lower hydrodynamic diameter values ​​are obtained while maintaining similar polydispersity and zeta potential values. After a second extrusion across a 100 nm pore, the average hydrodynamic diameter value is further reduced. The polydispersity is unchanged, however, the zeta potential has a reduced value while remaining positive.

[0075] These results can be interpreted in terms of surface area. When CAPC is formed, a finite concentration of cationic residues (lysine) is distributed on the colloid surface. As CAPC is extruded through the filter, the total number of suspended CAPCs increases, and so does the total surface area of ​​the suspended particles. As the total surface area of ​​the sample increases, the number of peptides on the surface of each CAPC necessarily decreases. The similarity between the starting CAPC preparation and the sample extruded at 200 nm suggests that the majority of the starting sample has a surface area similar to that of the extruded sample. Recognizing that a range of sizes are formed initially, the distribution should be asymmetric and biased towards the smaller CAPCs around 200 nm. When the sample is resized a second time through a 100 nm filter, increasing the total surface area, the zeta potential shows the expected decrease.

[0076] The table also contains data on sized CAPC that were freeze-dried and subsequently rehydrated. Although the rehydrated CAPC became slightly smaller, their polydispersity index and zeta potential did not change. Confocal microscopy of these samples is shown in the microscopy section (Figure 9). The ability to generate dried CAPC allows the preparation of more concentrated samples by reducing the amount of water upon rehydration.

[0077] [Table 4]

[0078] The use of DLS serves as a first step but does not provide information on size distribution and concentration. Nanoparticle Tracking Analysis (NTA) was used to examine the distribution of CAPCs immediately after formation (Figure 5A) and when resized again (Figure 5B). This technique allows for direct observation of diffusion events to be used to obtain high-resolution nanoparticle size distribution and particle concentration data information.

[0079] In the first preparation, 2.16 × 10 8 ±1.83×10 7Particles were obtained at 1.07 × 10 / mL with a mean of 188.2 nm and a standard deviation of 142.2 nm, with 90% of the particles less than 349.7 nm. After sizing through a 200 nm filter, 1.07 × 10 9 ±2.72×10 7 There were 146.1 nm particles / mL with a mean of 146.1 nm and a standard deviation of 75.5 nm, with 90% of the particles being less than 247.2 nm.

[0080] This data better represents the size distribution of the preparation before and after resizing compared to DLS analysis. Note that upon resizing, the number of particles increases by about 5-fold as larger particles are resized to smaller particles.

[0081] A thermal stability study was performed, replacing soybean oil with coconut oil, which undergoes a phase transition near room temperature. This test examined whether the liquid / solid phase of the oil affects the stability of the coconut oil / peptide colloids. CAPC were prepared at 37°C and then either cooled or heated in our temperature-controlled particle size analyzer. They were kept at the new temperature for 5 minutes before taking a reading. In this experiment, 200 nm sized CAPC were examined (Figure 6). At all temperatures the CAPC remained intact and the sizes of CAPC maintained at or above the phase transition temperature of coconut oil had similar diameters. Below the phase transition temperature, the diameters were roughly reduced by 7% and 10% for the 15°C and 5°C & 10°C samples, respectively. As the lipids transition to the solid state, the movement of the side chains is reduced, allowing for more uniform packing. Molecular dynamics simulations studying the phase transition of hydrated DPPC and DPPE bilayers showed that below the phase transition temperature, these lipids become ordered and have reduced molar volumes. Although not shown, the size of the colloids is reversible when the cold and hot CAPCs are returned to 30°C.

[0082] For the next study, we again prepared colloids with coconut oil. Coconut oil produces a phase transition near 24°C-25°C that can be tracked using differential scanning calorimetry. This property allowed us to evaluate CAPC stability when its contents are in solid or liquid phase. In Figure 7, we compared the phase transitions of coconut oil droplets sonicated in water with the peptide alone and with the peptide wrapped around Ac-FLIVIKKKKK-CONH (SEQ ID NO: 6) peptide. The peptide by itself did not show a phase transition in the temperature range used. The sonicated oil droplets show the expected major phase transition between 24°C-25°C. CAPC shows three transitions, with a minor broad phase transition between 17°C-20°C, a second major phase transition near 23°C-24°C, and a very broad phase transition between 30°C-42°C. This data suggests that the bulk of the encapsulated coconut oil is in a generally unbound transition from solid to liquid. The transition between 17 and 20 °C occurs because some of the oil weakly interacts with the peptide, lowering the peptide's melting temperature (T m The fractional transition between 30°C and 42°C may indicate a gel-like state in which some of the lipids dissociate at higher temperatures. At normal body temperature, coconut oil is almost entirely in a liquid state.

[0083] A recent paper investigating mixtures of short aromatic hydrophobic peptides with gasoline, kerosene or diesel fuels formed thermoreversible gels stabilized via Pi-Pi stacking and hydrophobic interactions. The presence of phenylalanine in each sequence may promote the self-assembly and stability of the CAPC peptides.

[0084] Stability testing - CAPC preparations were prepared to analyze long-term stability (Table 5). Test samples contained 100% mixture of two proprietary liquid fatty acid pheromones. Samples were monitored by particle size / zeta potential analyzer throughout the course of the experiment. Over the entire time course, particle size, polydispersity, and zeta potential remained remarkably constant. These results speak to the long-term stability of these particles and the fact that they do not coalesce into larger particles. Since no reactive active ingredients (AIs) were present, there is no data on the ability of CAPC to protect against light, redox, or other types of inactivation. Long-term stability experiments of AIs are currently underway. It is expected that freeze-dried and spray-dried material stored in the dark will have an even longer half-life as well as provide UV protection for light-sensitive active ingredients.

[0085] [Table 5]

[0086] Microscopic studies - Confocal microscopy images were obtained in water for 1) CF-labeled CAPC peptide sonicated in water (left), 2) sonicated soybean oil / Nile Red (middle), and 3) CF-labeled peptide sonicated in the presence of soybean oil / Nile Red (right) (Figure 8). In Figure 8 (left), the peptide (by itself) in water forms irregular spheroidal aggregates of different sizes, some approaching 10 nm in size. Some appear to be solid, while some appear to have water-filled cavities. The soybean oil / Nile Red mixture immediately after sonication in water forms larger irregular oil globules ranging in size from a few tens of μm to 100 μm. When the peptide is mixed with the Nile Red-containing soybean oil / peptide mixture and then sonicated in water, well-ordered polydisperse capsules are formed, ranging from approximately 3 microns to less than 1 micron in size. When analyzed for Nile Red fluorescence and then overlaid with the CF peptide signal (Figure 8, bottom), the soybean oil components are clearly encapsulated within CAPC. The peptide coating, although very thin, is visible as a green boundary surrounding the Nile Red dye-stained oil. Notably, Nile Red dye only fluoresces in hydrophobic environments, providing a good test case for hydrophobic active ingredients and therapeutic drugs.

[0087] Due to the size detection limit of confocal microscopy, we prepared CAPC using methyl-mercury labeled peptides to prepare electron-dense CAPC detectable by TEM (Figure 9). As shown in Table 2, labeling the peptide at the C-terminal water-exposed lysine residue does not affect the size, polydispersity or zeta potential of CAPC. Resized CAPC was used and the field of view selected for this image focused on smaller particle sizes. The diameters of some CAPC in this image range from 70 nm to 15 nm. The larger ones are approximately 200 nm.

[0088] As part of our stability study, we subjected CAPC colloidal particles containing soybean oil to freeze-drying followed by rehydration (Table 3). DLS studies showed that rehydration of freeze-dried samples was possible (see also Table 4). Confocal microscopy was used to verify whether the encapsulated oil was still present after the particles were returned to water (Figure 10). The colocalization of oil within the rehydrated CF-labeled CAPC indicates that these robust particles do not easily break down upon dehydration.

[0089] Cellular uptake studies - In designing the CAPC peptide, we wanted to maintain the cationic surface of the assembled particles identical to peptide bilayer-delimited branched amphiphilic capsules (BAPC). These water-filled capsules are readily taken up by cells in culture and in vivo. In vitro uptake of CF-CAPC encapsulating soybean oil containing Nile Red in fixed CHO cells (Figure 11). Confocal images show uptake after 2 hours. Carboxyfluorescein labeling (top left) is found throughout the cytoplasm with some concentration in the perinuclear region. DAPI stained cell nuclei are shown (top center). Nile Red-soybean oil mixture shows similar cellular distribution as CF-labeled peptide (top right). Brightfield images and a merge of the three fluorophores are shown (bottom left and bottom center, respectively). Bottom right is a blank. This uptake experiment does not demonstrate collapse of CAPC upon release of oil. In other early in vitro experiments, when CAPC containing soybean oil was used as a control, we found that cell growth was promoted and nitrogen-rich amino acids and oil were released and metabolized (data not shown).

[0090] conclusion CAPCs encapsulate low dielectric oils and solvents, producing colloidal suspensions in water with sizes ranging from tens of nanometers to microns. These particles are positively charged and are easily sized using polycarbonate sizing filters. In the case of coconut oil, much of the encapsulated oil retains the physical characteristics of the pure solvent. CAPCs are thermally stable up to 70°C and are stable at room temperature for over a year. The cationic nature of their surface facilitates their uptake by cells in culture. Lysine residues on their surface can be chemically modified for targeting purposes. From a drug delivery perspective, encapsulation of hydrophobic drugs within cell-targeting CAPCs prevents dilution of the active ingredient in the bloodstream.

[0091] Example 2 General protocol for CAPC formation 2 mg of peptide to encapsulate 10 μL of lipid Pipette the solution containing 2 mg of peptide into an Eppendorf tube and dry using, for example, a SpeedVac centrifuge (depending on the peptide concentration and liquid volume, this may take anywhere from 30 minutes to several hours). The dried tube should be at room temperature with a slight thin film on the bottom of the tube. To the dried peptide in the Eppendorf tube, add 10 μL of the desired lipid or oil and vortex for 30 seconds to mix the peptide and lipid. Add 990 μL of distilled water to the tube and place the tube in a 37 kHz, 100% water bath sonicator for 30 minutes to 1 hour. The water bath can be started at room temperature. Typically, within 30 minutes, the solution in the tube turns into a milky, homogenous suspension as colloidal particles form around the lipid droplets, sequestering them. The suspension is sonicated until it appears homogenous or uniform, and is subjected to sonication for an additional 15 minutes to 30 minutes at the same power until a homogenous colloidal suspension is formed. The suspension is then subjected to DLS and Zeta analysis for quality control purposes.

[0092] Example 3 Effect of sonication time To confirm the optimal period for CAPC formation by sonication, water (40 mL), peptide (5 mg) and oil (50 μL) were added together and sonicated for 10 s to obtain a solution containing them before the first reading was taken. The sample was then sonicated continuously for 30 min in a Helios Br Laser Diffraction instrument with a built-in probe sonicator (Sympatec GmbH, Clausthal-Zellerfeld, Germany). Size scans were recorded every 3 min.

[0093] No micron-sized particles are detected in the scan recorded after the first 10 second pulse. As shown in Figure 12, no broad distribution of particles is observed between the instrument's detection limit of 1 micron and over 16 microns until 6 minutes. At each time point up to 21 minutes, the particle size decreases and the particle population becomes more homogenous. At 27 minutes, the distribution density decreases slightly but the homogeneity increases. This experiment was repeated four times with nearly identical results. Based on this work, a 30 minute sonication time was used for all subsequent experiments.

[0094] Example 4 Colloidal particles encapsulating cannabidiol (CBD) Cannabidiol (CBD) is an active compound that is notoriously difficult to encapsulate and deliver. Cannabidiol is a BCS Class II drug with poor solubility and low bioavailability. This study was a comparative pharmacokinetic (PK) study utilizing two groups of seven dogs per group. In this example, CBD was formulated into various CAPCs and compared to MCT oil as a negative control. The nano-CBD formulation of CAPC was a solution containing water, CAPC peptides, and soybean oil in which CBD was dissolved, mixed together and continuously sonicated for 30 minutes. A total of 14 dogs were randomized into one of two treatment groups: "NANO" for dogs treated with nano-formulated CAPC, or "MCT" for dogs in the control group. Dogs were orally administered either formulation. In treatment period I, animals in group 1 were treated with CBD-MCT (2mg / Kg) and animals in group 2 were treated with CBD-NANO (2mg / Kg). Blood samples were collected pre-dose (0) and at 15 min, 30 min, and 1, 1.5, 2, 3, 4, 5, 6, 8, 12, and 24 h after treatment. CBD concentrations in plasma were analyzed using a certified analytical method validated for dog plasma. Results are shown in Figure 13. NANO vs. MCT values ​​were significantly different at P<0.05 (AUC 0-LOQ and C max (Only AUC was subjected to statistical analysis.) 0-LOQ and C max The values ​​were log-transformed prior to analysis. The back-transformed least squares means were as follows: MCT: AUC 0-LOQ and C max : 428hr each * ng / mL and 54ng / mL. NANO:AUC 0-LOQ and C max : 890hr each * CAPC's nano-CBD formulation significantly increased CBD bioavailability when administered orally to dogs compared to the conventional MTC oil formulation.

Claims

1. A dry composition comprising a plurality of freeze-dried or spray-dried peptide colloidal particles, wherein each colloidal particle comprises a peptide monolayer that encapsulates a droplet of a nonpolar excipient, optionally together with one or more hydrophobic and / or poorly water-soluble activators dispersed or distributed therein, and the peptide monolayer comprises a plurality of amphiphilic linear peptides having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment.

2. A kit comprising a unit dose form of the dried composition according to claim 1 in a container, and instructions for rehydrating the composition using an aqueous solvent system.

3. A method for preparing a suspension of colloidal particles, A step of mixing the dried composition according to claim 1 with an aqueous solvent system to rehydrate the freeze-dried or spray-dried peptide colloid particles, wherein the rehydrated peptide colloid particles remain uniformly suspended in the aqueous solvent system, and the nonpolar excipient remains encapsulated in the peptide monolayer of each particle after the rehydration step. A method that includes the following:

4. A method for resizing peptide-based colloidal particles, A step of providing a suspension of colloidal particles having a first average diameter, wherein each colloidal particle comprises a peptide monolayer encapsulating a droplet of a nonpolar excipient, optionally together with one or more hydrophobic and / or poorly water-soluble activators dispersed or distributed therein, and the peptide monolayer comprises a plurality of amphiphilic linear peptides having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment. A step of extruding the suspension of colloidal particles through an opening having dimensions smaller than the first average diameter, and A step of collecting extruded colloidal particles, wherein the extruded colloidal particles have a second average diameter smaller than the first average diameter. A method that includes the following:

5. The method according to claim 4, wherein the extrusion step includes passing the suspension of colloidal particles through the bore opening of a syringe and / or through the pores of a filter.

6. The method according to claim 4, wherein the second average diameter is 200 nm or less.

7. The composition according to claim 1 or the kit according to claim 2, wherein the nonpolar excipient is a lipid, fat, oil, wax, or oil.

8. The method according to claim 3 or 4, wherein the nonpolar excipient is a lipid, fat, oil, wax or oil.

9. The composition according to claim 1 or the kit according to claim 2, wherein the nonpolar excipient is a low dielectric solvent selected from the group consisting of alkanes (pentane, hexane, heptane, and n-decane), cycloalkanes (cyclohexane), diethyl ether, carbon tetrachloride, methylene chloride, aromatics (benzene, toluene, and xylene), phthalate esters (diethyl phthalate), piperonyl butoxide, and combinations thereof.

10. The method according to claim 3 or 4, wherein the nonpolar excipient is a low dielectric solvent selected from the group consisting of alkanes (pentane, hexane, heptane, and n-decane), cycloalkanes (cyclohexane), diethyl ether, carbon tetrachloride, methylene chloride, aromatics (benzene, toluene, and xylene), phthalate esters (diethyl phthalate), piperonyl butoxide, and combinations thereof.

11. A composition comprising a plurality of peptide-based colloidal particles, wherein each colloidal particle comprises a peptide monolayer that encapsulates a lipid, fat, oil, wax, or oil that is solid at room temperature, together optionally with one or more hydrophobic and / or poorly water-soluble activators dispersed or distributed therein, and the peptide monolayer comprises a plurality of amphiphilic linear peptides having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment.

12. The composition according to claim 11, wherein the lipids, fats, oils, waxes, or oils that are solid at room temperature are selected from the group consisting of coconut oil, glycerol based on stearic acid, triglycerides, partial glycerides, fatty acids, steroids, and waxes.

13. A method for preparing the composition according to claim 11, A step of heating a lipid, fat, oil, wax, or oil that is solid at room temperature to a temperature above its melting point, wherein the lipid, fat, oil, wax, or oil that is solid at room temperature transitions to a liquid state. Depending on the circumstances, the process may involve dissolving one or more hydrophobic and / or poorly water-soluble surfactants in the liquid state of lipids, fats, oils, waxes, or oils. A step of dispersing a plurality of the amphiphilic peptides in the liquid state of lipids, fats, oils, waxes, or oils to produce a mixture. The steps include adding an aqueous solvent system to the mixture and stirring for a sufficient time to obtain a suspension of peptide-based colloidal particles in the aqueous solvent system, and The step of cooling the suspension to at least room temperature. Includes, A method comprising a peptide monolayer in which the peptide colloidal particles encapsulate the lipids, fats, oils, waxes, or oils that have solidified after cooling, together with one or more hydrophobic and / or poorly water-soluble activators, if present, dispersed or distributed therein.

14. The composition according to claim 1 or 11, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment has a total chain length of 5 to 20 amino acid residues.

15. The amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment has a total chain length of 5 to 20 amino acid residues, according to the kit of claim 2 or the method of claim 13.

16. The method according to claim 3 or 4, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment has a total chain length of 5 to 20 amino acid residues.

17. The composition according to claim 1 or 11, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment each comprises a hydrophobic segment having a length of about 3 to about 11 residues.

18. The amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment each comprises a hydrophobic segment having a length of about 3 to about 11 residues, according to the kit of claim 2 or the method of claim 13.

19. The method according to claim 3 or 4, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment each comprises a hydrophobic segment having a length of about 3 to about 11 residues.

20. The composition according to claim 1 or 11, wherein each amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment comprises a hydrophobic segment comprising at least one phenylalanine residue and at least two further residues selected from the following: leucine, isoleucine, and / or valine.

21. The amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment each comprises a hydrophobic segment comprising at least one phenylalanine residue and at least two further residues selected from the following: leucine, isoleucine and / or valine, the kit according to claim 2 or the method according to claim 13.

22. The method according to claim 3 or 4, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment each comprises a hydrophobic segment comprising at least one phenylalanine residue and at least two or more further residues selected from the following residues: leucine, isoleucine and / or valine.

23. The composition according to claim 1 or 11, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment comprises a hydrophilic segment containing about 1 to about 7 hydrophilic amino acid residues, each of which is preferably lysine.

24. The kit according to claim 2 or the method according to claim 13, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment comprises a hydrophilic segment containing about 1 to about 7 hydrophilic amino acid residues, each of which is preferably lysine.

25. The method according to claim 3 or 4, wherein the amphiphilic linear peptide having an N-terminal hydrophobic segment and a C-terminal hydrophilic segment comprises a hydrophilic segment containing about 1 to about 7 hydrophilic amino acid residues, each preferably being lysine.

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