Lipid-encapsulated amphiphilic peptide

Abstract

Described herein are linear peptides for the preparation of colloidal particles and micelles for the delivery of active agents. The colloidal particles facilitate the encapsulation and delivery of hydrophobic and / or poorly water-soluble active agents within the hydrophobic core of the particles. The compositions are suitable for the delivery of active agents to plants and animals.

Description

Cross - reference to related applications

[0001] This application claims priority from U.S. Provisional Application Serial No. 62 / 822,370, filed on March 22, 2019, entitled "LIPID ENCASING AMPHIPATHIC PEPTIDES", and the entire disclosure content of such prior patent application is incorporated herein by reference.

[0002] Sequence Listing The following application includes a Sequence Listing in computer - readable format (CRF), which was submitted as an ASCII - formatted text file entitled "SequenceListing52234 - PCT" created on March 18, 2020, as 4KB. The content of the CRF is incorporated herein by reference.

Technical Field

[0003] Field of the Invention The present invention relates to, for example, peptide - based delivery of active agents via colloidal particles and / or micelles.

Background Art

[0004] Description of the Related Art Conventional approaches for preparing formulations containing hydrophobic active agents have numerous drawbacks, such as large structures that aggregate and ultimately lead to complete phase separation. Due to their physical instability and lack of homogeneity, these formulations also suffer from poor and variable absorption. There remains a need for improved techniques for formulating and delivering hydrophobic or poorly soluble active agents.

Summary of the Invention

[0005] The present invention relates broadly to a composition comprising a plurality of colloidal particles suspended in an aqueous carrier. Each of the colloidal particles comprises a peptide matrix having a hydrophobic core and a hydrophilic outer surface. The hydrophobic core advantageously sequesters (encapsulates) a nonpolar excipient, such as a lipid, an oil, or a nonpolar solvent, optionally together with one or more hydrophobic and / or poorly soluble active agents dispersed or distributed therein.

[0006] Also described herein is a method for delivering a hydrophobic and / or poorly soluble active agent to a subject in need thereof. The method comprises administering to the subject a composition according to various aspects described herein.

[0007] This application also relates to a method for delivering a hydrophobic and / or poorly soluble active agent to a plant. The method comprises spraying a composition according to various aspects described herein onto at least a part of the plant and / or the soil in which the plant is planted or to be planted. In some aspects, the active agent is sprayed onto the plant and / or the soil in which the plant is planted or to be planted for the purpose of delivering the active agent to pests.

[0008] Also described herein is a method for delivering a hydrophobic and / or poorly soluble active agent, specifically an insecticide, to an insect. The method comprises contacting the insect with a composition according to various aspects described herein.

[0009] Also described herein are peptide-based micelles that can encapsulate an active agent in their hydrophilic core and are formed from a peptide having the sequence XLIVIKKKKK (SEQ ID NO: 1), where X is F or V. BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

[0011] Description Novel colloidal particles that can be used to encapsulate active agents are described herein. More specifically, the inventors disclose the use of linear peptides that can encapsulate lipids, oils, and hydrophobic or (water) poorly soluble active ingredients, thereby enabling them to be dispersed as colloidal particles suspended in an aqueous carrier. Other carriers, adjuvants, synergists, dispersants, or solvents may also be included within / with the particles. Advantageously, the outer surface of the particles is hydrophilic, allowing them to be easily dispersed in an aqueous solution, promoting uptake by animal and plant tissues, thereby facilitating the delivery of lipophilic active ingredients into cells. The particles also shield the active agent from the external environment where it may be rapidly inactivated. As a drug delivery vehicle, the novel colloidal particles can also be used to alter the biological half-life of the active agent.

[0012] The colloidal particles comprise a peptide matrix having a hydrophobic core in which a nonpolar excipient, such as a lipid, an oil, or a nonpolar solvent, is optionally isolated together with one or more active agents dispersed or distributed therein. The particles are characterized by a hydrophilic outer surface formed from hydrophilic segments of the peptide that are oriented outwardly with respect to the external environment in each particle. Preferably, the peptide matrix is homogeneous, which means that it is composed of a plurality of the same type of peptide (same sequence).

[0013] The peptides used to prepare the particles are linear and without branch points, and comprise (consist essentially of, or consist of) a hydrophobic segment (first terminus) directly linked to a hydrophilic segment (second terminus). The hydrophobic head groups preferably each have a length of about 3 residues to about 11 residues, more preferably a length of about 4 to about 10 residues, and even more preferably a length of about 5 to about 9 residues. A preferred hydrophobic sequence is XLIVI (“h5”; SEQ ID NO: 2), where X is F or V, preferably F. The hydrophilic (polar) lysine tail sequence preferably has a length of about 1 to about 7 lysine residues, more preferably about 1 to about 6 lysine residues, and even more preferably about 1 to about 5 lysine residues. A particularly preferred lysine sequence is KKKKK (SEQ ID NO: 3). The peptides preferably have a molecular weight in the range of 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” for these peptides is the average weight calculated based on the total MW of the actual amino acids present divided by the number of residues. The linear peptides have an overall chain length in the range of 20 amino acid residues or less, preferably about 5 to about 20, more preferably a length of about 8 to about 15 residues, and even more preferably a length of about 8 to about 12 residues. The peptides can be synthesized using traditional Fmoc chemistry.

[0014] These peptides are referred to herein as lipid-encapsulated amphiphilic peptides (LEAPs) and include the following preferred sequences: FLIVIKKKKK (SEQ ID NO: 1, where X is F) and VLIVIKKKKK (SEQ ID NO: 1, where X is V). In one or more embodiments, the peptide comprises a cysteine residue added at the C-terminus of the peptide at the terminal lysine position to facilitate further functionalization. In some embodiments, the N-terminus of each hydrophobic segment may be capped with an acetyl group (Ac).

[0015] In one or more embodiments, a functional group and / or various moieties can be attached to the C-terminal lysine, or the C-terminal carboxyl group, or in the case of the C-terminal cysteine, the free sulfhydryl group. The term "functional moiety" is used herein to include functional groups, targeting moieties, and active agents that can bind to the outer surface of the particle. Exemplary functional moieties that can be attached include fluorophores, dyes, targeting moieties and ligands, antibodies, cysteine, cysteamine, biotin, biocytin, nucleic acids, polyethylene glycol (PEG), organometallic compounds (e.g., methyl mercury), radiolabels, conjugating chemistries, -COOH, -CONH2, -SH, and the like. A plurality of such moieties can also be attached in a sequential chain from the C-terminus using an aliphatic spacer to separate the different moieties. Thus, the present invention provides an opportunity to create multifunctionalized 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 comprising a portion of the assembled colloidal particles.

[0016] Figure 1 shows an illustration of particles according to an aspect of the present invention. In this figure, the outer shaded layer represents the bulk aqueous carrier surrounding the colloid. The wavy lines represent the amphiphilic peptides forming the peptide matrix, with their cationic lysine residues facing the aqueous external environment, and the peptides forming a monolayer at the oil-water interface. The hydrophobic amino acids in the sequence face the interior / core direction of the colloid and interact with some of the lipid, oil, or nonpolar solvent molecules. The colloidal particles remain stable as discrete colloidal particles in an aqueous solution for a long period of time without aggregating, coalescing, or decomposing (preferably for at least 3 months, more preferably for at least 6 months, even more preferably for at least 12 months). This is referred to herein as the "shelf life" or "storage stability" of the colloid.

[0017] The colloidal particles are prepared by mixing a lipid, oil, or nonpolar solvent (excipient) with the peptide in a reactor. In one or more aspects, the active agent is first dispersed or dissolved in the bulk excipient encapsulated by the peptide. Preferred lipids and oils include vegetable oils, GRAS vegetable oils, mineral oils, Miglyol oils, paraffin oils, Solutol®, etc., or combinations thereof. The oil may itself be an active ingredient or may contain the active ingredient. Preferred nonpolar solvents include cyclohexane, benzene, n-decane, piperonyl butoxide, diethyl phthalate, dimethyl sulfoxide, etc., or combinations thereof.

[0018] Add the peptide in an amount sufficient to encapsulate all of the excipients present, then leave it standing 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 the peptide to the excipient is about 1:50 to about 1:20, preferably about 1:25 to about 1:10. Then add an excess amount of distilled / deionized water and mix using a vortex mixer or a bath sonicator for at least about 5 minutes, preferably about 5 minutes to about 15 minutes. As the colloid forms, the solution becomes somewhat turbid and suspends in water. Upon centrifugation, the colloid migrates to the top of the water column and the oil layer is no longer visible. As shown in Figure 1, the hydrophobic amino acids in the peptide sequence are oriented towards the inside of the colloid and interact with the droplets of the encapsulated bulk excipient.

[0019] In one or more embodiments, the resulting colloid particles have a maximum surface-to-surface dimension of greater than about 100 nm, preferably about 200 nm to about 1000 nm, more preferably about 200 to about 800 nm. Advantageously, the particles have a low polydispersity with a PDI of less than 250%, preferably less than 100%, more preferably less than 50%, more preferably less than 40%, even more preferably about 2% to about 30%. Another important aspect of the design of the colloid particles is the cationic nature of the solvent-exposed surface. The particles have a zeta potential of about 1 mV to about 400 mV, preferably about 20 mV to about 100 mV.

[0020] Advantageously, the colloidal particles can be prepared to target specific cell surface receptors via the addition of different molecules or functional groups, such as cholesterol, mannose, TAT peptide, insulin, biotin, nucleotides, or any other suitable known surface targeting molecule, and combinations thereof, to the C-terminus lysine. Thus, the colloidal particles having such targeting moieties attached to their outer surface localize to specific cells or tissues of a patient and are selectively taken up by those cells or tissues. For this reason, the colloidal particles can be used for targeted therapy (such as gene therapy, cancer therapy, etc.) and nanodrug delivery by administering to a patient the colloidal particles having a targeting moiety. The targeting moiety is attached to the hydrophilic component of the peptide used to form the colloidal particles, and this hydrophilic component mainly occupies the outer layer of the particles. Thus, the targeting moiety is presented on the outer surface of the colloidal particles after formation. This moiety is recognized by the target region or tissue in a patient, and the colloidal particles will automatically localize to that region or tissue. By targeting these structures to specific cell types, the amount of the active ingredient required can be reduced and the off-target effect can be limited.

[0021] Colloidal particles are currently finding application in the cellular delivery of (water) poorly soluble compounds / drugs that are too hydrophobic to be effectively delivered. As used herein, reference to a "poorly soluble" active agent refers to compounds and materials that have low solubility in an aqueous solvent system and are contrasted with agents that can be fully dispersed or dissolved in an aqueous system. There are numerous synthetic and vegetable oils that can preferentially solubilize hydrophobic molecules. The above techniques appear to be useful, for example, in preparing pesticides, fungicides, anticancer agents, and improving the bioavailability of many lipophilic active ingredients.

[0022] Colloidal particles can be used in a pharmaceutically acceptable composition for delivering the colloidal particles to a subject. In one or more embodiments, the composition comprises a therapeutically effective amount of colloidal particles dispersed in a pharmaceutically acceptable carrier. As used herein, a "therapeutically effective" amount refers to an amount of colloidal particles that elicits a biological or medical response in a tissue, system, animal, or human being that is being investigated by a researcher or clinician, particularly, that elicits some desired therapeutic effect. One of ordinary skill in the art will recognize that the amount may be considered therapeutically effective even if the condition is not completely eradicated but is partially improved. As used herein, the term "pharmaceutically acceptable" means that it can be administered to a subject, cell, or tissue without causing excessive toxicity, irritation, or allergic reaction, and does not cause any undesirable biological effects or interact in a harmful manner with any other segment of the composition in which it is included, and is not biologically or otherwise undesirable. A pharmaceutically acceptable carrier will be naturally selected so as to minimize any degradation of the colloidal particles, functional groups, or active agent, and to minimize any harmful side effects in the subject, cell, or tissue, as would be well known to one of ordinary skill in the art. Pharmaceutically acceptable components include those acceptable for veterinary use and 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 distilled water for injection (DAW), water-in-oil or oil-in-water emulsions, and the like.

[0023] Also described herein is a method for targeting delivery of an active agent to a region of a patient, the method comprising administering to the patient the colloidal particles described herein comprising a target moiety on an outer surface. This moiety is recognized by a target region or tissue in the patient, and the colloidal particles will automatically localize to that region or tissue. The colloidal particles can be injected directly into the target tissue or can be administered systemically.

[0024] Methods for delivering an active agent to a plant, such as the leaves, stems, roots, or other tissues or cells of a plant, are also described herein. This method can be used to deliver a variety of active agents, including for treating and / or preventing pests, diseases, infections, etc. This method comprises spraying colloidal particles onto at least a part of the plant and / or the soil in which the plant is planted or to be planted.

[0025] Methods for delivering an active agent to an insect by contacting the insect with colloidal particles containing an active agent, such as an insecticide, are also described herein. This method can comprise spraying the colloidal particles onto the leaves, stems, roots, or other tissues or cells of a plant, or alternatively, placing the colloidal particles at a location where the insect / pest will come into contact with the colloidal particles. In some embodiments, the colloidal particles may be ingested by the insect. In some embodiments, the colloidal particles can be provided in the diet of the insect together with an edible insect attractant (sugar, carbohydrate, yeast, fat, oil, protein). The diet can be in the form of a liquid, gel, or solid tablet or granule.

[0026] One alternative embodiment relates to the formation of micelles using linear peptides. This embodiment relates to dispersing the peptides in an aqueous solution, at which time they spontaneously aggregate into relatively small (i.e., less than about 150 nm) micelles. Over time, the micelles aggregate into larger structures. The active agent can first be dispersed in an aqueous solution for subsequent encapsulation into micelles for delivery to a patient.

[0027] The techniques described herein can be used to deliver a wide variety of active agents including, but not limited to, fungicides, anti-cancer agents, insecticides, herbicides, metabolic inhibitors, etc.

[0028] Further advantages of various aspects of the present invention will become apparent to those skilled in the art upon review of the disclosure herein and the following examples. It will be recognized that the various aspects described herein are not necessarily mutually exclusive, unless otherwise specifically stated herein. For example, features described or depicted in one aspect may be included, but not necessarily included, in one or more other aspects. For this reason, the present invention encompasses various combinations and / or integrations of the specific aspects described herein.

[0029] As used herein, the phrase "and / or" when used in a listing of two or more items means that any one of the listed items can be used by itself or any combination of two or more of the listed items can be used. For example, if a composition is described as containing or excluding component A, B, and / or C, the composition can contain 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.

[0030] This specification also uses numerical ranges to quantify certain parameters relating to various aspects of the present invention. When a numerical range is provided, such a range should be understood to provide literal support for claim limitations that recite only the lower limit of the range and claim limitations that recite only the upper limit of the range. For example, the disclosed numerical range of about 10 to about 100 provides literal support for claims that recite "greater than about 10" (no upper limit) and claims that recite "less than about 100" (no lower limit).

Examples

[0031] Hereinafter, the method of the present invention will be described by way of examples. However, it should be understood that these examples are provided by way of illustration and that none of them should be construed as limiting the overall scope of the present invention.

[0032] Example 1 Peptides were synthesized using Fmoc chemistry. Two different cleavage protocols were tested, and two different counterions: trifluoroacetic acid counterion (TFA - ) and chloride counterion (Cl - ) were obtained. The chloride counterion was found to be superior for this application.

[0033] The first encapsulation test was performed using high-purity soybean oil containing the lipophilic dye Nile Red. The test was performed using FLIVI-KKKKK (SEQ ID NO: 1, where X is F). Increasing concentrations of the peptide (see Figure 2) were prepared in trifluoroethanol and vacuum dried. To each tube, 22 μL of soybean oil containing Nile Red dye (1 mg / mL) was added and left for 15 minutes. Then, distilled / deionized water (480 μL) was added and mixed repeatedly using a vortex mixer for at least a 5-minute period. The solution became somewhat turbid as the dye-containing colloids were formed at higher peptide concentrations. 25% was encapsulated at a peptide concentration of 0.5 mM, and 80% was encapsulated up to a peptide concentration of 1 mM. The colloids formed using the FLIVI-KKKKK (SEQ ID NO: 1, where X is F) sequence remained stable in aqueous solution for a long period (at least 12 months).

[0034] Example 2 Peptide colloids were prepared in high-purity soybean oil with Ac-FLIVI-KKKKK-CO-NH2 (SEQ ID NO: 1, where X is F, "h5F-L") or Ac-VLIVI-KKKKK-CO-NH2 (SEQ ID NO: 1, where X is V, "h5V-L") and tested for average size and zeta potential (surface charge) using light scattering on a ZetaPlus particle size measuring instrument. The results for the two peptides are shown in the following table. A control was prepared using high-purity soybean oil mixed with water and sonicated.

[0035]

Table 1

[0036] The h5F-L peptide had a polydispersity index (PDI) of 0.23, resulting in an average diameter of approximately 410 nm (upper panel), while the h5V-L peptide (lower panel) had a PDI of 0.2 and showed a smaller average diameter of approximately 240 nm. A PDI value of less than 0.3 is considered monodisperse in the pharmaceutical industry. The size of the suspended lipid droplets in the control sample was larger than that observed for the peptide colloids and increased over time as the lipid droplets further coalesced, and the lipid droplets eventually separated from the water, forming an oil layer on top of the water. The polydispersity index of the control was higher than 0.3.

[0037] Regarding the zeta potential, the larger h5F-L particles had a value of 46 ± 2.5 mV, while the smaller h5V-L particles had a value of 43 ± 1.6 mV. These highly positive values are the result of the highly cationic surface of the particles conferred by the presence of the solvent-exposed oligo-lysine segments. These values are also an indicator of particle stability in that they remain monodispersed. A positive zeta potential is also associated with efficient intracellular uptake of the nanoparticles. The values observed for these novel colloids are similar to those observed for a patented branched-chain amphiphilic peptide capsule that is readily taken up by different cell types.

[0038] Considering that the particle sizes of both peptides exceeded the 100 nm nanoscale cutoff, the samples were subjected to size adjustment by extrusion through a 100 nm polycarbonate filter. The h5F-L particles were size-adjusted to approximately 80 nm after extrusion (data not shown). The h5V-L particles could be size-adjusted to approximately 90 nm.

[0039] Example 3 The ability of LEAP to deliver a lipophilic active ingredient to an organism was tested using the antifungal agent captan: (3aR,7aS)-2-[(trichloromethyl)sulfanyl]-3a,4,7,7a-tetrahydro-1H-isoindole-1,3(2H)-dione. This compound is highly soluble in solvents such as acetone and xylene, but 4000-fold less soluble in water. This compound also has good solubility in the C3 organic acid, propionic acid. It should be noted that propionic acid itself has antifungal activity. In this experiment, two different peptides: h5F-L and h5V-L were tested.

[0040] A 0.5% solution of captan was prepared in a 1:1 mixture of propionic acid and soybean oil (final volume 10 μL for each peptide). The solution was added to 1 mg of dry peptide. After 15 minutes of incubation, water (490 μL, pH = 3) was added dropwise, followed by sonication in a bath sonicator at 37 °C for 5 minutes and then left standing for 20 minutes. The samples were then centrifuged at 14K × g for 10 minutes at room temperature. At this point, each peptide-lipid complex formed a whitish thin film on the surface of the water. The volume of water was reduced using a pipetteman to a final volume of 150 μL. The solution was vortexed again to resuspend the colloid. An example of colloidal particles with the active ingredient distributed in the colloidal body is shown in Figure 3. A number of different controls were tested with all components: soybean oil alone, soybean oil + propionic acid, soybean oil + propionic acid + captan together with the peptides prepared. Volumes of 5 - 40 μL were spotted onto plates seeded with spores of Aspergillus nidulans ( Aspergillus nidulans ) such that 0.33 and 1.33 μg of the active agent were delivered respectively (Figure 4).

[0041] In Figure 4, Aspergillus nidulans ( Aspergillus nidulans) The fungal colony was seeded immediately before the test. The actively growing fungus appears greenish-yellow in color. For the control (Figure 5A), soybean oil and soybean oil + propionic acid (1:1, v / v) were applied directly to the plate in the indicated volumes. 40 μL of soybean oil (Figure 5A, column 1) shows some antifungal activity, but when it is diluted by half in 1:1 soybean + propionic acid (Figure 5A, column 2), this effect is significantly reduced. When 1:1 soybean + propionic acid was encapsulated separately using two peptides (Figure 5B, columns 1 and 2), minimal antifungal activity was observed. In Figure 5C, column 1, captan was dissolved in propionic acid before mixing with soybean oil. The topical application of this mixture was not very effective as an antifungal agent. When this mixture was encapsulated in the h5V-L peptide (Figure 5C, column 2), a small amount of antifungal activity was observed. However, when the mixture was encapsulated in the h5F-L peptide (Figure 5C, column 3), complete growth inhibition of the fungus was observed as judged by the appearance of the white milk agar surface. This result indicates that captan is most effective for killing when combined with oil and is internally translocated by the organism using the LEAP system.

[0042] Example 4 In this example, the antifungal activity of captan alone or captan encapsulated in LEAP colloidal particles against Aspergillus nidulans A. nidulans) The effectiveness against the growth of

[0043] LEAP colloidal particles containing the active ingredient were significantly more efficient in delivering the inhibitory concentration compared to applying the compound alone. They showed almost complete growth inhibition even at the lowest dose of the active ingredient. Captan by itself showed strong growth inhibition only at the highest concentration (left column). At the 0.1% concentration, only slight inhibition was observed. As a result of using LEAP to prepare colloidal particles containing the active agent, growth inhibition was shown at all the concentrations tested (right column). These results may be due to the improved intracellular uptake of LEAP colloidal particles. It will be recognized that this technology allows growers to use significantly reduced amounts of the active ingredient without affecting the effectiveness of the treatment.

[0044] Example 5 In this test, the inventors examined the effect of captan fungicide formulations with and without the LEAP peptide when sprayed on the leaves of Vicia faba )(Fava bean). Three formulations were prepared for this experiment:

[0045]

Table 2

[0046] Deionized water was added to each formulation to a final volume of 1 mL and left at room temperature for 60 - 90 minutes. Using each of these formulations, they were sprayed onto the leaves of Vigna unguiculata ( V. faba ), and the effects were recorded 10 minutes after spraying onto the plants and 48 hours after treatment.

[0047] As expected, leaves treated with water alone or soybean oil in water showed no harmful effects. However, propionic acid in water caused black spots on the leaves after 24 hours (data not shown). Leaves sprayed with the formulation containing soybean oil combined with propionic acid also showed extreme damage after 24 hours, and leaf death was observed approximately 48 hours later (Figure 7A). Almost the same results were observed in leaves treated with a solution containing soybean oil, propionic acid, and captan (Figure 7B), where the leaves showed visible damage recognized as black spots and leaf death was observed approximately 96 hours later (not shown). On the other hand, the same components when encapsulated in colloidal particles via LEAP showed only healthy leaves (Figure 7C), and no signs of damage were observed even after 5 days (not shown).

[0048] When the formulation solution that requires propionic acid to dissolve captan came into contact with the leaves, it caused visible damage recognized as black spots or in some cases total leaf damage. Analysis of each component of the isolated formulation confirmed that propionic acid was the component causing the toxic effect on the leaves. Interestingly, when combined with the LEAP formulation, the damage caused by propionic acid was suppressed, suggesting that LEAP colloidal particles can promote protective results.

[0049] Example 6 In this example, Nicotiana tabacumi (tobacco plants) on the treated leaf pieces of Manduca sexta)(The lethality of the active ingredient alone or the active ingredient encapsulated in LEAP colloidal particles against the tobacco hornworm (Manduca sexta) was examined.)

[0050] )(Different lipophilic active ingredients (novaluron (1-[3-chloro-4-phenyl]-3-urea)) were dissolved in a novel formulation that could encapsulate them into colloidal particles with LEAP peptides. Diethyl phthalate (DEP), 10% DMSO, and 20 μL of soybean oil were mixed with novaluron and then combined with 2 mg of the h5F-L peptide. The mixture was incubated for 15 minutes, then deionized distilled water (490 μL, pH = 5.5) was added dropwise and sonicated in a bath sonicator at 37 °C for 5 minutes. Then the mixture was left standing for 20 minutes. Immediately before use, Tween-80 (polyoxyethylene (80) sorbitan monooleate, 0.05%) was added as a wetting agent.)

[0051] )(For the test, 3-cm leaf pieces were cut from the leaves of living plants. The leaf pieces were washed, dried, then immersed in different test solutions and dried for 60 minutes. After drying, they were placed on 1.5% agar in a 6-well polystyrene culture plate. The leaves were divided into five test groups:)

[0052] [Table 3]

[0053] )(Regarding the first experiment, 1-day-old (1st instar) tobacco hornworms ( M. sexta )) larvae were placed on the treated leaves and observed for 8 days. The insects were exposed to 12 hours of light followed by 12 hours of darkness in an incubator at 26 °C and 85% RH. Each day, the number of dead larvae was counted. In this experiment, no statistical difference was observed between novaluron in water and novaluron encapsulated with LEAP colloidal particles (data not shown).)

[0054] Subsequently, a persistence assay was conducted to confirm whether the LEAP colloidal particles could protect the active ingredient from environmental degradation. Two tests were performed, with the waiting period before adding first-instar larvae being 3 days for one test (Figure 8) and 5 days for the other test (Figure 9). The method used to prepare the LEAP colloidal particle encapsulation material and the preparation of the coated leaves were the same as above, except that the leaf pieces were left in the incubator for the specified period under the previously described conditions outlined above. After the specified waiting period, first-instar (1-day-old) tobacco budworms ( M. sexta ) larvae were placed on the treated leaves. The number of dead larvae was counted each day. Examination of Figures 8 and 9 reveals that it is clear that the LEAP colloidal particles containing the active ingredient novaluron initiated death faster than the novaluron control. During both waiting periods, the LEAP colloidal particles initiated death more quickly and with higher efficiency. Since novaluron itself remained active, it appears that the peptide coating of novaluron containing oil droplets was ingested more efficiently, such that the lethal dose was achieved more rapidly.

[0055] Example 7 In this test, the ability of the LEAP peptide to encapsulate different nonpolar solvents and oils (excipients) was examined: benzene, cyclohexane, n-decane, (heavy) mineral oil paraffin, soybean oil, and Miglyol® 812N (C8 / C10 triglyceride excipient). The nonpolar reagent (20 μL) was added to water (480 μL) and sonicated for 15 minutes. Next, 1 mg of the peptide was added to each mixture and sonicated again for 10 minutes. The samples were then left for 24 hours and analyzed for size, polydispersity by dynamic light scattering, and zeta potential. All of these steps were carried out at room temperature. The measured particle diameter was derived from the evaluation of the DLS intensity peak.

[0056]

Table 4

[0057] The results of this test indicate that LEAP was able to generate stable colloids with a positive zeta potential. Different solvents or oils produced colloids of different sizes ranging from 169 nm for benzene to 804 nm for n-decane. For each of the solvents or oils tested, except cyclohexane, a standard / single colloid size was produced. Except for mineral oil, all the colloids appear to be monodisperse with values less than 30% (0.3 PDI). The presence of a positive zeta potential in the nonpolar mixtures indicates that the LEAP peptides are oriented in the colloids with their positive oligo-lysyl epsilon-amino groups exposed to the solvent, with their hydrophobic segments directed towards the core of the colloid particles.

[0058] Being positively charged and monodisperse enables these colloid structures to be taken up by cells both in vitro and in vivo. It should be noted that the ability of LEAP to emulsify and deliver such a variety of nonpolar solvents and oils (excipients) suggests that a platform can be used to deliver a wide variety of hydrophobic active agents that are soluble in this and similar nonpolar excipients.

[0059] As a second part of this study, the structure of the peptides associating with nonpolar excipients was determined. Circular dichroism was used to analyze the percentage of alpha helix, beta sheet, and random coil in the peptides used to form colloidal particles. Only those colloidal solutions that were clear could be used for these measurements (benzene, cyclohexane, n-decane, and mineral oil). Free peptides were included as a control. The results are shown in Figure 10. The samples were left standing for 5 days before the test. Examination of the spectra of these different samples revealed that all of the colloid-bound peptides retained the structure of the free peptides, indicating that the peptides did not associate with each other to form beta sheets through the formation of intermolecular hydrogen bonds. Hydrophobic forces appear to be the main stabilizing forces for maintaining these structures. These 5-day-old samples were analyzed by dynamic light scattering, and it was shown that they maintained their 24-hour size and polydispersity values.

[0060] Discussion The first tests on colloidal particles containing soybean oil (SO) examined the lipid-to-peptide ratio required to encapsulate the lipophilic dye (Nile red). This was the first formulation (FS1) developed using the LEAP system, as it was shown to be effective to combine approximately 40 - 50 μL of soybean oil with 1 mM peptide in 1 mL of distilled water with a final volume. Its first application involving the encapsulation of lipophilic active ingredients was the use of the fungicide (captan) for the growth inhibition of Aspergillus nidulans A. nidulans ) spores in culture. To improve the solubility of captan, propionic acid was added to the formulation containing soybean oil in a 1:1 ratio to obtain a second formulation (FS2).

[0061] Subsequent experiments required a higher concentration of the active ingredient captan and incorporated a synergist (piperonyl butoxide, PBO) into the formulation, creating a third formulation (FS3) containing propionic acid, soybean oil, and PBO (1:1:2). This formulation has been shown to be effective for the assembly of LEAP as well as the encapsulation and delivery of lipophilic fungicides.

[0062] To determine any potential effects on the leaves, each component of the formulation was tested separately, and it was shown that propionic acid could cause leaf damage, but that this effect was suppressed in the presence of the LEAP peptide component.

[0063] In further tests, peptides were used to deliver the insecticide novaluron. To improve the stability of this active ingredient, a new basic formulation was created, leading to the development of a fourth formulation (FS4) containing diethyl phthalate, 10% DMSO, and soybean oil combined with 2 mg of peptide. This new formulation was tested for its lethal effect against the larvae of tobacco thrips ( M. sexta ) on tobacco plants, which are its commonly affected crops. This formulation demonstrated favorable colloid formation, showed no apparent cytotoxicity, exhibited efficient intracellular uptake, and improved the delivery of components used for the biological control of insects without causing any environmental effects.

[0064] These positive results of these tests currently support the preferred use of peptides for the cellular delivery of hydrophobic compounds / drugs that are too hydrophobic to be effectively delivered. The LEAP system has shown that it can be reformulated to accommodate two different hydrophobic active ingredients and in various non-polar excipients. Also, having lysine groups readily added in the aqueous phase enables the addition to various cell / tissue target moieties. This could be achieved by adding cysteine residues to the terminal lysine residues of the peptide for attachment to other chemical moieties via free sulfhydryl groups. By targeting these structures to specific cell types, the amount of the active ingredient required can be reduced and off-target effects can be limited.

[0065] Example 8 1 mg of LEAP (Ac-FLIVIKKKK-CONH2, SEQ ID NO: 1, where X is F) was dissolved in 500 μL of DDI-RO water. The solution was sonicated for 10 minutes and then left to stand at room temperature for approximately 60 minutes. This solution was further diluted to 10 mL with DDI-RO water and then subjected to dynamic light scattering analysis. The light scattering data was recorded using an Anton-Parr Litesizer 500 instrument and processed using the proprietary “Kalliope” analysis software. As a result of the DLS calculation, particles with a hydrodynamic diameter of 928.3 nm having a peak intensity value at 120.09 nm and a polydispersity index of 229.1% were shown. This data indicates that the peptides self-associate to form relatively small micelles on average, however, it has become clear from the polydispersity index that they are likely to aggregate into aggregates of many different sizes.

Claims

1. A composition comprising a plurality of colloidal particles suspended in an aqueous carrier, wherein each of the colloidal particles comprises a peptide matrix having a hydrophobic core and a hydrophilic outer surface, the peptide matrix comprising a plurality of linear peptides forming a peptide monolayer, each of the linear peptides comprising a hydrophobic segment directly bonded to a hydrophilic segment containing 1 to 7 lysine residues, wherein the hydrophilic segment is oriented outwardly in each particle to form the hydrophilic outer surface, the hydrophobic segment is oriented towards the hydrophobic core, and the hydrophobic core isolates a lipid, an oil, or a nonpolar solvent.

2. The composition according to claim 1, wherein the lipid, oil, or nonpolar solvent isolates one or more hydrophobic and / or poorly soluble active agents dispersed or distributed therein.

3. The composition according to claim 1 or 2, wherein the hydrophobic segment interacts with the lipid, oil, or nonpolar solvent in the hydrophobic core.

4. The composition according to claim 1, wherein the hydrophobic segment comprises XLIVI (SEQ ID NO: 2), where X is F or V, preferably F.

5. The composition according to claim 4, wherein the hydrophobic segment is capped with an acetyl group at the N-terminus.

6. The composition according to claim 1, wherein the hydrophilic segment comprises KKKKK (SEQ ID NO: 3).

7. The composition according to claim 6, further comprising a cysteine residue added to the C-terminal lysine residue.

8. The composition according to claim 1, wherein the peptide is FLIVIKKKKK (SEQ ID NO: 1, where X is F) or VLIVIKKKKK (SEQ ID NO: 1, where X is V).

9. The composition according to claim 1, wherein the outer surface comprises one or more functional moieties extending therefrom.

10. The composition according to claim 1, wherein the peptide matrix is homogeneous.

11. The composition according to claim 1, wherein the colloidal particles have a maximum surface-to-surface dimension of greater than about 100 nm.

12. The composition according to claim 1, having a polydispersity index of less than 30%.

13. The composition according to claim 1, wherein the composition has an effective period of at least 12 months.

14. The composition according to claim 2, or claim 3 which cites claim 2, wherein the active agent is present and is selected from the group consisting of fungicides, anti-cancer agents, insecticides, herbicides, and metabolic inhibitors.

15. The composition according to claim 1, which is a therapeutic composition administered to a patient.

16. A method for delivering a hydrophobic and / or poorly soluble active agent to a plant, the method comprising spraying the composition according to claim 2, or claim 3 which cites claim 2, onto at least a part of the plant and / or the soil in which the plant is planted or to be planted.

17. The method according to claim 16, wherein the active agent is an insecticide, and wherein the composition is sprayed onto at least a part of the plant and / or the soil in which the plant is planted or to be planted, where it comes into contact with or is ingested by pests.

18. A method for delivering a hydrophobic and / or poorly soluble active agent to an insect, the method comprising contacting the insect with the composition according to claim 2, or claim 3 which cites claim 2, the composition comprising an insecticidal active agent.

19. The method according to claim 18, wherein the composition is sprayed onto at least a part of the plant and / or the soil in which the plant is planted or to be planted such that the composition comes into contact with the insect.

20. A composition comprising a plurality of micelles suspended in an aqueous carrier, each micelle comprising a peptide monolayer membrane enclosing a liquid-receiving internal space, wherein the peptide is a linear peptide, and each linear peptide comprises a hydrophobic segment directly linked to a hydrophilic segment containing 1 to 7 lysine residues, wherein the hydrophilic segment is oriented outwardly in each particle to form a hydrophilic outer surface, and the hydrophobic segment faces the internal space of the micelle.

21. The composition according to claim 20, wherein the hydrophobic segment comprises XLIVI (SEQ ID NO: 2), wherein X is F or V, preferably F.

22. The composition according to claim 20, wherein the hydrophilic segment comprises KKKKK (SEQ ID NO: 3).

23. The composition according to claim 20, wherein the peptide is FLIVIKKKKKK (SEQ ID NO: 1, where X is F) or VLIKIKKKKKK (SEQ ID NO: 1, where X is V).

Images