Polypeptide-encapsulated complex lipid nanoparticles and uses thereof

Abstract

Disclosed herein are complex lipid particles encapsulating one or more exogenous peptides, polypeptides, or proteins, and methods of producing complex lipid formulations comprising exogenous peptides, polypeptides, or proteins. Also disclosed herein are modified plant messenger package (PMP) formulations encapsulating one or more exogenous peptides, polypeptides, or proteins, and methods of producing modified PMP formulations comprising exogenous peptides, polypeptides, or proteins.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 339,043, filed on May 6, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Polypeptides (e.g., proteins or peptides) are used in therapy (e.g., for treating a disease or condition), for diagnostic purposes, and as pathogen control agents. However, current methods for delivering polypeptides to cells may be limited by the delivery mechanism, such as the efficiency of polypeptide delivery to cells. Therefore, there is a need in the art for methods and compositions for delivering polypeptides to cells. Summary of the invention

[0004] One aspect of the present invention relates to a method for delivering a therapeutic peptide or protein to a human subject in need thereof. The method comprises orally or enterally administering to the human subject a pharmaceutical formulation comprising:

[0005] (a) a plurality of complex lipid particles characterized by: (i) comprising at least 10 plant lipids extracted from one or more plant sources; (ii) comprising sterols that are exogenous to the one or more plant sources; (iii) comprising polyethylene glycol (PEG) conjugated lipids; (iii) containing less than 10% w / w proteinaceous material that is endogenous to the one or more plant sources; and (iv) containing less than 10 mol% of exogenous ionizable lipids; and

[0006] (b) the therapeutic peptide or protein encapsulated in the composite lipid particle.

[0007] In some embodiments, the therapeutic peptide or protein is a hormone or a glucagon-like peptide 1 (GLP-1) agonist. In some embodiments, the therapeutic peptide or protein is insulin, exenatide, semaglutide, or tirzepatide.

[0008] In some embodiments, the therapeutic peptide or protein is delivered to brain tissue of the human subject.

[0009] In some embodiments, the complex lipid particle contains ten or more lipids belonging to one or more subclasses selected from the group consisting of acyl steryl glycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerols, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyldiacylglycerols, and sterols.

[0010] In some embodiments, the composite lipid particle contains lipids from at least five, at least six, at least seven, at least eight, at least nine, or at least ten different subclasses.

[0011] In some embodiments, the complex lipid particle contains less than 5% w / w proteinaceous material endogenous to the one or more plant sources.

[0012] In some embodiments, the composite lipid particle contains less than 5 mol % of exogenous ionizable lipids.

[0013] In some embodiments, at least one of the plant sources is grapefruit, lemon, pitaya, spinach, kale, strawberry, broccoli, or soybean.

[0014] In some embodiments, the complex lipid particle comprises:

[0015] about 85-95% w / w of said vegetable lipid,

[0016] about 5-8% w / w of said sterol,

[0017] About 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipid in the complex lipid formulation.

[0018] Another aspect of the present invention relates to a complex lipid formulation, comprising: a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and one or more exogenous peptides, polypeptides or proteins encapsulated in the complex lipid particles. The complex lipid particles are characterized by one or more of the following characteristics:

[0019] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0020] ii) contains less than 50 mol % ionizable lipids.

[0021] In certain embodiments, the composite lipid particle contains 5-1000 kinds of lipids extracted from one or more plant sources. In certain embodiments, the composite lipid particle contains at least 10 kinds of plant lipids, and the plant lipids belong to one or more of the categories selected from the group consisting of: glycerolipids, sphingolipids and sterols. For example, the composite lipid particle contains at least 15 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds, at least 60 kinds, at least 70 kinds, at least 80 kinds, at least 90 kinds, at least 100 kinds, at least 150 kinds, at least 200 kinds, at least 250 kinds, at least 300 kinds, at least 400 kinds, at least 500 kinds, at least 600 kinds, at least 700 kinds or at least 800 kinds of plant lipids, and the plant lipids belong to one or more of the categories selected from the group consisting of: glycerolipids, sphingolipids and sterols. In certain embodiments, the composite lipid particle contains lipids from at least two or at least three categories in these different categories.

[0022] In some embodiments, the composite lipid particles contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG) and sulfatides (SL). In some embodiments, the composite lipid particles contain one or more sphingolipids selected from the group consisting of glycosyl inositolphosphoceramide (GIPC), glucosylceramide (GCer), ceramide (Cer) and free long chain base (LCB). In some embodiments, the composite lipid particles contain one or more phytosterols selected from the group consisting of campesterol, stigmasterol and sitosterol.

[0023] In some embodiments, the complex lipid particle contains one or more lipids belonging to one or more subclasses selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesterol ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acids, fatty acid esters of hydroxy fatty acids, hemi-monoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine. Choline, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidyl, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, sterol ester, stigmasterol, sulfatide, sulfonic acid lipid, sphingomyelin, sulfoisorhamnosyldiacylglycerol, sterol and triacylglycerol. In certain embodiments, the composite lipid particle contains at least 10 plant lipids, and the plant lipids belong to one or more of the subclasses selected from the group consisting of the subclasses listed above. For example, the composite lipid particle contains at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 plant lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.

[0024] In some embodiments, the complex lipid particle contains ten or more lipids, the ten or more lipids belonging to one or more subclasses selected from the group consisting of acyl steryl glycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyldiacylglycerols, and sterols. For example, the composite lipid particle contains at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 plant lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.

[0025] In some embodiments, the composite lipid particle contains lipids from at least five, at least six, at least seven, at least eight, at least nine, or at least ten different subclasses listed above.

[0026] The composite lipid particles contain less than 50% w / w of protein substances that are endogenous to the one or more plant sources. For example, the composite lipid particles contain less than 45% w / w, less than 40% w / w, less than 35% w / w, less than 30% w / w, less than 25% w / w, less than 20% w / w, less than 15% w / w, less than 10% w / w, less than 9% w / w, less than 8% w / w, less than 7% w / w, less than 6% w / w, less than 5% w / w, less than 4% w / w, less than 3% w / w, less than 2% w / w, less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w or are substantially free of protein substances that are endogenous to the one or more plant sources. In certain embodiments, the composite lipid particles contain less than 30% w / w of protein substances that are endogenous to the one or more plant sources. In certain embodiments, the composite lipid particles contain less than 20% w / w of protein substances that are endogenous to the one or more plant sources. In some embodiments, the complex lipid particle contains less than 10% w / w proteinaceous material endogenous to the one or more plant sources. In some embodiments, the complex lipid particle contains less than 5% w / w proteinaceous material endogenous to the one or more plant sources.

[0027] The composite lipid particles can include reduced or minimized residual dsDNA materials that are endogenous to the one or more plant sources. For example, the composite lipid particles can contain less than 15% w / w, less than 10% w / w, less than 5% w / w, less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, less than 0.01% w / w, less than 0.005% w / w, less than 0.001% w / w or substantially free of residual dsDNA materials that are endogenous to the one or more plant sources. In some cases, the lipid bilayer of the composite lipid particles does not contain residual dsDNA. In certain embodiments, the composite lipid particles contain less than 1% w / w of residual dsDNA materials that are endogenous to the one or more plant sources. In certain embodiments, the composite lipid particles contain less than 0.1% w / w of residual dsDNA materials that are endogenous to the one or more plant sources. In some embodiments, the complex lipid particles contain less than 0.01% w / w residual dsDNA material endogenous to the one or more plant sources.

[0028] The composite lipid particles contain less than 50mol% ionizable lipids (e.g., ionizable lipids that are exogenous to one or more plant sources). For example, the composite lipid particles contain less than 45mol%, less than 40mol%, less than 35mol%, less than 30mol%, less than 25mol%, less than 20mol%, less than 15mol%, less than 10mol%, less than 9mol%, less than 8mol%, less than 7mol%, less than 6mol%, less than 5mol%, less than 4mol%, less than 3mol%, less than 2mol%, less than 1mol%, less than 0.5mol%, less than 0.1mol% or substantially free of ionizable lipids (e.g., ionizable lipids that are exogenous to one or more plant sources). In certain embodiments, the composite lipid particles contain less than 20mol% exogenous ionizable lipids. In certain embodiments, the composite lipid particles contain less than 5mol% exogenous ionizable lipids. In certain embodiments, the composite lipid particles are substantially free of exogenous ionizable lipids.

[0029] In some embodiments, the complex lipid formulation does not contain exogenous nucleic acid.

[0030] In some embodiments, at least one of the plant sources is a citrus fruit. For example, the citrus fruit can be a grapefruit or a lemon.

[0031] In some embodiments, at least one of the plant sources is a non-citrus plant. For example, the non-citrus plant can be pitaya, spinach, kale, strawberry, broccoli, or soybean.

[0032] In some embodiments, the one or more plant sources can be citrus fruits, non-citrus plants, or a combination thereof. In some embodiments, the one or more plant sources can be grapefruit, lemon, pitaya, spinach, kale, strawberry, broccoli, soybean, or a combination thereof.

[0033] In some embodiments, the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0034] In some embodiments, the exogenous lipid further comprises a lipid selected from the group consisting of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, a second sterol, and an additive synthetic lipid. In some embodiments, the exogenous lipid further comprises phosphatidylglycerol (PS), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), soybean PS, soybean PC, soybean PG, soybean PE, arachidonic acid, glucosylsitosterol, glucosylceramide, MGDG, DOPC, DLPC, DLPE, DGTS, DGDG, or a mixture thereof.

[0035] In some embodiments, the composite lipid particles comprise about 10-95% w / w of vegetable lipids. For example, based on the amount of total lipids in the composite lipid formulation, the composite lipid particles comprise about 25-95% w / w, about 30-95% w / w, about 35-95% w / w, about 40-95% w / w, about 45-95% w / w, about 50-95% w / w, about 55-95% w / w, about 60-95% w / w, about 65-95% w / w, about 70-95% w / w, about 75-95% w / w, about 80-95% w / w, or about 85-95% w / w of vegetable lipids.

[0036] In some embodiments, the complex lipid particle comprises:

[0037] about 10-95% w / w of said vegetable lipid,

[0038] about 5-60% w / w of said sterol,

[0039] About 0.5-15% w / w of the polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipids in the complex lipid formulation.

[0040] In some embodiments, the complex lipid particle comprises:

[0041] about 85-95% w / w of said vegetable lipid,

[0042] about 5-8% w / w of said sterol,

[0043] About 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipid in the complex lipid formulation.

[0044] Another aspect of the present invention relates to a modified plant messenger packet (PMP) formulation, comprising one or more PMPs modified with one or more sterols and one or more polyethylene glycol (PEG)-lipid conjugates, wherein the modified PMPs are formulated with one or more exogenous peptides, polypeptides or proteins, and wherein the one or more exogenous peptides, polypeptides or proteins are encapsulated by the modified PMPs.

[0045] In some embodiments, the PMP comprises a purified plant extracellular vesicle (EV) or a fragment or extract thereof. In some embodiments, the EV or a fragment or extract thereof is obtained from a citrus fruit, such as a grapefruit or a lemon.

[0046] In some embodiments, the PMP is obtained from citrus fruit such as grapefruit or lemon.

[0047] In some embodiments, the modified PMP is a lipophilic moiety selected from the group consisting of: lipid complexes, liposomes, lipid nanoparticles, polymer-based carriers, exosomes, lamellar bodies, micelles, and emulsions. In one embodiment, the modified PMP is a liposome selected from the group consisting of: cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In one embodiment, the modified PMP is a lipid nanoparticle.

[0048] In any of the above aspects of the invention relating to complex lipid formulations or modified PMP formulations, the following examples will be applicable.

[0049] The exogenous peptide, polypeptide or protein may be a therapeutic agent.

[0050] In some embodiments, the exogenous peptide, polypeptide or protein is an enzyme. In some embodiments, the enzyme is a recombinase or an editing enzyme.

[0051] In some embodiments, the exogenous peptide, polypeptide or protein is an antibody or antibody fragment.

[0052] In some embodiments, the exogenous peptide, polypeptide or protein is an Fc fusion protein.

[0053] In some embodiments, the exogenous peptide, polypeptide or protein is a hormone. In some embodiments, the exogenous peptide, polypeptide or protein is insulin.

[0054] In some embodiments, the exogenous peptide, polypeptide or protein is a peptide.

[0055] In some embodiments, the exogenous peptide, polypeptide or protein is a receptor agonist or a receptor antagonist. In some embodiments, the exogenous peptide, polypeptide or protein is a glucagon-like peptide 1 (GLP-1) agonist. In some embodiments, the exogenous peptide, polypeptide or protein is exenatide, semaglutide or tesiparatide.

[0056] In some embodiments, the exogenous peptide, polypeptide or protein is an antibody of Table 1, a peptide of Table 2, an enzyme of Table 3 or a protein of Table 4.

[0057] In some embodiments, the size of the exogenous peptide, polypeptide, or protein is less than 100 kD, less than 90 kD, less than 80 kD, less than 70 kD, less than 60 kD, less than 50 kD, less than 40 kD, less than 30 kD, less than 20 kD, or less than 10 kD. In some embodiments, the size of the exogenous peptide, polypeptide, or protein is less than 50 kD. In some embodiments, the size of the exogenous peptide, polypeptide, or protein is at least 3 kD, at least 4 kD, or at least 5 kD. In some embodiments, the size of the exogenous peptide, polypeptide, or protein is at least 3 kD. In some embodiments, the size of the exogenous peptide, polypeptide, or protein is at least 5 kD.

[0058] In some embodiments, the exogenous peptide, polypeptide or protein comprises at least 10, at least 20, at least 30, at least 40 or at least 50 amino acid residues. In some embodiments, the exogenous peptide, polypeptide or protein comprises at least 30 amino acid residues. In some embodiments, the exogenous peptide, polypeptide or protein comprises at least 50 amino acid residues.

[0059] In some embodiments, the overall charge of the exogenous peptide, polypeptide, or protein is neutral. In some embodiments, the exogenous peptide, polypeptide, or protein has been modified to be neutrally charged. In some embodiments, the overall charge of the exogenous peptide, polypeptide, or protein is positive. In some embodiments, the overall charge of the exogenous peptide, polypeptide, or protein is negative.

[0060] In some embodiments, the exogenous peptide, polypeptide or protein can be modified (e.g., lipid-modified, such as a lipid tail). In some embodiments, the exogenous peptide, polypeptide or protein can be a lipopeptide. In some embodiments, the exogenous peptide, polypeptide or protein can be synthetic or contain synthetic amino acids.

[0061] In some embodiments, the sterol is cholesterol or sitosterol.

[0062] In some embodiments, the PEG-lipid conjugate is C14-PEG2k or C18-PEG2k. In some embodiments, the PEG-lipid conjugate is PEG-DMG or PEG-PE. In some embodiments, PEG-DMG is PEG2000-DMG or PEG2000-PE. In some embodiments, the PEG-lipid conjugate is PEG2000-PE, PEG2000-DMG, PEG2000-DSPE or a derivative thereof. In some embodiments, the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof. For example, the PEG-lipid conjugate is DSPE-PEG2000.

[0063] In some embodiments, the sterol is cholesterol or sitosterol, and the PEG-lipid conjugate is C18-PEG2000PE or a derivative thereof. In some embodiments, the sterol is cholesterol or sitosterol, and the PEG-lipid conjugate is DSPE-PEG2000.

[0064] In some embodiments, the concentration of sterol in the composite lipid particle or in the modified PMP ranges from about 5 to 60% w / w, such as about 5 to 50% w / w, about 5 to 40% w / w, about 5 to 30% w / w, about 5 to 20% w / w, about 5, about 15% w / w, about 0.5 to 15% w / w, about 5 to 8% w / w, or about 6 to 7% w / w, based on the amount of total lipid in the composite lipid particle or in the modified PMP. In some embodiments, the sterol ranges from about 15 to 20% w / w, about 20 to 30% w / w, about 30 to 40% w / w, about 40 to 50% w / w, or about 50 to 60% w / w, based on the amount of total lipid in the composite lipid particle or in the modified PMP.

[0065] In some embodiments, the concentration of the PEG-lipid conjugate ranges from about 0.5 to 5% w / w, about 0.5 to 3.5% w / w, about 1 to 3.5% w / w, about 0.5 to 3% w / w, about 1 to 3% w / w, about 0.5 to 2.5% w / w, about 1 to 2.5% w / w, about 1.5 to 2.5% w / w, or about 2 to 2.5% w / w, based on the amount of total lipids in the complex lipid particle or in the modified PMP. In some embodiments, the concentration of the PEG-lipid conjugate ranges from about 0.5 to 15% w / w, about 1 to 15% w / w, about 2 to 5% w / w, about 5 to 8% w / w, about 8 to 12% w / w, or about 12 to 15% w / w, based on the amount of total lipids in the complex lipid particle or in the modified PMP.

[0066] In some embodiments, the sterol is cholesterol or sitosterol in a concentration range of about 5 to 50% w / w based on the amount of total lipids in the complex lipid particle or the modified PMP; and the PEG-lipid conjugate is PEG2000-PE, PEG2000-DMG, PEG2000-DSPE or a derivative thereof in a concentration range of about 1 to 3.5% w / w based on the amount of total lipids in the complex lipid particle or the modified PMP.

[0067] In some embodiments, the sterol is cholesterol or sitosterol at a concentration ranging from about 5 to 8% w / w based on the amount of total lipids in the complex lipid particle or modified PMP; and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof at a concentration ranging from about 1 to 3.5% w / w based on the amount of total lipids in the complex lipid particle or modified PMP.

[0068] In some embodiments, the sterol is cholesterol or sitosterol at a concentration ranging from about 20 to 25% w / w based on the amount of total lipids in the complex lipid particle or the modified PMP; and the PEG-lipid conjugate is DSPE-PEG2000 or a derivative thereof at a concentration ranging from about 1 to 3.5% w / w based on the amount of total lipids in the complex lipid particle or the modified PMP.

[0069] In some embodiments, the sterol is cholesterol or sitosterol at a concentration ranging from about 5 to 8% w / w based on the amount of total lipids in the complex lipid particle or modified PMP; and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof at a concentration ranging from about 1 to 3.5% w / w based on the amount of total lipids in the complex lipid particle or modified PMP.

[0070] The average size of the composite lipid particles or modified PMPs can be less than about 400nm, less than about 350nm, less than about 300nm, less than about 250nm, or less than about 200nm. In one embodiment, the average size of the composite lipid particles or modified PMPs is less than about 200nm. In one embodiment, the average size of the composite lipid particles or modified PMPs is about 100 to 180nm. In one embodiment, the average size of the composite lipid particles or modified PMPs is about 100 to 160nm.

[0071] The dispersibility index (PDI) of the composite lipid particle or modified PMP can be less than about 0.7, less than about 0.6, less than about 0.5 or less than about 0.4. For example, the range of the PDI of the composite lipid particle or modified PMP can be about 0.1 to about 0.7, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, about 0.2 to about 0.7, about 0.2 to about 0.6, about 0.2 to about 0.5 or about 0.2 to about 0.4. In one embodiment, the PDI of the composite lipid particle is about 0.1 to about 0.5. In one embodiment, the PDI of the composite lipid particle is about 0.2 to about 0.4.

[0072] In some embodiments, the composite lipid particle formulation or modified PMP formulation, such as aqueous phase, further comprises phosphate, citrate, sodium bicarbonate, HEPES, TAE or TRIS buffer. In some embodiments, the composite lipid particle formulation or modified PMP formulation further comprises water, PBS or bicarbonate. In some embodiments, the composite lipid particle formulation or modified PMP formulation further comprises a bicarbonate buffer having a molarity of 0.001M to 0.1M. The pH of the buffer solution can be about 3.0 to about 8.5. The pH of the HEPES or TRIS buffer can be about 7.0 to about 8.5. The concentration of the HEPES or TRIS buffer can be about 7mg / mL to about 15mg / mL. The aqueous phase can further comprise NaCl of about 2.0mg / mL to about 4.0mg / mL. In one embodiment, the aqueous phase comprises a citrate buffer having a pH of about 3.0 to about 3.2. In one embodiment, the aqueous phase comprises a sodium bicarbonate buffer having a pH of about 8.0 to about 8.2.

[0073] In some embodiments, the complex lipid particle formulation or modified PMP formulation further comprises one or more cryoprotectants. The one or more cryoprotectants can be sucrose, glycerol, mannitol, or a combination thereof. In one embodiment, the modified PMP formulation comprises 2-5% sucrose, 2-5% mannitol, or a combination thereof. In one embodiment, the modified PMP formulation comprises 0-0.5% sucrose, 0-0.5% mannitol, or a combination thereof. In one embodiment, the modified PMP comprises 0.5-2% sucrose, 0.5-2% mannitol, or a combination thereof.

[0074] In some embodiments, the composite lipid particle formulation or modified PMP formulation is a lyophilized composition. The lyophilized composition may include one or more lyoprotectants. The lyophilized composition may include poloxamer, potassium sorbate, sucrose or any combination thereof. In one embodiment, the lyophilized composition includes poloxamer, such as poloxamer 188.

[0075] In some embodiments, the complex lipid particle formulation is not lyophilized. In some embodiments, the complex lipid formulation is a liquid composition.

[0076] In some embodiments, the composite lipid particle formulation or modified PMP formulation is stable at room temperature for at least one day, and / or at least one day, at least one week, or at least one month at 4°C, with or without lyophilization. In some embodiments, the composite lipid particle formulation or modified PMP formulation is stable at 4°C for at least 24 hours, 48 ​​hours, seven days, or 30 days, with or without lyophilization. In one embodiment, the composite lipid formulation is stable at room temperature and / or at 4°C for at least two weeks without lyophilization. In some embodiments, the composite lipid particle formulation or modified PMP formulation is stable at a temperature of at least 20°C, 24°C, or 37°C.

[0077] Some embodiments provide a composition comprising a plurality of modified PMP formulations of any of the above embodiments. In some embodiments, the concentration of the modified PMP formulation in the composition is effective to increase the adaptability of the mammal.

[0078] Some embodiments provide a composition comprising a plurality of complex lipid particle formulations of any of the above embodiments. In some embodiments, the concentration of the complex lipid particle formulation in the composition is effective to increase the fitness of a mammal.

[0079] In some embodiments, the concentration of the exogenous peptide, polypeptide or protein in the complex lipid particle formulation is at least 0.01, 0.1, 0.2, 0.3, 0.4, 0.5 or 1 μg / mL. In some embodiments, the concentration of the exogenous peptide, polypeptide or protein is at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or 200 μg / mL.

[0080] In some embodiments, at least 15% of the composite lipid particles in the multiple composite lipid particle formulations encapsulate exogenous peptides, polypeptides or proteins. In some embodiments, at least 50% of the composite lipid particles in the multiple composite lipid particle formulations encapsulate exogenous peptides, polypeptides or proteins. In some embodiments, at least 95% of the composite lipid particles in the multiple composite lipid particle formulations encapsulate exogenous peptides, polypeptides or proteins.

[0081] In some embodiments, at least 15% of the modified PMPs in the plurality of modified PMP formulations encapsulate exogenous peptides, polypeptides, or proteins. In some embodiments, at least 50% of the modified PMPs in the plurality of modified PMP formulations encapsulate exogenous peptides, polypeptides, or proteins. In some embodiments, at least 95% of the modified PMPs in the plurality of modified PMP formulations encapsulate exogenous peptides, polypeptides, or proteins.

[0082] Another aspect of the present invention relates to a pharmaceutical composition or pharmaceutical formulation comprising the complex lipid formulation as described herein and a pharmaceutically acceptable vehicle, carrier or excipient.

[0083] In some embodiments, the pharmaceutical composition or pharmaceutical preparation is in an oral dosage form, such as a capsule dosage form or a tablet dosage form.

[0084] All of the above descriptions and all of the embodiments discussed in the above aspects relating to complex lipid formulations or complex lipid particles are applicable to these aspects of the invention relating to pharmaceutical formulations comprising complex lipid formulations.

[0085] In another aspect, the disclosure features a pharmaceutical composition comprising a modified PMP formulation according to any one of the above embodiments and a pharmaceutically acceptable vehicle, carrier, or excipient.

[0086] In some embodiments, the pharmaceutical composition or pharmaceutical formulation is formulated for administration to a mammal, such as a human subject. In some embodiments, the pharmaceutical composition or pharmaceutical formulation is formulated for administration to a mammalian cell.

[0087] In any of the above aspects of the invention relating to pharmaceutical compositions or pharmaceutical formulations, the following examples will be applicable.

[0088] Another aspect of the present invention relates to a method for producing a complex lipid formulation comprising a plurality of complex lipid particles encapsulating an exogenous peptide, polypeptide or protein. The method comprises:

[0089] extracting at least five lipids from one or more plant sources;

[0090] mixing at least two exogenous lipids with the extracted plant lipids to form composite lipid particles; and

[0091] The complex lipid particles are loaded with the exogenous peptide, polypeptide or protein, wherein the loading causes the exogenous peptide, polypeptide or protein to be encapsulated by the complex lipid particles, thereby forming the complex lipid formulation.

[0092] All of the above descriptions and all of the embodiments discussed in the above aspects relating to complex lipid formulations or complex lipid particles are applicable to these aspects of the invention relating to methods of producing complex lipid formulations.

[0093] In some embodiments, the lipids are extracted from the plant source by adding an extraction solvent to one or more plant sources, the extraction solvent comprising methanol, ethanol, propanol, 1-butanol, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, methyl tert-butyl ether, chloroform, ethyl acetate, or a mixture thereof. In some embodiments, the extraction solvent is dichloromethane:methanol; chloroform:methanol; methanol:methyl tert-butyl ether (MTBE); dimethylformamide:methanol; acetonitrile:methanol; acetone:methanol; tetrahydrofuran:methanol; dimethyl sulfoxide:methanol; acetonitrile:ethanol; or ethyl acetate:ethanol.

[0094] In some embodiments, the extracting step further comprises reducing proteinaceous material endogenous to the one or more plant sources to less than 50% w / w, less than 45% w / w, less than 40% w / w, less than 35% w / w, less than 30% w / w, less than 25% w / w, less than 20% w / w, less than 15% w / w, less than 10% w / w, less than 9% w / w, less than 8% w / w, less than 7% w / w, less than 6% w / w, less than 5% w / w, less than 4% w / w, less than 3% w / w, less than 2% w / w, less than 1% w / w, less than 0.5% w / w, or less than 0.1% w / w, or substantially completely eliminating proteinaceous material endogenous to the one or more plant sources.

[0095] The composite lipid particles can include reduced or minimized residual dsDNA materials that are endogenous to the one or more plant sources. For example, the composite lipid particles can contain less than 15% w / w, less than 10% w / w, less than 5% w / w, less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, less than 0.01% w / w, less than 0.005% w / w, less than 0.001% w / w or substantially free of residual dsDNA materials that are endogenous to the one or more plant sources. In some cases, the lipid bilayer of the composite lipid particles does not contain residual dsDNA. In certain embodiments, the composite lipid particles contain less than 1% w / w of residual dsDNA materials that are endogenous to the one or more plant sources. In certain embodiments, the composite lipid particles contain less than 0.1% w / w of residual dsDNA materials that are endogenous to the one or more plant sources. In some embodiments, the complex lipid particles contain less than 0.01% w / w residual dsDNA material endogenous to the one or more plant sources.

[0096] In some embodiments, the mixing step is performed by thin film mixing or microfluidic mixing.

[0097] In some embodiments, the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0098] In some embodiments, the exogenous lipids do not include ionizable lipids. Thus, the complex lipid formulation is prepared without adding exogenous ionizable lipids.

[0099] In another aspect, the disclosure features a method of producing a modified PMP formulation comprising an exogenous peptide, polypeptide, or protein, the method comprising: (a) providing a solution comprising a modified PMP, wherein the modified PMP contains one or more PMPs, one or more sterols, and one or more polyethylene glycol (PEG)-lipid conjugates; providing a solution comprising the exogenous peptide, polypeptide, or protein; and (b) loading the modified PMP with the exogenous peptide, polypeptide, or protein, wherein the loading causes the exogenous peptide, polypeptide, or protein to be encapsulated by the modified PMP.

[0100] In some embodiments, the exogenous peptide, polypeptide, or protein is soluble in solution.

[0101] In some embodiments, loading comprises one or more of ultrasonic treatment, electroporation and lipid extrusion. In some embodiments, loading comprises ultrasonic treatment and lipid extrusion. In some embodiments, loading comprises lipid extrusion. In some embodiments, the PMP lipid is separated before lipid extrusion. In some embodiments, the PMP lipid separated comprises glycosyl inositol phosphorylceramide (GIPC).

[0102] Another aspect of the present invention relates to a method for delivering a peptide, polypeptide or protein to a mammalian cell or a mammal. The method comprises contacting the mammalian cell with the complex lipid formulation or administering the complex lipid formulation to the mammal under conditions sufficient to allow the mammalian cell or the mammal to take up the complex lipid formulation. The complex lipid formulation comprises:

[0103] a plurality of complex lipid particles, each complex lipid particle of the plurality of complex lipid particles comprising at least five plant lipids and at least two exogenous lipids; and

[0104] One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties:

[0105] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0106] ii) contains less than 50 mol % ionizable lipids.

[0107] In some embodiments, the mammalian cell is a cell in a human body, or the mammal is a human. In some embodiments, the uptake of the exogenous peptide, polypeptide or protein encapsulated by the composite lipid particle is increased by the mammalian cell or the mammal relative to the uptake of the exogenous peptide, polypeptide or protein not encapsulated by the composite lipid particle.

[0108] In some embodiments, the methods are for delivering a peptide, polypeptide or protein to a mammal and the administration is via an oral, enteral, intranasal, intrarectal (including intracolonic) or intrajejunal route.

[0109] In some embodiments, the mammalian cell is a brain cell.

[0110] Another aspect of the present invention relates to a method for treating or preventing a disease or condition for which a therapeutic agent is suitable for use in a subject. The method comprises administering to the subject in need thereof an effective amount of a complex lipid formulation comprising:

[0111] a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and

[0112] One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties:

[0113] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0114] ii) contains less than 50 mol % ionizable lipids.

[0115] In some embodiments, administration is via an oral, enteral, intranasal, intrarectal (including intracolonic), or intrajejunal route.

[0116] In some embodiments, the disease is diabetes and the exogenous peptide, polypeptide or protein is insulin, exenatide, semaglutide or tesiparatide.

[0117] In another aspect, the disclosure features a method for delivering a peptide, polypeptide or protein to a mammalian cell, the method comprising contacting the cell with a modified PMP formulation according to any of the above embodiments, wherein the contacting is performed in an amount and for a time sufficient to allow the cell to take up the modified PMP formulation. In some embodiments, the cell is a cell in a subject.

[0118] In some embodiments, the exogenous peptide, polypeptide or protein is released from the modified PMP formulation in the mammalian cell contacted with the modified PMP formulation. In some embodiments, the exogenous peptide, polypeptide or protein is active in the cytoplasm of the mammalian cell. In some embodiments, the exogenous peptide, polypeptide or protein is translocated to the nucleus of the mammalian cell. In some embodiments, the exogenous peptide, polypeptide or protein is active in the nucleus of the mammalian cell.

[0119] In another aspect, the disclosure features a method for delivering a peptide, polypeptide, or protein to a mammal, the method comprising administering to the mammal a modified PMP formulation according to any of the above embodiments, wherein the administration is performed under conditions sufficient to allow the mammal to ingest the modified PMP formulation.

[0120] In another aspect, the disclosure features the modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, wherein the mammal is a human.

[0121] In another aspect, the disclosure features a modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, wherein cellular uptake of an exogenous peptide, polypeptide, or protein encapsulated by the modified PMP formulation is increased relative to uptake of an exogenous peptide, polypeptide, or protein not encapsulated by the modified PMP formulation.

[0122] In another aspect, the disclosure features a modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, wherein the effectiveness of an exogenous peptide, polypeptide, or protein encapsulated by the modified PMP formulation is increased relative to the effectiveness of an exogenous peptide, polypeptide, or protein not encapsulated by the modified PMP formulation.

[0123] In another aspect, the disclosure features a method for treating or preventing a disease or condition for which a therapeutic agent is indicated in a subject, the method comprising administering to the subject in need thereof an effective amount of a modified PMP formulation according to any of the above embodiments, wherein the therapeutic agent is an exogenous peptide, polypeptide or protein encapsulated by the modified PMP in the modified PMP formulation.

[0124] In some embodiments, the disease or condition is diabetes. In some embodiments, administration of the modified PMP formulation reduces blood glucose in a subject. In some embodiments, the exogenous peptide, polypeptide or protein is insulin.

[0125] In another aspect, the disclosure features a modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, wherein the modified PMP formulation is not significantly degraded by gastric fluid, e.g., not significantly degraded by fasting gastric fluid.

[0126] In another aspect, the disclosure features the method of any of the above embodiments, wherein the administering is via an oral, enteral, intranasal, or intrarectal (including intracolonic) route. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] FIG. 1 is a scheme showing an exemplary formulation method for preparing composite lipid particles containing naturally derived lipids and exogenous lipids, as described in Example 2.

[0128] Figure 2 is a scheme illustrating an exemplary method for loading biologically active molecules into composite lipid particles containing naturally derived lipids and exogenous lipids, as described in Example 2.

[0129] Figure 3 is a graph showing the insulin concentration in mouse plasma one hour after intrarectal administration of an insulin-loaded complex lipid formulation containing natural-origin lipids and exogenous lipids. The dashed line reflects the baseline for fusogenic liposomes containing insulin administered directly into the colon (10 U / kg, 12 minutes after injection).

[0130] Figure 4 is a graph showing insulin concentrations in the mouse brain two hours after intranasal administration of a complex lipid formulation containing insulin-loaded natural lipids and exogenous lipids. The dashed line reflects the concentration of insulin in the mouse brain 90 minutes after intranasal co-administration. Cell penetrating peptides and Free insulin is used as a benchmark for brain delivery of 10 U / kg insulin enhanced by insulin. DETAILED DESCRIPTION

[0131] I. Definitions

[0132] As used herein, the term "encapsulate or encapsulated" refers to a closed lipid membrane structure, such as a portion (e.g., an exogenous peptide, polypeptide, or protein as defined herein) within a lipid bilayer. The lipid membrane structure can be, for example, a plant messenger packet (PMP) or a plant extracellular vesicle (EV), or can be obtained from a plant EV or derived from a plant EV. The encapsulated portion (e.g., an encapsulated exogenous peptide, polypeptide, or protein) is enclosed by a lipid membrane structure, for example, such an encapsulated portion is located in the lumen of a closed lipid membrane structure (e.g., the lumen of a PMP). In some cases, the encapsulated portion (e.g., an encapsulated peptide, polypeptide, or protein) can interact or associate with the inner face of the lipid membrane structure. In some cases, an exogenous peptide, polypeptide, or protein can be embedded in a lipid membrane structure. In some cases, an exogenous peptide, polypeptide, or protein has an extraluminal portion. Alternatively, the term "encapsulate" or "encapsulated" can be used in the context of using a composite lipid particle to enclose a portion (e.g., an exogenous peptide, polypeptide, or protein as defined herein) within the composite lipid particle. In some cases, "encapsulation" can be used in the context of using a modified PMP to enclose a portion (e.g., an exogenous peptide, polypeptide, or protein as defined herein).

[0133] As used herein, the term "exogenous peptide, polypeptide or protein" refers to a peptide, polypeptide or protein (as defined herein) encapsulated by a complex lipid particle or a modified PMP (e.g., a PMP derived from a plant extracellular vesicle and modified with one or more exogenous lipids), which is not naturally present in a plant lipid vesicle (e.g., not naturally present in a plant extracellular vesicle) or is encapsulated in a complex lipid particle or a modified PMP, and its content is not found in a naturally occurring plant extracellular vesicle. In some cases, the exogenous peptide, polypeptide or protein may be naturally present in the plant from which the plant lipid or PMP is extracted. In other cases, the exogenous peptide, polypeptide or protein is not naturally present in the plant from which the plant lipid or PMP is extracted. The exogenous peptide, polypeptide or protein can be artificially expressed in the plant from which the plant lipid or PMP is extracted, for example, it can be a heterologous polypeptide. The exogenous peptide, polypeptide or protein can be derived from another organism. In some embodiments, exogenous peptides, polypeptides or proteins are loaded into complex lipid particles or modified PMP formulations, for example, using one or more of sonication, electroporation, lipid extraction and lipid extrusion. Exogenous peptides, polypeptides or proteins can be, for example, therapeutic agents, enzymes (e.g., recombinant enzymes or editing enzymes), hormones (e.g., insulin), receptor agonists or receptor antagonists (e.g., GLP-1 agonists, such as exenatide, semaglutide or tesiparatide) or pathogen control agents.

[0134] As used herein, "delivery" or "contacting" refers to providing or administering a complex lipid particle formulation or a modified PMP formulation (e.g., a modified PMP formulation comprising an exogenous protein or peptide) to an organism, such as an animal. Delivery to an animal can be, for example, oral or enteral delivery (e.g., delivery by feeding or into the GI tract, such as by gavage) or systemic delivery (e.g., delivery by injection). The modified PMP formulation can be delivered to the digestive tract, such as the stomach, small intestine, or large intestine. The complex lipid particle formulation or the modified PMP formulation can be stable in the digestive tract.

[0135] As used herein, the term "animal" refers to humans and non-human animals (including, for example, dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, chickens, and non-human primates).

[0136] As used herein, the term "formulated for delivery to an animal" refers to a complex lipid particle formulation or a modified PMP formulation that includes a pharmaceutically acceptable carrier.

[0137] As used herein, the term "infection" refers to the presence or colonization of a pathogen in an animal (e.g., in one or more parts of an animal), on an animal (e.g., on one or more parts of an animal), or in the habitat surrounding an animal, particularly where the infection reduces the fitness of the animal, such as by causing disease, disease symptoms, or an immune (e.g., inflammatory) response.

[0138] As used herein, the term "pathogen" refers to an organism, such as a microorganism or invertebrate, that causes disease or disease symptoms in an animal by, for example: (i) directly infecting an animal, (ii) producing an agent that causes disease or disease symptoms in an animal (e.g., bacteria that produce a pathogenic toxin, etc.), and / or (iii) by eliciting an immune (e.g., inflammatory response) in an animal (e.g., biting insects, e.g., bed bugs). As used herein, pathogens include, but are not limited to, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids, and viruses, or any combination thereof, each of which is capable of causing disease or symptoms in humans by itself or in conjunction with another pathogen.

[0139] As used herein, the terms "polypeptide," "peptide," or "protein" encompass any chain of naturally or non-naturally occurring amino acids (D-amino acids or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more than 1000 amino acids), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of, for example, one or more non-amino acyl groups (e.g., sugars, lipids, etc.) covalently attached to the polypeptide, and include, for example, native polypeptides, synthetic or recombinant polypeptides, hybrid molecules, peptoids, or peptidomimetics. The size of the polypeptide can be, for example, at least 0.1, at least 1, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50 or greater than 50 kD. The polypeptide can be a full-length protein. Alternatively, the polypeptide can comprise one or more domains of a protein.

[0140] As used herein, the term "antibody" encompasses immunoglobulins that can specifically bind to an antigen, whether natural or partially or completely synthetically produced, and fragments thereof. The term also encompasses any protein having a binding domain homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources, or partially or completely synthetically produced. "Antibody" further includes polypeptides that specifically bind to antigens and recognize antigens comprising framework regions from immunoglobulin genes or fragments thereof. The use of the term "antibody" is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies and recombinant antibodies, fragments thereof, and further includes single-chain antibodies (nano antibodies), humanized antibodies, mouse antibodies, chimeric antibodies, mouse-human antibodies, mouse-primate antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments, such as scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, bifunctional antibodies and antibody-related polypeptides. "Antibody" further includes bispecific antibodies and multispecific antibodies.

[0141] As used herein, the term "antigen binding fragment" refers to a fragment of a complete immunoglobulin, and any part of a polypeptide, including an antigen binding region with the ability to specifically bind to an antigen. For example, an antigen binding fragment may be a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, or a scFv fragment, but is not limited thereto. The Fab fragment has an antigen binding site and contains the variable regions of the light chain and the heavy chain, the constant region of the light chain, and the first constant region CH1 of the heavy chain. The difference between the Fab' fragment and the Fab fragment is that the Fab' fragment additionally includes the hinge region of the heavy chain, including at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 region. Produce a F(ab')2 fragment, whereby the cysteine ​​residues of the Fab' fragment are connected by disulfide bonds at the hinge region. The Fv fragment is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and the recombinant technology for producing Fv fragments is well known in the art. The double-chain Fv fragment may have a structure in which the heavy chain variable region is connected to the light chain variable region by a non-covalent bond. Single-chain Fv (scFv) fragments can generally have a dimer structure, such as in a double-chain Fv fragment, in which the heavy chain variable region is covalently bound to the light chain variable region via a peptide linker, or the heavy chain variable region and the light chain variable region are directly connected to each other at their C-termini. Antigen-binding fragments can be obtained using proteases (e.g., digesting whole antibodies with papain to obtain Fab fragments, and digesting with pepsin to obtain F(ab')2 fragments), and can be prepared by genetic recombination techniques. dAb fragments consist of VH domains.

[0142] A single-chain antibody molecule may comprise a polymer having many individual molecules, for example, a dimer, trimer or other polymer.

[0143] As used herein, the term "heterologous" refers to an agent (e.g., a polypeptide) that is (1) exogenous to the plant (e.g., derived from a source other than the plant or plant part that produces the PMP) (e.g., an agent added to the PMP using the loading methods described herein) or (2) endogenous to the plant cells or tissues that produce the PMP, but is present in the PMP at a concentration greater than that found in nature (e.g., greater than that found in naturally occurring plant extracellular vesicles) (e.g., added to the PMP using the loading methods, genetic engineering, and in vitro or in vivo methods described herein).

[0144] As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, which is described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0145] As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds and their offspring. Plant cells include, but are not limited to, cells from seeds, suspension cultures, plumules, meristem zones, calluses, leaves, roots, buds, gametophytes, sporophytes, pollen and microspores. Plant parts include differentiated and undifferentiated tissues including, but not limited to, roots, stems, buds, leaves, pollen, seeds, fruits, harvests, tumor tissues and various forms of cells and cultures (e.g., unicellular, protoplasts, plumules and calluses). Plant tissues may be in plants or in plant organs, tissues or cell cultures. In addition, plants may be genetically engineered to produce heterologous proteins or RNA.

[0146] As used herein, the term "complex lipid particles" refers to lipid particles with complexity, characterized in that it comprises a variety of lipids, including lipids extracted from one or more plant sources. The composite lipid particles can include 10% w / w to 99% w / w of the lipid structure derived from one or more plant sources, for example, can contain at least 10% w / w, at least 20% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w or about 99% w / w of the lipid structure derived from one or more plant sources. The composite lipid particles can contain 5-1000 kinds of lipids extracted from one or more plant sources. The composite lipid particles can contain at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 different classes or subclasses of the lipid from plant sources. The composite lipid particle can comprise all or a portion of lipid species present in the lipid structure from plant origin, for example, the composite lipid particle can contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or almost 100% lipid species present in the lipid structure from plant origin. The composite lipid particle can comprise reduction or minimized protein substances that are endogenous to the one or more plant sources, for example, can contain 0%w / w, be less than 1%w / w, be less than 5%w / w, be less than 10%w / w, be less than 15%w / w, be less than 20%w / w, be less than 30%w / w, be less than 40%w / w or be less than 50%w / w protein substances that are endogenous to the one or more plant sources. In some cases, the lipid bilayer of the composite lipid particle does not contain protein.

[0147] The compound lipid particle can further comprise at least two exogenous lipids.The compound lipid particle can comprise the exogenous lipid of at least 1%w / w, at least 2%w / w, at least 5%w / w, at least 10%w / w, at least 15%w / w, at least 20%w / w, at least 25%w / w, at least 30%w / w, at least 40%w / w, at least 50%w / w, at least 60%w / w, at least 70%w / w, at least 80%w / w or about 90%w / w.Exemplary exogenous lipid comprises sterol and PEG-lipid conjugate.The compound lipid particle can be used for encapsulating exogenous peptide, polypeptide or protein so that exogenous peptide, polypeptide or protein can be delivered to target cell or tissue.

[0148] As used herein, the term "plant extracellular vesicle", "plant EV" or "EV" refers to a closed lipid bilayer structure naturally present in plants. Optionally, plant EVs include one or more plant EV markers. As used herein, the term "plant EV marker" refers to a component naturally associated with a plant, such as a plant protein, a plant nucleic acid, a plant small molecule, a plant lipid, or a combination thereof, including but not limited to any plant EV marker listed in the appendix disclosed in WO 2021 / 041301 (which is incorporated herein by reference in its entirety). In some cases, a plant EV marker is an identification marker for a plant EV, but is not a pesticide. In some cases, a plant EV marker is an identification marker for a plant EV and is also a pesticide (e.g., associated with or encapsulated by multiple PMPs, or not directly associated with or encapsulated by multiple PMPs).

[0149] As used herein, the term "plant messenger packet" or "PMP" refers to a lipid structure (e.g., a lipid bilayer, a monolayer, a multilayer structure; e.g., a vesicular lipid structure) having a diameter of about 5-2000 nm (e.g., at least 5-1000 nm, at least 5-500 nm, at least 400-500 nm, at least 25-250 nm, at least 50-150 nm, or at least 70-120 nm) derived from a plant source or a segment, part, or extract thereof (e.g., enriched, isolated, or purified therefrom), which includes lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated therewith that have been enriched, isolated, or purified from a plant, plant part, or plant cell, wherein the enrichment or isolation removes one or more contaminants or undesirable components from the source plant. The PMP can be a highly purified preparation of naturally occurring EVs. Preferably, at least 1% of one or more contaminants or undesirable components from a source plant are removed (e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99% or 100%), wherein the source plant is, for example, a plant cell wall component; pectin; a plant organelle (e.g., mitochondria; plastids such as chloroplasts, leucoplasts or amyloplasts; and a nucleus); a plant chromatin (e.g., a plant chromosome); or a plant molecular aggregate (e.g., a protein aggregate, a protein-nucleic acid aggregate, a lipoprotein aggregate or a lipid-protein structure). Preferably, the PMP is at least 30% pure (e.g., at least 40% pure, at least 50% pure, at least 60% pure, at least 70% pure, at least 80% pure, at least 90% pure, at least 99% pure, or 100% pure) relative to one or more contaminants or undesirable components from the source plant as measured by weight (w / w), spectral imaging (% transmittance), or conductivity (S / m).

[0150] The PMP can be modified to include exogenous lipids, such as lipids that are (1) exogenous to the plant (e.g., derived from a source other than the plant or plant part that produces the PMP) (e.g., added using the methods described herein) or (2) endogenous to the plant cell or tissue that produces the PMP, but present in the PMP (e.g., added to the PMP using the methods described herein, genetic engineering, in vitro, or in vivo methods) at a concentration greater than that found in nature (e.g., greater than that found in naturally occurring plant extracellular vesicles). The lipid composition of the PMP can include 0%, less than 1%, or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% exogenous lipids. Exemplary exogenous lipids include cationic lipids, ionizable lipids, zwitterionic lipids, and lipidoids.

[0151] PMPs can be modified to optionally include additional agents, such as polypeptides (e.g., peptides or proteins), therapeutic agents, polynucleotides or small molecules. PMPs can carry additional agents (e.g., polypeptides) or associate with additional agents in a variety of ways to enable delivery of the agents to target plants, such as by encapsulating the agents, incorporating the agents into lipid bilayer structures, or associating the agents (e.g., by conjugation) with the surface of lipid bilayer structures. Heterologous functional agents can be incorporated into PMPs in vivo (e.g., in plants) or in vitro (e.g., in tissue culture, in cell culture, or synthetically incorporated).

[0152] As used herein, the term "pure" refers to a PMP preparation from which at least a portion (e.g., at least 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99% or 100%) of plant cell wall components, plant organelles (e.g., mitochondria, chloroplasts and nuclei) or plant molecular aggregates (protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates or lipid-protein structures) have been removed, relative to an initial sample isolated from a plant or part thereof.

[0153] As used herein, the term "exogenous lipid" refers to a lipid that is exogenous to a plant, that is, a lipid that is derived from a source that is not a plant-derived source of lipid extraction (e.g., a lipid that is added to a composite lipid particle formulation using methods described herein). The term "exogenous lipid" does not exclude plant-derived lipids (e.g., plant-derived sterols). In other words, the exogenous lipid can be a plant-derived lipid (e.g., a plant-derived sterol that is exogenous to the plant-derived source of lipid extraction, e.g., an exogenous lipid can be a plant-derived sterol that is added to a composite lipid particle formulation). The exogenous lipid can be a cell penetrating agent that can increase the delivery of a peptide, polypeptide, or protein to a cell through a composite lipid formulation and / or can increase the loading (e.g., loading efficiency or loading capacity) of a peptide, polypeptide, or protein. In certain embodiments, the exogenous lipid can be a stable lipid. In certain embodiments, the exogenous lipid can be a structural lipid. Exemplary exogenous lipids include sterols and PEGylated lipids.

[0154] As used herein, the term "treat" refers to administering a pharmaceutical composition to an animal for preventive and / or therapeutic purposes. "Preventing an infection" refers to the preventive treatment of an animal that does not yet have a disease or condition but is susceptible to or otherwise at risk for a particular disease or condition. "Treating an infection" refers to administering a treatment to an animal that already has a disease to improve or stabilize the animal's condition.

[0155] As used herein, the term "treatment infection" refers to the application of treatment to an individual (e.g., animal) already suffering from a disease to improve the individual's condition or stabilize the individual's condition. This may be related to reducing the colonization of pathogens in, on, or around animals relative to the initial amount (e.g., reducing by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) and / or allow individual benefits (e.g., reducing colonization with an amount sufficient to resolve symptoms). In such cases, the infection treated may be manifested as a reduction in symptoms (e.g., reducing by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). In some cases, the treated infection can be effective to increase the likelihood of survival of an individual (e.g., an increase in the likelihood of survival of about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or to increase the overall survival of a population (e.g., an increase in the likelihood of survival of about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, the compositions and methods can be effective to "substantially eliminate" an infection, which means reducing the infection by an amount sufficient to persistently resolve symptoms in the animal (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0156] As used herein, the term "preventing infection" refers to preventing an increase in colonization of one or more pathogens in, on, or around an animal (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to untreated animals) in an amount sufficient to maintain the initial pathogen population, prevent the onset of infection, and / or prevent symptoms or conditions associated with infection (e.g., about the amount found in a healthy individual). For example, in immunocompromised individuals (e.g., individuals with cancer, individuals with HIV / AIDS, or individuals taking immunosuppressants), or in individuals undergoing long-term antibiotic therapy, individuals (e.g., animals, such as humans) can receive prophylactic treatment to prevent fungal infection when preparing for an invasive medical procedure (e.g., preparing for surgery, such as receiving a transplant, stem cell therapy, a graft, a prosthesis, receiving long-term or frequent intravenous catheters, or receiving treatment in an intensive care unit).

[0157] As used herein, the expression that a complex lipid formulation or a modified PMP formulation is "stable" refers to the stability of the complex lipid formulation or the modified PMP formulation relative to the complex lipid formulation or the modified PMP formulation, optionally at a defined temperature range (e.g., a temperature of at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C or 23°C), at least 4°C (e.g., at least 5°C, 10°C or 15°C), at least -20°C (e.g., at least -20°C, -15°C, -10°C, -5°C or 0°C), or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C or -30°C). The amount of lipid particles or modified PMPs (e.g., when produced or formulated), the complex lipid formulation or modified PMP composition retains at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of the initial amount of complex lipid particles or modified PMPs (e.g., complex lipid particles or modified PMPs per mL of solution) over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days).

[0158] Alternatively, the expression refers to the temperature of the complex lipid formulation, optionally at a defined temperature range (e.g., a temperature of at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C or 23°C), at least 4°C (e.g., at least 5°C, 10°C or 15°C), at least -20°C (e.g., at least -20°C, -15°C, -10°C, -5°C or 0°C) or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C or -30°C)). or the initial activity of a modified PMP formulation (e.g., when produced or formulated), the complex lipid formulation or modified PMP composition retains at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of its activity over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days).

[0159] Alternatively, the expression refers to the initial size of the complex lipid particle or modified PMP, optionally at a defined temperature range (e.g., a temperature of at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C or 23°C), at least 4°C (e.g., at least 5°C, 10°C or 15°C), at least -20°C (e.g., at least -20°C, -15°C, -10°C, -5°C or 0°C) or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C or -30°C)). The complex lipid formulation or modified PMP formulation retains its particle size (e.g., upon production or formulation), i.e., the particle size does not increase, or increases by no more than 5% (e.g., no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 2-fold, 2.5-fold, or 3-fold) over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days).

[0160] In some embodiments, the stable complex lipid formulation or modified PMP continues to encapsulate or maintain association with the exogenous peptide, polypeptide, or protein to which the complex lipid formulation or modified PMP has been loaded, for example, continues to encapsulate or maintain association with the exogenous peptide, polypeptide, or protein for at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, at least 90 days, or 90 or more days.

[0161] As used herein, the term "vector" refers to an insect that can carry or transmit an animal pathogen from a reservoir to an animal. Exemplary vectors include insects, such as those with piercing-sucking mouthparts, such as those found in the order Hemiptera and some orders Hymenoptera and Diptera, such as mosquitoes, bees, wasps, mosquitoes, lice, cabbage flies, fleas, and ants, and members of the class Arachnidae, such as ticks and mites.

[0162] As used herein, the term "juice cell" or "juice sac" refers to the juice-containing membrane-bound component of the inner pericarp (carpel) of a citrus fruit (e.g., citrus fruit). In some embodiments, the juice cell is separated from other parts of the fruit, such as the pericarp (exocarp or inocarp), the inner pericarp (mesocarp, limon or pith), the central column (placenta), the node wall, or the seed. In some embodiments, the juice cell is a juice cell of a grapefruit, lemon, lime, or orange.

[0163] II. Polypeptide-encapsulated composite lipid particles or modified PMPs

[0164] One aspect of the present invention relates to a complex lipid formulation, comprising: a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and one or more exogenous peptides, polypeptides or proteins encapsulated in the complex lipid particles. The complex lipid particles are characterized by one or more of the following characteristics:

[0165] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0166] ii) contains less than 50 mol % ionizable lipids.

[0167] Another aspect of the present invention relates to plant messenger packets (PMPs) modified with one or more exogenous lipids.

[0168] The complex lipid formulations or modified PMP formulations described herein include exogenous peptides, polypeptides or proteins, such as those described in Section III herein. A plurality of complex lipid particles or modified PMPs can be loaded with exogenous peptides, polypeptides or proteins such that at least 5%, at least 10%, at least 15%, at least 25%, at least 50%, at least 75%, at least 90% or at least 95% of the complex lipid particles or modified PMPs encapsulate exogenous peptides, polypeptides or proteins.

[0169] The exogenous peptide, polypeptide, or protein can be, for example, a therapeutic agent, a pathogen control agent (e.g., an agent having antipathogenic activity (e.g., antibacterial, antifungal, antinematicidal, antiparasitic, or antiviral activity)), or an enzyme (e.g., a recombinase or editing enzyme.

[0170] A.CLP

[0171] Composite lipid particles (CLP) described herein comprise the multiple lipids extracted from one or more plant sources. Composite lipid particles can comprise the lipid of the lipid structure being derived from one or more plant sources of 10%w / w to 99%w / w, for example, can contain at least 10%w / w, at least 20%w / w, at least 30%w / w, at least 40%w / w, at least 50%w / w, at least 60%w / w, at least 70%w / w, at least 80%w / w, at least 90%w / w, at least 95%w / w or about 99%w / w of the lipid structure being derived from one or more plant sources.

[0172] In some embodiments, the composite lipid particles comprise about 10-95% w / w of vegetable lipids. For example, based on the amount of total lipids in the composite lipid formulation, the composite lipid particles comprise about 25-95% w / w, about 30-95% w / w, about 35-95% w / w, about 40-95% w / w, about 45-95% w / w, about 50-95% w / w, about 55-95% w / w, about 60-95% w / w, about 65-95% w / w, about 70-95% w / w, about 75-95% w / w, about 80-95% w / w, or about 85-95% w / w of vegetable lipids.

[0173] The composite lipid granule can contain 5-1000 kinds of lipids extracted from one or more plant sources. In certain embodiments, the composite lipid granule contains at least 10 kinds of plant lipids, and the plant lipids belong to one or more of the categories selected from the group consisting of: glycerolipids, sphingolipids and sterols. For example, the composite lipid granule contains at least 15 kinds, at least 20 kinds, at least 25 kinds, at least 30 kinds, at least 35 kinds, at least 40 kinds, at least 45 kinds, at least 50 kinds, at least 60 kinds, at least 70 kinds, at least 80 kinds, at least 90 kinds, at least 100 kinds, at least 150 kinds, at least 200 kinds, at least 250 kinds, at least 300 kinds, at least 400 kinds, at least 500 kinds, at least 600 kinds, at least 700 kinds or at least 800 kinds of plant lipids, and the plant lipids belong to one or more of the categories selected from the group consisting of: glycerolipids, sphingolipids and sterols. In certain embodiments, the composite lipid granule contains lipids from at least two or at least three categories in these different categories.

[0174] The composite lipid particles may contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG) and sulfatides (SL). In some embodiments, the composite lipid particles contain one or more sphingolipids selected from the group consisting of glycosyl inositolphosphoceramide (GIPC), glucosylceramide (GCer), ceramide (Cer) and free long chain base (LCB). In some embodiments, the composite lipid particles contain one or more phytosterols selected from the group consisting of campesterol, stigmasterol and sitosterol.

[0175] The complex lipid particle may contain one or more lipids, the one or more lipids belonging to one or more subclasses selected from the group consisting of: acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesterol ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acids, fatty acid esters of hydroxy fatty acids, hemi-dimonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, Lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidyl, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, sterol ester, stigmasterol, sulfatide, sulfonic acid lipid, sphingomyelin, sulfoisorhamnosyldiacylglycerol, sterol and triacylglycerol. In certain embodiments, the composite lipid particle contains at least 10 kinds of plant lipids, and the plant lipids belong to one or more of the subclasses selected from the group consisting of the subclasses listed above. For example, the composite lipid particle contains at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 plant lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.

[0176] The complex lipid particle may contain 10 or more lipids, the ten or more lipids belonging to one or more subclasses selected from the group consisting of: acyl steryl glycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerols, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyldiacylglycerols and sterols. For example, the composite lipid particle contains at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 400, at least 500, at least 600, at least 700, or at least 800 plant lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.

[0177] The complex lipid particle may contain at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of plant lipids from plant-derived lipids.

[0178] By lipidomic analysis, by dissolving lipid extract or composite lipid particles in a compatible solvent and analyzing by mass spectrometry (e.g., MS / MS), the identity (and class and subclass) and amount of the lipid extracted from plant sources can be analyzed. The example of the lipidomic analysis based on MS / MS of composite lipid particles is shown in Example 2.

[0179] The composite lipid particle may comprise all or a portion of the lipid species present in the lipid structure from a plant source, for example, the composite lipid particle may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or nearly 100% of the lipid species present in the lipid structure from a plant source.

[0180] The composite lipid granule can comprise reduction or minimized protein substances that are endogenous to the one or more plant sources.For example, the composite lipid granule can contain less than 50%w / w, less than 45%w / w, less than 40%w / w, less than 35%w / w, less than 30%w / w, less than 25%w / w, less than 20%w / w, less than 15%w / w, less than 10%w / w, less than 9%w / w, less than 8%w / w, less than 7%w / w, less than 6%w / w, less than 5%w / w, less than 4%w / w, less than 3%w / w, less than 2%w / w, less than 1%w / w, less than 0.5%w / w, less than 0.1%w / w or be substantially free of protein substances that are endogenous to the one or more plant sources.In some cases, the lipid bilayer of composite lipid granule does not contain protein. To calculate the % w / w of residual protein material that is endogenous to the one or more plant sources, the protein concentration is divided by the concentration of the plant lipid extract and then multiplied by 100. Alternatively, the % w / w is calculated as a percentage based on the mass of the total lipid extract to the mass of total protein that is endogenous to the one or more plant sources.

[0181] The composite lipid particles can include reduced or minimized residual dsDNA materials that are endogenous to the one or more plant sources. For example, the composite lipid particles can contain less than 15% w / w, less than 10% w / w, less than 5% w / w, less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, less than 0.01% w / w, less than 0.005% w / w, less than 0.001% w / w or substantially free of residual dsDNA materials that are endogenous to the one or more plant sources. In some cases, the lipid bilayer of the composite lipid particles does not contain residual dsDNA. In order to calculate the %w / w of residual dsDNA materials that are endogenous to one or more plant sources, the dsDNA concentration of the total adjustment is divided by the concentration of the plant lipid extract, and then multiplied by 100. Alternatively, %w / w is calculated as the percentage of the mass of the total residual dsDNA that is endogenous to one or more plant sources based on the mass of the total lipid extract.

[0182] The compound lipid granule can further comprise at least two kinds of exogenous lipids.The compound lipid granule can comprise the exogenous lipid of at least 1%w / w, at least 2%w / w, at least 5%w / w, at least 10%w / w, at least 15%w / w, at least 20%w / w, at least 25%w / w, at least 30%w / w, at least 40%w / w, at least 50%w / w, at least 60%w / w, at least 70%w / w, at least 80%w / w or about 90%w / w.For example, the compound lipid granule can contain sterol and PEG-lipid conjugate.The other exogenous lipid that is suitable for being included in the compound lipid granule is described hereinafter.

[0183] B.PMP

[0184] The PMPs may include plant EVs or segments, parts or extracts thereof, wherein the diameter of the plant EVs is about 5-2000 nm. For example, the PMPs may include plant EVs or segments, parts or extracts thereof having an average diameter of about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 500-550 nm, about 500-50 ...250 nm About 550-600nm, about 600-650nm, about 650-700nm, about 700-750nm, about 750-800nm, about 800-850nm, about 850-900nm, about 900-950nm, about 950-1000nm, about 1000-1250nm, about 1250-1500nm, about 1500-1750nm or about 1750-2000nm. In some cases, the PMPs include plant EVs or segments, parts or extracts thereof having an average diameter of about 5-950 nm, about 5-900 nm, about 5-850 nm, about 5-800 nm, about 5-750 nm, about 5-700 nm, about 5-650 nm, about 5-600 nm, about 5-550 nm, about 5-500 nm, about 5-450 nm, about 5-400 nm, about 5-350 nm, about 5-300 nm, about 5-250 nm, about 5-200 nm, about 5-150 nm, about 5-100 nm, about 5-50 nm or about 5-25 nm. In some cases, the average diameter of plant EVs or segments, parts or extracts thereof is about 50-200 nm. In some cases, the average diameter of plant EVs or segments, parts or extracts thereof is about 50-300 nm. In some cases, the average diameter of the plant EV, or a segment, part, or extract thereof, is about 200-500 nm. In some cases, the average diameter of the plant EV, or a segment, part, or extract thereof, is about 30-150 nm.

[0185] In some cases, the PMPs may include plant EVs or fragments, portions, or extracts thereof having an average diameter of at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, or at least 1000 nm. In some cases, the PMPs include plant EVs or segments, parts or extracts thereof having an average diameter of less than 1000 nm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm or less than 50 nm. The particle diameter of plant EVs or segments, parts or extracts thereof can be measured using a variety of standard methods in the art (e.g., dynamic light scattering methods).

[0186] In some cases, the PMP may include plant EVs or fragments, portions, or extracts thereof having an average surface area of: 77 nm 2 Up to 3.2x 10 6 nm 2 (For example, 77-100nm 2 , 100-1000nm 2 , 1000-1x 10 4 nm 2 , 1x 10 4 -1x10 5 nm 2 , 1x 10 5 -1x 10 6 nm 2 or 1x 10 6 -3.2x 10 6 nm 2 In some cases, the PMP may include plant EVs or fragments, portions, or extracts thereof having an average volume of: 65 nm 3 Up to 5.3x 10 8 nm 3 (e.g., 65-100nm 3 , 100-1000nm 3 , 1000-1x 10 4 nm 3, 1x 10 4 -1x 10 5 nm 3 , 1x 10 5 -1x 10 6 nm 3 , 1x 10 6 -1x 10 7 nm 3 , 1x10 7 -1x 10 8 nm 3 , 1x 10 8 -5.3x 10 8 nm 3 In some cases, the PMP may include plant EVs or fragments, portions, or extracts thereof having an average surface area of ​​at least 77 nm. 2 (For example, at least 77nm 2 , at least 100nm 2 , at least 1000nm 2 , at least 1x 10 4 nm 2 , at least 1x10 5 nm 2 , at least 1x 10 6 nm 2 or at least 2x 10 6 nm 2 In some cases, the PMP may include plant EVs or fragments, portions, or extracts thereof having an average volume of at least 65 nm. 3 (For example, at least 65nm 3 , at least 100nm 3 , at least 1000nm 3 , at least 1x 10 4 nm 3 , at least 1x 10 5 nm 3 , at least 1x 10 6 nm 3 , at least 1x 10 7 nm 3 , at least 1x10 8 nm 3 , at least 2x 10 8 nm 3 , at least 3x 10 8 nm 3 , at least 4x 10 8 nm 3 , or at least 5x 10 8 nm 3 ).

[0187] In some cases, the size of the PMP can be the same as the size of the plant EV or a segment, extract or portion thereof. Alternatively, the size of the PMP can be different from the size of the initial plant EV from which the PMP is produced. For example, the diameter of the PMP can be about 5-2000 nm. For example, the average diameter of the PMP can be about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-600 nm. In some embodiments, the present invention relates to a nanostructured carbonyl group comprising: a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, a nanostructured carbonyl group, In some cases, the average diameter of the PMP can be at least 5nm, at least 50nm, at least 100nm, at least 150nm, at least 200nm, at least 250nm, at least 300nm, at least 350nm, at least 400nm, at least 450nm, at least 500nm, at least 550nm, at least 600nm, at least 650nm, at least 700nm, at least 750nm, at least 800nm, at least 850nm, at least 900nm, at least 950nm, at least 1000nm, at least 1200nm, at least 1400nm, at least 1600nm, at least 1800nm ​​or about 2000nm. The particle diameter of the PMP can be measured using a variety of standard methods in the art (e.g., dynamic light scattering methods). In some cases, the size of the PMP is determined after loading a heterologous functional agent or after other modifications are performed on the PMP.

[0188] In some cases, the average surface area of ​​the PMP can be 77 nm 2 Up to 1.3x 10 7 nm 2 (For example, 77-100nm 2 , 100-1000nm 2 , 1000-1x 10 4 nm 2 , 1x 10 4 -1x 10 5 nm 2 , 1x 10 5 -1x 10 6 nm2 or 1x 10 6 -1.3x10 7 nm 2 In some cases, the average volume of the PMP can be 65 nm. 3 Up to 4.2x 10 9 nm 3 (e.g., 65-100nm 3 , 100-1000nm 3 , 1000-1x 10 4 nm 3 , 1x 10 4 -1x 10 5 nm 3 , 1x 10 5 -1x 10 6 nm 3 , 1x 10 6 -1x 10 7 nm 3 , 1x10 7 -1x 10 8 nm 3 , 1x 10 8 -1x 10 9 nm 3 or 1x 10 9 -4.2x10 9 nm 3 In some cases, the average surface area of ​​the PMP is at least 77 nm 2 (For example, at least 77nm 2 , at least 100nm 2 , at least 1000nm 2 , at least 1x 10 4 nm 2 , at least 1x10 5 nm 2 , at least 1x 10 6 nm 2 or at least 1x 10 7 nm 2 ). In some cases, the average volume of the PMP is at least 65 nm 3 (For example, at least 65nm 3 , at least 100nm 3 , at least 1000nm 3 , at least 1x 10 4 nm 3 , at least 1x 10 5 nm 3 , at least 1x 10 6 nm3 , at least 1x 10 7 nm 3 , at least 1x 10 8 nm 3 , at least 1x 10 9 nm 3 , at least 2x 10 9 nm 3 , at least 3x 10 9 nm 3 or at least 4x10 9 nm 3 ).

[0189] In some cases, the PMP may include a whole plant EV. Alternatively, the PMP may include a segment, part, or extract of the total surface area of ​​a vesicle of a plant EV (e.g., a segment, part, or extract comprising less than 100% (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 10%, less than 5%, or less than 1%) of the total surface area of ​​the vesicle). The segment, part, or extract may have any shape, such as a circumferential segment, a spherical segment (e.g., a hemispherical segment), a curved segment, a straight segment, or a flat segment. In the case where the segment is a spherical segment of a vesicle, the spherical segment may represent a spherical segment produced by splitting a spherical vesicle along a pair of parallel lines, or a spherical segment produced by splitting a spherical vesicle along a pair of non-parallel lines. Therefore, a plurality of PMPs may include a plurality of whole plant EVs, a plurality of plant EV segments, parts, or extracts, or a mixture of whole plant EVs and their segments. Those skilled in the art will appreciate that the ratio of intact to fragmented plant EVs will depend on the specific isolation method used. For example, grinding or blending a plant or its parts can produce PMPs containing a higher percentage of plant EV fragments, parts or extracts compared to non-destructive extraction methods such as vacuum infiltration.

[0190] In cases where the PMPs include segments, parts, or extracts of plant EVs, the average surface area of ​​the EV segments, parts, or extracts can be less than the average surface area of ​​intact vesicles (e.g., an average surface area of ​​less than 77 nm). 2 , 100nm 2 , 1000nm 2 , 1x 10 4 nm 2 , 1x 10 5 nm 2 , 1x 10 6 nm 2 or 3.2x 10 6 nm 2). In some cases, the surface area of ​​the EV segment, portion, or extract is less than 70 nm 2 、60nm 2 , 50nm 2 、40nm 2 、30nm 2 , 20nm 2 or 10nm 2 In some cases, the PMPs may include plant EVs or fragments, portions, or extracts thereof having an average volume that is less than the average volume of intact vesicles (e.g., an average volume less than 65 nm). 3 , 100nm 3 , 1000nm 3 , 1x 10 4 nm 3 , 1x 10 5 nm 3 , 1x 10 6 nm 3 , 1x 10 7 nm 3 , 1x 10 8 nm 3 or 5.3x10 8 nm 3 ).

[0191] In the case where the PMP comprises an extract of plant EVs, for example, in the case where the PMP comprises lipids extracted from plant EVs (e.g., with chloroform), the PMP may comprise at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or greater than 99% of lipids extracted from plant EVs (e.g., with chloroform). The PMPs in the plurality may comprise plant EV segments and / or lipids extracted from plant EVs or mixtures thereof.

[0192] C. Method for producing PMP

[0193] PMPs can be produced from plant EVs or segments, parts or extracts (e.g., lipid extracts) naturally present in plants or parts thereof (including plant tissues or plant cells). An exemplary method for producing PMPs comprises (a) providing an initial sample from a plant or part thereof, wherein the plant or part thereof comprises EVs; and (b) isolating a crude PMP fraction from the initial sample, wherein the crude PMP fraction has a reduced level of at least one contaminant or undesirable component from the plant or part thereof relative to the level in the initial sample. The method may further comprise an additional step (c), comprising purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the plurality of pure PMPs have a reduced level of at least one contaminant or undesirable component from the plant or part thereof relative to the level in the crude EV fraction. Each production step is discussed in further detail below. Exemplary methods for the isolation and purification of PMPs are found in, for example, Rutter and Innes, Plant Physiol. 173(1):728-741, 2017; Rutter et al., Bio. Protoc. 7(17):e2533, 2017; Regente et al., J of Exp. Biol. 68(20):5485-5496, 2017; Mu et al., Mol. Nutr. Food Res., 58, 1561–1573, 2014, and Regente et al., FEBS Letters. 583:3363-3366, 2009, each of which is incorporated herein by reference.

[0194] For example, a plurality of PMPs can be isolated from a plant by a method comprising the steps of: (a) providing an initial sample from a plant or a part thereof, wherein the plant or part thereof comprises EVs; (b) isolating a crude PMP fraction from the initial sample, wherein the crude PMP fraction has a reduced level of at least one contaminant or undesirable component from the plant or part thereof relative to the level in the initial sample (e.g., a level reduced by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 90%, 95%, 96%, 98%, 99% or 100%); and (c) purifying the crude PMP fraction to produce a plurality of pure PMPs, wherein the plurality of pure PMPs have a reduced level of at least one contaminant or undesirable component from a plant or a portion thereof relative to the level in the crude EV fraction (e.g., a level reduced by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99% or 100%).

[0195] The PMPs provided herein may include plant EVs isolated from a variety of plants or their segments, parts or extracts. PMPs may be isolated from plants of any genus (vascular or non-vascular), including but not limited to angiosperms (monocots and dicots), gymnosperms, ferns, selaginella, equisetum, naked ferns, lycophytes, algae (e.g., unicellular or multicellular algae, such as primitive chromatophores) or bryophytes. In some cases, PMPs may be produced by vascular plants, such as monocots or dicots or gymnosperms. For example, PMPs can be produced from alfalfa, apple, Arabidopsis, banana, barley, canola, castor bean, chicory, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, yam, eucalyptus, fescue, flax, gladiolus, lily, linseed, millet, melon, mustard, oats, oil palm, rapeseed, papaya, peanut, pineapple, ornamental plants, beans, potatoes, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugar beet, sugar cane, sunflower, strawberry, tobacco, tomato, turf Grass, wheat or vegetable crops such as lettuce, celery, cabbage, cauliflower, gourd; fruit and nut trees such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazelnut; vines such as grape, kiwi, hops; fruit shrubs and thorns such as raspberry, blackberry, gooseberry; forest trees such as ash, pine, fir, maple, oak, chestnut, poplar; with alfalfa, canola, castor bean, corn, cotton, sea kale, flax, linseed, mustard, oil palm, rapeseed, peanut, potato, rice, safflower, sesame, soybean, sugar beet, sunflower, tobacco, tomato or wheat. In some embodiments, PMP can be produced by pitaya, kale, spinach or strawberry.

[0196] The PMP may be produced by the whole plant (e.g., the whole inflorescence or seedling), or alternatively by one or more plant parts (e.g., leaves, seeds, roots, fruits, vegetables, pollen, phloem sap or xylem sap). For example, the PMP may be produced by a bud vegetative organ / structure (e.g., leaves, stems or tubers), a root, a flower and a flower organ / structure (e.g., pollen, bracts, sepals, petals, stamens, carpels, anthers or ovules), a seed (including an embryo, endosperm or seed coat), a fruit (mature ovary), a sap (e.g., phloem or xylem sap), a plant tissue (e.g., vascular tissue, root tissue, tumor tissue, etc.), and a cell (e.g., a single cell, a protoplast, an embryo, a callus, a guard cell, an egg cell, etc.), or its progeny. For example, the separation step may involve (a) providing a plant or part thereof, wherein the plant part is an Arabidopsis leaf. The plant may be at any developmental stage. For example, PMPs can be produced by seedlings, such as 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks old seedlings (e.g., Arabidopsis seedlings). Other exemplary PMPs can include PMPs produced by roots (e.g., ginger root), juices (e.g., grapefruit juice), vegetables (e.g., broccoli), pollens (e.g., olive pollen), phloem sap (e.g., Arabidopsis phloem sap), or xylem sap (e.g., tomato xylem sap). In some embodiments, PMPs are produced by citrus fruits such as grapefruit or lemon.

[0197] PMPs can be produced from plants or parts thereof by a variety of methods. Any method that allows the release of a plant's apoplast fraction containing EVs or other extracellular fractions (e.g., cell culture media) containing PMPs containing secreted EVs is suitable for the methods of the present invention. EVs can be separated from plants or plant parts by destructive methods (e.g., grinding or blending plants or any plant parts) or non-destructive methods (washing or vacuum infiltration of plants or any plant parts). For example, plants or parts thereof can be vacuum infiltrated, ground, blended, or a combination thereof to separate EVs from the plants or plant parts, thereby producing PMPs. For example, the separation step can involve (b) separating a crude PMP fraction from an initial sample (e.g., a plant, a plant part, or a sample derived from a plant or a plant part), wherein the crude PMP fraction has a reduced level of at least one contaminant or undesirable component from a plant or part thereof relative to the level in the initial sample; wherein the separation step involves vacuum infiltration of the plant (e.g., using a vesicle isolation buffer) to release and collect the apoplast fraction. Alternatively, the separation step may involve (b) grinding or blending the plants to release the EVs, thereby producing PMPs.

[0198] When isolating plant EVs and thereby producing PMPs, the PMPs can be separated or collected into a crude PMP fraction (e.g., apoplast fraction). For example, the separation step can involve separating multiple PMPs into a crude PMP fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the PMP-containing fraction from large contaminants including plant tissue fragments, plant cells, or plant cell organelles (e.g., nuclei or chloroplasts). In this way, the number of large contaminants including, for example, plant tissue fragments, plant cells, or plant organelles (e.g., nuclei, mitochondria, or chloroplasts) in the crude PMP fraction will be reduced as compared to the initial sample from the source plant or plant part.

[0199] The crude PMP fraction can be further purified by additional purification methods to produce a plurality of pure PMPs. For example, the crude PMP fraction can be separated from other plant components by ultracentrifugation, for example using a density gradient (iodixanol or sucrose), size exclusion, and / or using other methods for removing aggregated components (e.g., precipitation or size exclusion chromatography). The resulting pure PMP can have a reduced level of contaminants or undesirable components from the source plant (e.g., one or more non-PMP components, such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins or lipid-protein structures, cell nuclei, cell wall components, organelles, or a combination thereof) relative to one or more fractions produced during an earlier separation step, or relative to a pre-established threshold level, such as a commercial release specification. For example, the level of plant organelles or cell wall components of the pure PMP may be reduced relative to the level in the initial sample (e.g., reduced by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%; or reduced by about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x, or more than 100x). In some cases, the pure PMP is substantially free of (e.g., having undetectable levels of) one or more non-PMP components, such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof. Other examples of release and separation steps can be found in WO 2021 / 041301, which is incorporated herein by reference in its entirety. The concentration of the PMP can be, for example, 1x 10 9 , 5x 10 9 , 1x 10 10 , 5x 10 10 , 5x 10 10 , 1x 10 11 , 2x 10 11 , 3x 10 11 , 4x 1011 , 5x 10 11 , 6x 10 11 , 7x 10 11 , 8x 10 11 , 9x 10 11 , 1x 10 12 , 2x 10 12 , 3x 10 12 , 4x 10 12 , 5x 10 12 , 6x10 12 , 7x 10 12 , 8x 10 12 , 9x 10 12 , 1x 10 13 or greater than 1x 10 13 PMP / mL.

[0200] For example, protein aggregates can be removed from the separated PMP. For example, the separated PMP solution can be taken out through a certain range of pH (e.g., as measured using a pH probe) to precipitate protein aggregates in the solution. The pH can be adjusted to, for example, pH 3, pH 5, pH 7, pH 9, or pH 11 by adding, for example, sodium hydroxide or hydrochloric acid. Once the solution is at a specified pH, it can be filtered to remove the particles. Alternatively, the separated PMP solution can be flocculated by adding a charged polymer such as Polymin-P or Praestol 2640. In short, Polymin-P or Praestol 2640 is added to the solution and mixed with an impeller. The solution can then be filtered to remove the particles. Alternatively, the aggregates can be dissolved by increasing the salt concentration. For example, NaCl can be added to the separated PMP solution until it reaches, for example, 1 mol / L. The solution can then be filtered to separate the PMP. Alternatively, the aggregates are dissolved by increasing the temperature. For example, the separated PMPs can be heated with mixing until the solution reaches a uniform temperature of, for example, 50°C for 5 minutes. The PMP mixture can then be filtered to separate the PMPs. Alternatively, soluble contaminants can be separated from the PMP solution by an exclusion chromatography column according to standard procedures, wherein the PMPs are eluted in the first fraction, while proteins and ribonucleoproteins and some lipoproteins are subsequently eluted. The efficiency of protein aggregate removal can be determined by BCA / Bradford protein quantitative measurement and comparison of protein concentrations before and after removal of protein aggregates. In some embodiments, protein aggregates are removed before exogenous peptides, polypeptides or proteins are encapsulated by PMPs. In other embodiments, protein aggregates are removed after exogenous peptides, polypeptides or proteins are encapsulated by PMPs.

[0201] Any of the production methods described herein can be supplemented with any quantitative or qualitative methods known in the art to characterize or identify PMPs in any step of the production process. PMPs can be characterized by a variety of analytical methods to estimate PMP yield, PMP concentration, PMP purity, PMP composition or PMP size. PMPs can be evaluated by a variety of methods known in the art that can achieve visualization, quantitative or qualitative characterization (e.g., identification of composition) of PMPs, such as microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectral analysis (e.g., Fourier transform infrared analysis (Fourier transforminfrared analysis)) or mass spectrometry (protein and lipid analysis). In some cases, methods (e.g., mass spectrometry) can be used to identify plant EV markers present on PMPs, such as markers disclosed in the appendix disclosed in WO 2021 / 041301 (which is incorporated herein by reference in its entirety). In order to facilitate the analysis and characterization of PMP fractions, PMPs can be additionally labeled or stained. For example, PMP can be iodinated with 3,3'-dihexyloxycarbocyanine (DIOC6) (fluorescent lipophilic dye), PKH67 (Sigma Aldrich), Alexa Fluor 488 (Thermo Fisher Scientific) or DyLight TM 800 (Thermo Fisher Scientific) for staining. In the absence of complex forms of nanoparticle tracking, this relatively simple method quantifies total membrane content and can be used to indirectly measure the concentration of PMPs (Rutter and Innes, Plant Physiology 173 (1): 728-741, 2017; Rutter et al., Bioprotocol 7 (17): e2533, 2017). In order to make more accurate measurements and evaluate the size distribution of PMPs, nanoparticle tracking, nanoflow cytometry or tunable resistive pulse sensing can be used.

[0202] During the production process, the PMPs can be optionally prepared such that the concentration of the PMPs is increased relative to the EV levels in a control or initial sample (e.g., an increase of about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%; or an increase of about 2x, 4x, 5x, 10x, 20x, 25x, 50x, 75x, 100x, or more than 100x). The isolated PMPs can comprise about 0.1% to about 100% of the PMP composition, such as any of about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, or about 50% to about 99%. In some cases, the composition includes at least any one of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher percentages of PMP, for example, as measured by weight / volume, PMP protein composition percentage and / or lipid composition percentage (for example, by measuring fluorescently labeled lipids). In some cases, concentrates are used as commercial products, for example, end users can use diluents with significantly lower concentrations of active ingredients. In some embodiments, the composition is formulated into a PMP concentrated formulation, for example, an ultra-low volume concentrated formulation. In some embodiments, the concentration of the PMP in the composition can effectively increase the adaptability of an organism, such as a plant, an animal, an insect, a bacterium or a fungus. In other aspects, the concentration of the PMP in the composition can effectively reduce the adaptability of an organism, such as a plant, an animal, an insect, a bacterium or a fungus.

[0203] PMP can be produced by a variety of plants or parts thereof (e.g., leaf apoplast, seed apoplast, root, fruit, vegetable, pollen, phloem or xylem sap). For example, PMP can be released from the apoplast of a plant, such as the apoplast of a leaf (e.g., Arabidopsis thaliana leaf apoplast) or the apoplast of a seed (e.g., the apoplast of a sunflower seed). Other exemplary PMPs are produced by roots (e.g., ginger root), juice (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem sap (e.g., Arabidopsis phloem sap), xylem sap (e.g., tomato plant xylem sap) or cell culture supernatant (e.g., BY2 tobacco cell culture supernatant). WO 2021 / 041301, incorporated by reference as a whole, further confirms that PMP is produced by these various plant sources.

[0204] PMPs can be produced and purified by a variety of methods, for example, by using a density gradient (iodixanol or sucrose) combined with ultracentrifugation and / or methods for removing aggregated contaminants, such as precipitation or size exclusion chromatography. Further description of the production, purification and characterization of PMPs can be found in WO 2021 / 041301, which is incorporated by reference in its entirety.

[0205] In some cases, the PMP of the compositions and methods of the invention can be isolated from a plant or part thereof and used without further modification of the PMP. In other cases, the PMP can be modified prior to use, as further outlined herein.

[0206] D. Plant EV markers

[0207] The PMP may have a series of markers that identify the PMP as being produced by plant EVs and / or including segments, portions, or extracts thereof. As used herein, the term "plant EV marker" refers to a component naturally associated with a plant and incorporated into or on a plant EV in the plant, such as a plant protein, a plant nucleic acid, a plant small molecule, a plant lipid, or a combination thereof. Examples of plant EV markers can be found, for example, in Rutter and Innes, Plant Physiology 173(1):728-741, 2017; Raimondo et al., Oncotarget. 6(23):19514, 2015; Ju et al., Mol. Therapy 21(7):1345-1357, 2013; Wang et al., Molecular Therapy 22(3):522-534, 2014; and Regente et al., Journal of Experimental Biology 68(20):5485-5496, 2017; each of which is incorporated herein by reference. Additional examples of plant EV markers are listed in the appendix disclosed in WO 2021 / 041301 (which is incorporated herein by reference in its entirety) and are further summarized herein.

[0208] Plant EV markers may include plant lipids. Examples of plant lipid markers that may be found in PMPs include phytosterols, campesterol, β-sitosterol, stigmasterol, avenasterol, glycosyl inositol phosphorylceramide (GIPC), glycolipids (e.g., monogalactosyldiacylglycerol (MGDG) or digalactosyldiacylglycerol (DGDG) or a combination thereof. For example, PMPs may include GIPC, which represents the major sphingolipid class in plants and is one of the most abundant membrane lipids in plants. Other plant EV markers may include lipids that accumulate in plants in response to abiotic or biotic stressors (e.g., bacterial or fungal infections), such as phosphatidic acid (PA) or phosphatidylinositol-4-phosphate (PI4P).

[0209] Alternatively, the plant EV marker may include a plant protein. In some cases, the protein plant EV marker may be an antimicrobial protein naturally produced by the plant, including a defense protein secreted by the plant in response to abiotic or biotic stressors (e.g., bacterial or fungal infections). Plant pathogen defense proteins include soluble N-ethylmaleimide-sensitive factor-associated protein receptor proteins (SNARE) proteins (e.g., syntaxin-121 (SYP121; GenBank Accession No.: NP_187788.1 or NP_974288.1), penetration 1 (PEN1; GenBank Accession No.: NP_567462.1)) or ABC transporter penetration 3 (PEN3; GenBank Accession No.: NP_191283.2). Other examples of plant EV markers include proteins that facilitate long-distance transport of RNA in plants, including phloem proteins (e.g., phloem protein 2-A1 (PP2-A1), GenBank Accession No.: NP_193719.1), calcium-dependent lipid binding proteins, or lectins (e.g., jacaranda-related lectins, such as Helianthus annuus jacalin (Helja; GenBank Accession No.: AHZ86978.1). For example, the RNA binding protein may be glycine-rich RNA binding protein-7 (GRP7; GenBank Accession No.: NP_179760.1). In addition, in some cases, proteins that regulate plasmodesmata function can be found in plant EVs, including proteins such as Synap-Totgamin. AA (GenBank Accession No.: NP_565495.1). In some cases, plant EV markers may include proteins involved in lipid metabolism, such as phospholipase C or phospholipase D. In some cases, plant protein EV markers are cellular transport proteins in plants. In some cases where the plant EV marker is a protein, the protein marker may lack a signal peptide that is typically associated with a secretory protein. Unconventional secretory proteins appear to have several common features, such as (i) lack of a leader sequence, (ii) absence of PTMs specific to the ER or Golgi apparatus, and / or (iii) secretion is not affected by brefeldin A, which blocks the classical ER / Golgi-dependent secretory pathway. A person skilled in the art can use a variety of tools freely available to the public (e.g., SecretomeP database; SUBA3 (Subcellular Localization Database of Arabidopsis Proteins)) to evaluate the signal sequence or lack of a signal sequence for a protein.

[0210] In the case where the plant EV marker is a protein, the protein may have an amino acid sequence having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with a plant EV marker, such as any of the plant EV markers listed in the Appendix disclosed in WO2021 / 041301 (which is incorporated herein by reference in its entirety). For example, the protein may have an amino acid sequence having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with PEN1 from Arabidopsis thaliana (GenBank Accession No.: NP_567462.1).

[0211] In some cases, plant EV markers include nucleic acids encoded in plants, such as plant RNA, plant DNA or plant PNA. For example, PMPs may include dsRNA, mRNA, viral RNA, microRNA (miRNA) or small interfering RNA (siRNA) encoded by plants. In some cases, nucleic acids may be nucleic acids associated with proteins that promote long-distance transport of RNA in plants, as discussed herein. In some cases, nucleic acid plant EV markers may be markers involved in host-induced gene silencing (HIGS), which is a process in which plants silence exogenous transcripts of plant pests (e.g., pathogens, such as fungi). For example, nucleic acids may be nucleic acids that silence bacterial or fungal genes. In some cases, nucleic acids may be microRNAs, such as miR159 or miR166, which target genes in fungal pathogens (e.g., Verticillium dahliae). In some cases, the protein can be a protein involved in carrying plant defense compounds, such as a protein involved in the transport and metabolism of glucosinolates (GSLs), including glucosinolate transporter-1 (GTR1; GenBank Accession No.: NP_566896.2), glucosinolate transporter-2 (GTR2; NP_201074.1), or episulfide-specific modifier 1 (ESM1; NP_188037.1).

[0212] In the case where the plant EV marker is a nucleic acid, the nucleic acid may have a nucleotide sequence having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with the plant EV marker, such as those encoding the plant EV markers listed in the Appendix disclosed in WO 2021 / 041301 (which is incorporated herein by reference in its entirety). For example, the nucleic acid may have a polynucleotide sequence having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with miR159 or miR166.

[0213] In some cases, plant EV markers include compounds produced by plants. For example, the compound can be a defense compound produced in response to abiotic or biotic stressors such as secondary metabolites. One such secondary metabolite found in PMPs is glucuronate (GSL), which is a nitrogen and sulfur secondary metabolite mainly found in cruciferous plants (Brassicaceae plant). Other secondary metabolites may include allelopathic substances.

[0214] In some cases, PMPs can also be identified as being produced by plant EVs based on the lack of certain markers (e.g., lipids, polypeptides, or polynucleotides) that are not usually produced by plants but are usually associated with other organisms (e.g., markers of animal EVs, bacterial EVs, or fungal EVs). For example, in some cases, PMPs lack lipids that are usually seen in animal EVs, bacterial EVs, or fungal EVs. In some cases, PMPs lack lipids that are specific to animal EVs (e.g., sphingomyelin). In some cases, PMPs do not contain lipids that are specific to bacterial EVs or bacterial membranes (e.g., LPS). In some cases, PMPs lack lipids that are specific to fungal membranes (e.g., ergosterol).

[0215] Plant EV markers can be identified using any method known in the art capable of identifying small molecules (e.g., mass spectrometry, mass spectrometry), lipids (e.g., mass spectrometry, mass spectrometry), proteins (e.g., mass spectrometry, immunoblotting), or nucleic acids (e.g., PCR analysis). In some cases, the PMP compositions described herein include a detectable amount of a plant EV marker described herein, such as a predetermined threshold amount.

[0216] E. Exogenous lipids

[0217] In some embodiments, the complex lipid particle contains not only lipids extracted from one or more plant sources, but also two or more exogenous lipids.

[0218] In some embodiments, the PMP is modified to contain two or more exogenous lipids.

[0219] Exogenous lipids can be cell penetrating agents, can be capable of increasing the delivery of peptides, polypeptides or proteins to cells through complex lipid formulations and / or can be capable of increasing the loading (e.g., loading efficiency or loading capacity) of peptides, polypeptides or proteins. In certain embodiments, exogenous lipids can be stable lipids. In certain embodiments, exogenous lipids can be structural lipids. Exemplary exogenous lipids include sterols and PEGylated lipids.

[0220] In some embodiments, the complex lipid particle includes other components (eg, lipids, eg, sterols, eg, cholesterol; or small molecules).

[0221] In some embodiments, the PMP can be modified with other components (e.g., lipids, e.g., sterols, e.g., cholesterol; or small molecules) to further alter the functional and structural properties of the PMP. For example, the PMP can be further modified with a stabilizing molecule that increases the stability of the PMP (e.g., for at least one day at room temperature, and / or for at least one week at 4°C).

[0222] In some embodiments, the complex lipid particles include a sterol, such as sitosterol, dihydrositosterol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., sheep cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucoxantranol, or any sterol analog (e.g., glycoside, ester or peptide).

[0223] In some embodiments, the PMP is modified with a sterol, such as sitosterol, dihydrositosterol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., sheep cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucoxantranol, or any sterol analog (e.g., glycoside, ester or peptide).

[0224] In some examples, exogenous sterols are added to the formulation prior to a mixing step, such as step (b), for example, before step (b) and mixed with the extracted lipids. The amount of exogenous sterols added can be, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% (w / w) of the total lipids and sterols in the formulation.

[0225] In some embodiments, the concentration of the sterol in the composite lipid particle ranges from about 5 to 60% w / w, such as about 5 to 50% w / w, about 5 to 40% w / w, about 5 to 30% w / w, about 5 to 20% w / w, about .5 to 15% w / w, about 0.5 to 15% w / w, about 5 to 8% w / w, or about 6 to 7% w / w, based on the amount of total lipid in the composite lipid particle. In some embodiments, the sterol ranges from about 15 to 20% w / w, about 20 to 30% w / w, about 30 to 40% w / w, about 40 to 50% w / w, or about 50 to 60% w / w, based on the amount of total lipid in the composite lipid particle.

[0226] In some embodiments, the concentration of the PEG-lipid conjugate ranges from about 0.5 to about 5% w / w, about 0.5 to 3.5% w / w, about 1 to 3.5% w / w, about 0.5 to 3% w / w, about 1 to 3% w / w, about 0.5 to 2.5% w / w, about 1 to 2.5% w / w, about 1.5 to 2.5% w / w, or about 2 to 2.5% w / w, based on the amount of total lipid in the composite lipid particle. In some embodiments, the concentration of the PEG-lipid conjugate ranges from about 0.5 to 15% w / w, about 1 to 15% w / w, about 2 to 5% w / w, about 5 to 8% w / w, about 8 to 12% w / w, or about 12 to 15% w / w, based on the amount of total lipid in the composite lipid particle.

[0227] In some embodiments, the sterol is cholesterol or sitosterol. In some cases, the composite lipid particles or modified PMPs include a molar ratio of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more than 60% of the sterol (e.g., cholesterol or sitosterol), for example, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50% or 50%-60% of the sterol. In some embodiments, the composite lipid particles or modified PMPs include a molar ratio of about 35%-50% of the sterol (e.g., cholesterol or sitosterol), for example, about 36%, 38.5%, 42.5% or 46.5% of the sterol. In some embodiments, the composite lipid particles or modified PMPs include a molar ratio of about 20%-40% of the sterol.

[0228] In some embodiments, the stability of the PMP modified with a sterol is altered (eg, the stability is increased) relative to the PMP not modified with a sterol. In some embodiments, the PMP modified with a sterol has a greater fusion rate with the membrane of a target cell relative to the PMP not modified with a sterol.

[0229] In some cases, the complex lipid particle or modified PMP comprises an exogenous lipid and an exogenous sterol.

[0230] In some embodiments, the composite lipid particle comprises a PEGylated lipid.

[0231] In some embodiments, the PMP is modified with a PEGylated lipid.

[0232] The polyethylene glycol (PEG) length can vary between 1 kDa and 10 kDa; in some aspects, a PEG of 2 kDa in length is used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k.

[0233] In some cases, the complex lipid particle or modified PMP comprises a molar ratio of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.5%, 4% or more. , 4.5%, 5%, 10%, 20%, 30%, 40%, 50% or more of PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k or DMPE-PEG2k), for example, 0.1%-0.5%, 0.5%-1%, 1%-1.5%, 1.5%-2.5%, 2.5%-3.5%, 3.5%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40% or 40%-50% of PEGylated lipid. In some embodiments, the composite lipid particle or modified PMP comprises a molar ratio of about 0.1%-10% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k or DMPE-PEG2k), for example, about 1%-3% PEGylated lipid, for example, about 1.5% or about 2.5% PEGylated lipid.

[0234] In some embodiments, the stability of a PMP modified with a PEGylated lipid is altered (e.g., stability is increased) relative to a PMP not modified with a PEGylated lipid. In some embodiments, the particle size of a PMP modified with a PEGylated lipid is altered relative to a PMP not modified with a PEGylated lipid.

[0235] In some embodiments, a complex lipid particle or modified PMP containing a PEGylated lipid is less likely to be phagocytosed than a complex lipid particle or modified PMP not containing a PEGylated lipid.

[0236] The addition of PEGylated lipids may also affect stability in the GI tract and enhance particle migration through mucus.PEG can be used as a method of attaching targeting moieties.

[0237] Cellular uptake of the complex lipid particles or modified PMPs can be measured by a variety of methods known in the art. For example, the complex lipid particles or modified PMPs or components thereof can be labeled with markers (e.g., fluorescent markers) that can be detected in isolated cells to confirm uptake.

[0238] The composite lipid particles contain less than 50mol% ionizable lipids (e.g., ionizable lipids that are exogenous to one or more plant sources). For example, the composite lipid particles contain less than 45mol%, less than 40mol%, less than 35mol%, less than 30mol%, less than 25mol%, less than 20mol%, less than 15mol%, less than 10mol%, less than 9mol%, less than 8mol%, less than 7mol%, less than 6mol%, less than 5mol%, less than 4mol%, less than 3mol%, less than 2mol%, less than 1mol%, less than 0.5mol%, less than 0.1mol% or are substantially free of ionizable lipids (e.g., ionizable lipids that are exogenous to one or more plant sources). In some embodiments, the composite lipid particles contain less than 20mol% of exogenous ionizable lipids. In some embodiments, the composite lipid particles contain less than 5mol% of exogenous ionizable lipids.

[0239] In some embodiments, the complex lipid formulations provided herein comprise two or more different types of complex lipid particles, for example, complex lipid particles derived from two or more different plant sources, and / or complex lipid particles comprising exogenous lipids such as sterols and / or PEGylated lipids of different species and / or different ratios.

[0240] In some embodiments, the modified PMP formulations provided herein comprise two or more different modified PMPs, e.g., comprise modified PMPs derived from different unmodified PMPs (e.g., unmodified PMPs from two or more different plant sources) and / or comprise modified PMPs comprising exogenous lipids such as sterols and / or PEGylated lipids of different species and / or different ratios.

[0241] In some cases, the organic solvent in which the lipid film is dissolved is chloroform, ethanol or dimethylformamide:methanol (DMF:MeOH). Alternatively, the organic solvent or solvent combination can be, for example, acetonitrile, acetone, chloroform, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethyl sulfoxide:methanol or dimethylformamide:methanol.

[0242] F. Drug Formulations

[0243] Included herein are pharmaceutical compositions that can be formulated, for example, for administration to animals, such as complex lipid formulations or modified PMP formulations of humans. The pharmaceutical composition can be administered to animals together with a pharmaceutically acceptable diluent, carrier, and / or excipient. Depending on the mode of administration and dosage, the pharmaceutical composition of the methods described herein will be formulated into a suitable pharmaceutical composition to allow for easy delivery. A single dose can be in unit dosage form as needed.

[0244] The complex lipid formulation or modified PMP formulation can be formulated for, for example, oral administration, enteral administration, intravenous administration (e.g., injection or infusion) or subcutaneous administration to animals (e.g., humans). For injectable formulations, various effective drug carriers are known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, (2012) and ASHP Handbook on injectable Drugs, 18th edition, (2014)).

[0245] The pharmaceutically acceptable carriers and excipients in the compositions of the present invention are non-toxic to the recipient at the doses and concentrations employed. Acceptable carriers and excipients may include: buffers such as phosphate, citrate, HEPES and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyl dimethylbenzyl ammonium chloride, resorcinol and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine and lysine; and carbohydrates such as glucose, mannose, sucrose and sorbitol. The composition may be formulated according to conventional pharmaceutical practice. The concentration of the compound in the formulation will vary according to many factors, including the dose of the active agent to be administered (e.g., an exogenous peptide, polypeptide or protein encapsulated by a complex lipid formulation or a modified PMP), and the route of administration.

[0246] For oral administration to animals, the complex lipid formulation or modified PMP formulation can be prepared in the form of an oral formulation. Formulations for oral use can include tablets, caplets, capsules, syrups or oral liquid dosage forms containing the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives (including microcrystalline cellulose), starch (including potato starch), cross-linked carboxymethyl cellulose sodium, alginate or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffers, etc. Formulations for oral use may also be provided in unit dosage form as chewable tablets, non-chewable tablets, caplets, capsules (e.g., as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium). The compositions disclosed herein may further include immediate release, extended release, or delayed release formulations.

[0247] For parenteral administration to animals, the complex lipid formulation or modified PMP composition can be formulated in the form of a liquid solution or suspension and administered by a parenteral route (e.g., topical, subcutaneous, intravenous or intramuscular). The pharmaceutical composition can be formulated for injection or infusion. The pharmaceutical composition for parenteral administration can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a medium. Pharmaceutically acceptable media include, but are not limited to, sterile water, normal saline or cell culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-modified Eagle medium (α-MEM), F-12 culture medium). Preparation methods are known in the art, see, for example, Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd Edition) Taylor & Francis Group, CRC Press (2009).

[0248] III. Exogenous peptides, polypeptides or proteins

[0249] The present invention includes composite lipid formulations or modified PMP formulations, wherein the composite lipid particles or modified PMPs encapsulate exogenous peptides, polypeptides or proteins. Exogenous peptides, polypeptides or proteins can be enclosed in composite lipid particles or modified PMPs, such as being located inside the lipid membrane structure, such as being separated from surrounding materials or solutions by two leaflets of a lipid bilayer. In certain embodiments, the encapsulated exogenous peptides, polypeptides or proteins can interact or associate with the inner lipid membrane of a composite lipid particle or modified PMP. In certain embodiments, the encapsulated exogenous peptides, polypeptides or proteins can interact or associate with the outer lipid membrane of a composite lipid particle or modified PMP. In some cases, exogenous peptides, polypeptides or proteins can be embedded in lipid membrane structures. In some cases, exogenous peptides, polypeptides or proteins have an extraluminal portion. In some cases, exogenous peptides, polypeptides or proteins are conjugated to the outer surface of a lipid membrane structure, such as using click chemistry.

[0250] An exogenous peptide, polypeptide or protein may be a peptide, polypeptide or protein that is not naturally present in a plant EV. Alternatively, an exogenous peptide, polypeptide or protein may be naturally present in a plant EV, but it is encapsulated in a composite lipid particle or a modified PMP in an amount that is not found in a naturally occurring plant extracellular vesicle. In some cases, an exogenous peptide, polypeptide or protein may be naturally present in a plant from which plant lipids or PMPs are extracted. In other cases, an exogenous peptide, polypeptide or protein is not naturally present in a plant from which plant lipids or PMPs are extracted. An exogenous polypeptide may be artificially expressed in a plant from which plant lipids or PMPs are extracted, for example, a heterologous polypeptide. An exogenous peptide, polypeptide or protein may be derived from another organism. In some embodiments, an exogenous peptide, polypeptide or protein is loaded into a composite lipid particle or a modified PMP, for example, using one or more of ultrasonic treatment, electroporation, lipid extraction and lipid extrusion.

[0251] The peptides, polypeptides or proteins included herein may include naturally occurring peptides, polypeptides or proteins or variants produced by recombinant production. In some cases, they may be functional fragments or variants thereof (e.g., enzymatically active fragments or variants thereof). For example, peptides, polypeptides or proteins may be functionally active variants of any one of peptides, polypeptides or proteins described herein, which may have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% consistency with its sequence described herein or naturally occurring peptides, polypeptides or proteins in a specified region or entire sequence. In some cases, a peptide, polypeptide, or protein can be at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or more) identical to a peptide, polypeptide, or protein of interest.

[0252] Peptides, polypeptides or proteins described herein can be formulated in compositions for any use in the uses described herein. Compositions disclosed herein can include peptides, polypeptides or proteins of any quantity or type (e.g., species), such as at least about any one of 1, 2, 3, 4, 5, 10, 15, 20 or more. The suitable concentration of each peptide, polypeptide or protein in the composition depends on factors such as the stability of efficacy, peptides, polypeptides or proteins, the number of different species in the composition, formulations and the method of application of the composition. In some cases, each peptide, polypeptide or protein in the liquid composition is about 0.1 ng / mL to about 100 mg / mL. In some cases, each peptide, polypeptide or protein in the solid composition is about 0.1 mg / g to about 100 mg / g.

[0253] Methods for preparing peptides, polypeptides or proteins are routine in the art. See generally Smales and James (eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar and Meibohm (eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0254] Methods for producing peptides, polypeptides or proteins involve expression in plant cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, mammalian cells or other cells under the control of appropriate promoters. Mammalian expression vectors can contain non-transcribed elements such as replication origins, suitable promoters and enhancers, as well as other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites and termination sequences. DNA sequences derived from the SV40 viral genome, such as the SV40 origin, early promoter, enhancer, splicing and polyadenylation sites can be used to provide other genetic elements required for expressing heterologous DNA sequences. Suitable cloning and expression vectors used with bacteria, fungi, yeast and mammalian cell hosts are described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press) (2012).

[0255] Various mammalian cell culture systems can be used to express and manufacture recombinant polypeptide agents. Examples of mammalian expression systems include CHO cells, COS cells, HeLA and BHK cell lines. The process of host cell culture for producing protein therapeutics is described in, for example, Zhou and Kantardjieff (eds.), Mammalian Cell Cultures for Biologics Manufacturing (Progress in Biochemical Engineering / Biotechnology), Springer (2014). Protein purification is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). The formulation of protein therapeutics is described in Meyer (ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012). Alternatively, the peptide, polypeptide or protein can be a chemically synthesized peptide, polypeptide or protein.

[0256] In some cases, the complex lipid formulation or modified PMP includes an antibody or an antigen-binding fragment thereof. For example, the agent described herein can be an antibody that blocks or enhances the activity and / or function of a component of a pathogen. The antibody can act as an antagonist or agonist of a polypeptide (e.g., an enzyme or a cell receptor) in a pathogen. The preparation and use of antibodies against target antigens in pathogens are known in the art. See, for example, Zhiqiang An (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, 1st edition, Wiley, 2009, and Greenfield (ed.), Antibodies: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 2013, for methods of preparing recombinant antibodies, including antibody engineering, using degenerate oligonucleotides, 5'-RACE, phage display and mutagenesis; Antibody testing and characterization; Antibody pharmacokinetics and pharmacodynamics; Antibody purification and storage; and screening and labeling techniques.

[0257] Exogenous peptides, polypeptides or proteins can be released from the complex lipid formulation or modified PMP in the target cell. In some embodiments, the exogenous peptides, polypeptides or proteins are active in the cytoplasm of the target cell or in the nucleus of the target cell. The exogenous peptides, polypeptides or proteins can be translocated to the nucleus of the target cell.

[0258] In some embodiments, cellular uptake of an exogenous peptide, polypeptide, or protein encapsulated by the complex lipid particle or modified PMP is increased relative to uptake of an exogenous peptide, polypeptide, or protein not encapsulated by the complex lipid particle or modified PMP.

[0259] In some embodiments, the effectiveness of an exogenous peptide or protein encapsulated by the complex lipid particle or modified PMP is increased relative to the effectiveness of an exogenous peptide, polypeptide or protein not encapsulated by the complex lipid particle or modified PMP.

[0260] A. Therapeutic agents

[0261] The exogenous peptide, polypeptide or protein can be a therapeutic agent, such as an agent for preventing or treating a condition or disease. In some embodiments, the disease is cancer, an autoimmune condition or a metabolic disorder.

[0262] In some examples, the therapeutic agent is a peptide (eg, a naturally occurring peptide, a recombinant peptide, or a synthetic peptide) or a protein (eg, a naturally occurring protein, a recombinant protein, or a synthetic protein). In some embodiments, the protein is a fusion protein.

[0263] In some examples, the peptide, polypeptide or protein is endogenous to the organism (eg, mammal) to which the complex lipid formulation or modified PMP is delivered. In other examples, the peptide, polypeptide or protein is not endogenous to the organism.

[0264] In some examples, the therapeutic agent is an antibody (e.g., a monoclonal antibody, such as a monospecific, bispecific, or multispecific monoclonal antibody) or an antigen-binding fragment thereof (e.g., scFv, (scFv)2, Fab, Fab' and F(ab')2, F(ab1)2, Fv, dAb and Fd fragments, or bifunctional antibodies), nanobodies, conjugated antibodies, or antibody-related polypeptides.

[0265] In some examples, the therapeutic agent is an antimicrobial, antibacterial, antifungal, antinematicide, antiparasitic, or antiviral polypeptide.

[0266] In some instances, the therapeutic agent is an allergen, allergen, or antigen.

[0267] In some examples, the therapeutic agent is a vaccine (e.g., a conjugate vaccine, an inactivated vaccine, or a live attenuated vaccine),

[0268] In some instances, the therapeutic agent is an enzyme, such as a metabolic recombinase, a helicase, an integrase, an RNase, a DNase, a ubiquitinated protein. In some instances, the enzyme is a recombinase.

[0269] In some examples, the therapeutic agent is a gene editing protein, such as a component of a CRISPR-Cas system, a TALEN, or a zinc finger.

[0270] In some examples, the therapeutic agent is any of a cytokine, a hormone, a signaling ligand, a transcription factor, a receptor, a receptor antagonist, a receptor agonist, a blocking or neutralizing polypeptide, a riboprotein, or a chaperone protein.

[0271] In some examples, the therapeutic agent is a pore-forming protein, a cell-penetrating peptide, a cell-penetrating peptide inhibitor, or a proteolysis-targeting chimera (PROTAC).

[0272] In some examples, the therapeutic agent is any of an aptamer, a blood derivative, a cell therapy, or an immunotherapy (eg, a cellular immunotherapy).

[0273] In some embodiments, the therapeutic agent is a protein or peptide therapeutic with enzymatic activity, regulatory activity, or targeting activity, such as a protein or peptide with activity that affects one or more of the following: endocrine and growth regulation, metabolic enzyme deficiencies, hematopoiesis, hemostasis, and thrombosis; gastrointestinal disorders; pulmonary disorders; immunodeficiency and / or immunoregulation; fertility; aging (e.g., anti-aging activity); autophagy modulation; epigenetic regulation; oncology; or infectious diseases (e.g., antimicrobial peptides, antifungal or antiviral drugs).

[0274] In some embodiments, the therapeutic agent is a protein vaccine, such as a vaccine used to protect against harmful foreign agents, treat autoimmune diseases, or treat cancer (eg, neoantigens).

[0275] In some examples, the peptide, polypeptide, or protein is globular, fibrous, or disordered.

[0276] In some examples, the size of the peptide, polypeptide, or protein is less than 1, less than 2, less than 5, less than 10, less than 15, less than 20, less than 30, less than 40, less than 50, less than 60, less than 70, less than 80, less than 90, or less than 100 kD, such as 1-50 kD (e.g., 1-10, 10-20, 20-30, 30-40, or 40-50 kD) or 50-100 kD (e.g., 50-60, 60-70, 70-80, 80-90, or 90-100 kD).

[0277] In some examples, the overall charge of a peptide, polypeptide or protein is positive, negative or neutral. A peptide, polypeptide or protein can be modified to change the overall charge, for example by adding one or more charged amino acids, such as one or more (e.g., 1-10 or 5-10) positively or negatively charged amino acids, such as an arginine tail (e.g., 5-10 arginine residues) to the N-terminus or C-terminus of the peptide, polypeptide or protein.

[0278] In some embodiments, the disease is diabetes, such as diabetes mellitus, such as type 1 diabetes. In some embodiments, diabetes is treated by administering to the patient an effective amount of a composition comprising a plurality of composite lipid particles or modified PMPs, wherein one or more exogenous peptides, polypeptides or proteins are encapsulated by the composite lipid particles or modified PMPs. In some embodiments, administration of a plurality of composite lipid particles or modified PMPs reduces blood glucose in the subject. In some embodiments, the therapeutic agent is insulin. In some embodiments, the therapeutic agent is exenatide, semaglutide or tesiparatide.

[0279] In some examples, the therapeutic agent is an antibody shown in Table 1, a peptide shown in Table 2, an enzyme shown in Table 3, or a protein shown in Table 4.

[0280] Table 1. Antibodies

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311] Table 2. Peptides

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] Table 3. Enzymes

[0336]

[0337]

[0338]

[0339]

[0340] Table 4. Protein

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354] B. Enzyme

[0355] The exogenous peptide, polypeptide or protein can be an enzyme, such as an enzyme that catalyzes a biological reaction useful for preventing or treating a condition or disease, preventing or treating infection by a pathogen, diagnosing a disease, or diagnosing a disease or condition.

[0356] The enzyme can be a recombinase, such as Cre recombinase. In some embodiments, Cre recombinase is delivered to cells comprising a Cre reporter construct via a complex lipid formulation or a modified PMP.

[0357] The enzyme can be an editing enzyme, such as a gene editing enzyme. In some embodiments, the gene editing enzyme is, for example, a component of a CRISPR-Cas system (e.g., a Cas9 enzyme), a TALEN, or a zinc finger nuclease.

[0358] C. Pathogen Control Agents

[0359] The exogenous peptide, polypeptide or protein can be a pathogen control agent, such as an antibacterial, antifungal, insecticidal, nematicidal, antiparasitic or virucidal peptide, polypeptide or protein for human health. In some cases, the complex lipid formulations or modified PMP formulations described herein include peptides, polypeptides or proteins or functional fragments or derivatives thereof that target pathways in pathogens. The complex lipid formulations or modified PMP formulations comprising peptides, polypeptides or proteins as described herein can be applied to pathogens, their carriers in an amount and time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of peptide, polypeptide or protein concentration; and (b) reduce or eliminate pathogens. In some cases, the complex lipid formulations or modified PMP formulations comprising peptides, polypeptides or proteins as described herein can be applied to animals suffering from pathogen infection or at risk of pathogen infection in an amount and time sufficient to: (a) reach a target level (e.g., a predetermined or threshold level) of peptide, polypeptide or protein concentration in the animal; and (b) reduce or eliminate pathogens. The peptides, polypeptides or proteins described herein can be formulated in, and in some cases, associated with, complex lipid formulations or modified PMPs thereof for use in any of the methods described herein.

[0360] Examples of peptides, polypeptides or proteins that can be used herein can include enzymes (e.g., metabolic recombinases, helicases, integrases, RNases, DNases or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (e.g., CRISPR-Cas systems, TALENs or zinc fingers), riboproteins, protein aptamers or chaperones.

[0361] The complex lipid formulations or modified PMP formulations described herein can include bacteriocins. In some cases, bacteriocins are naturally produced by Gram-positive bacteria, such as Pseudomonas, Streptomyces, Bacillus, Staphylococcus, or lactic acid bacteria (LAB, such as Lactococcus lactis). In some cases, bacteriocins are naturally produced by Gram-negative bacteria, such as Hafnia alvei, Citrobacter freundii, Klebsiella oxytoca, Klebsiella pneumonia, Enterobacter cloacae, Serratia plymithicum, Xanthomonas campestris, Erwinia carotovora, Ralstonia solanacearum, or Escherichia coli. Exemplary bacteriocins include, but are not limited to, Class I-IV LAB antibiotics (such as prebiotics), colibacins, microcins, and pyocyanin.

[0362] The complex lipid formulations or modified PMP formulations described herein may include antimicrobial peptides (AMPs). Any AMP suitable for inhibiting microorganisms may be used. AMPs are a group of different molecules that are divided into subgroups based on their amino acid composition and structure. AMPs may be derived from or produced from any organism that naturally produces AMPs, including AMPs derived from plants (e.g., copsin), insects (e.g., wasp toxins, poneratoxins, cecropins, silkworm antimicrobial peptides, melittin), frogs (e.g., bombycin, dermatostagmin, aurein) and mammals (e.g., rabbit antimicrobial peptides, defensins and protegrins).

[0363] IV. Methods for Producing Complex Lipid Formulations Comprising Exogenous Polypeptides or Modified PMPs

[0364] Another aspect of the present invention relates to a method for producing a complex lipid formulation comprising a plurality of complex lipid particles encapsulating an exogenous peptide, polypeptide or protein. The method comprises:

[0365] extracting at least five lipids from one or more plant sources;

[0366] mixing at least two exogenous lipids with the extracted plant lipids to form composite lipid particles; and

[0367] The complex lipid particles are loaded with the exogenous peptide, polypeptide or protein, wherein the loading causes the exogenous peptide, polypeptide or protein to be encapsulated by the complex lipid particles, thereby forming the complex lipid formulation.

[0368] Additional description of general procedures and exemplary methods for producing complex lipid particles and encapsulating the complex lipid particles with exogenous peptides, polypeptides or proteins can be found in Examples 1-2.

[0369] In another aspect, the disclosure generally features a method of producing a modified PMP comprising an exogenous peptide, polypeptide, or protein. The method accordingly comprises: (a) providing a solution comprising an exogenous peptide, polypeptide, or protein; and (b) loading the modified PMP with the exogenous peptide, polypeptide, or protein, wherein the loading causes the exogenous peptide, polypeptide, or protein to be encapsulated by the modified PMP.

[0370] The exogenous peptide, polypeptide or protein can be placed in a solution, such as a phosphate buffered saline (PBS) solution. The exogenous peptide, polypeptide or protein may or may not be soluble in the solution. If the peptide, polypeptide or protein is insoluble in the solution, the pH of the solution can be adjusted until the polypeptide is soluble in the solution. Insoluble peptides, polypeptides or proteins can also be used for loading.

[0371] Loading the composite lipid particles or modified PMPs with exogenous peptides, polypeptides or proteins may comprise or consist of sonicating a solution comprising an exogenous peptide, polypeptide or protein (e.g., a soluble or insoluble exogenous polypeptide) and a plurality of composite lipid particles or modified PMPs to induce pores in the composite lipid particles or modified PMPs and diffusion of the peptide, polypeptide or protein into the composite lipid particles or modified PMPs, such as sonication according to the protocol described in Wang et al., Nature Comm., 4:1867, 2013.

[0372] Alternatively, loading the composite lipid particles or modified PMPs with exogenous peptides, polypeptides or proteins may comprise or consist of electroporating a solution comprising an exogenous peptide, polypeptide or protein (e.g., a soluble or insoluble exogenous polypeptide) and a plurality of composite lipid particles or modified PMPs, such as electroporation according to the protocol described in Wahlgren et al., Nucl. Acids. Res., 40(17), e130, 2012.

[0373] Alternatively, small amounts of detergents (e.g., saponin) can be added to increase loading of exogenous peptides, polypeptides, or proteins into complex lipid particles or modified PMPs, for example, as described in Fuhrmann et al., J Control Release., 205:35-44, 2015.

[0374] Loading composite lipid particles or modified PMP with exogenous peptides, polypeptides or proteins may include or consist of the following: lipid extraction and lipid extrusion. In short, plant lipids can be separated by adding MeOH:CHCl3 (e.g., 3.75mL 2:1 (v / v) MeOH:CHCl3) to a PBS solution containing PMP (e.g., PBS containing 1mL PMP) and vortexing the mixture. Then sequentially add and vortex CHCl3 (e.g., 1.25mL) and ddHO (e.g., 1.25mL). Then the mixture is centrifuged at 2,000rpm for 10 minutes in a glass tube at 22°C to separate the mixture into two phases (aqueous phase and organic phase). The organic phase sample containing plant lipids is dried by heating under nitrogen (2psi). In order to load peptides, the separated plant lipids are mixed with peptides, polypeptides or protein solutions and passed through a lipid extruder, such as according to the scheme from Haney et al., " Controlled Release Journal ", 207:18-30,2015.

[0375] It is also possible to separate plant lipids using methods for separating other plant lipid classes, such as glycosyl inositol phosphorylceramide (GIPC), such as Casas et al., Plant Physiology, 170: 367-384, described in 2016. In brief, in order to extract plant lipids including GIPC, chloroform: methanol: HCl (e.g., 3.5 mL chloroform: methanol: HCl (200: 100: 1, v / v / v)) plus butylated hydroxytoluene (e.g., 0.01% (w / v) butylated hydroxytoluene) is added to PMP and incubated with PMP. Next, NaCl (e.g., 2 mL 0.9% (w / v) NaCl) is added and vortexed for 5 minutes. The sample is then centrifuged to induce the organic phase to aggregate at the bottom of the glass tube, and the organic phase is collected. The upper phase can be re-extracted with chloroform (e.g., 4 mL pure chloroform) to separate lipids. The organic phases are combined and dried. After drying, the aqueous phase is resuspended in water (e.g., 1 mL pure water), and GIPC is stripped twice using butanol-1 (e.g., 1 mL butanol-1). In order to load exogenous peptides, polypeptides or proteins, according to the scheme from Haney et al., " Controlled Release Journal ", 207: 18-30, 2015, the separated plant lipid phase is mixed with a peptide, polypeptide or protein solution and passed through a lipid extruder. Alternatively, lipids can be extracted with methyl tert-butyl ether (MTBE): methanol: water plus butylated hydroxytoluene (BHT) or with propan-2-ol: hexane: water.

[0376] In some embodiments, isolated GIPCs may be added to isolated plant lipids.

[0377] In some embodiments, loading the complex lipid particles or modified PMPs with exogenous peptides, polypeptides, or proteins comprises sonication and lipid extrusion, as described above.

[0378] In some embodiments, exogenous peptides, polypeptides, or proteins may be pre-complexed (eg, using protamine sulfate), or cationic lipids (eg, DOTAP) may be added to facilitate encapsulation of negatively charged proteins.

[0379] Before use, the loaded composite lipid particles or the loaded modified PMP can be purified to remove peptides, polypeptides or proteins that are not combined with or encapsulated by the composite lipid particles or modified PMPs. The loaded composite lipid particles or the loaded modified PMPs can be characterized, and their stability can be tested. The loading of exogenous peptides, polypeptides or proteins can be quantified by protein quantification methods known in the art. For example, Pierce Quantitative Colorimetric Peptide Assay can be used for loading and unloaded composite lipid particles or small samples of modified PMPs, or Western blotting using specific antibodies can be used to detect exogenous peptides, polypeptides or proteins. Alternatively, peptides, polypeptides or proteins can be fluorescently labeled, and fluorescence can be used to determine the exogenous peptides, polypeptides or protein concentrations of the labels in the loaded and unloaded composite lipid particles or modified PMPs. Further descriptions of the purification, characterization, stability and loading of PMPs can be found in WO 2021 / 041301, which is incorporated by reference as a whole.

[0380] V. Treatment Methods

[0381] The complex lipid formulations or modified PMP formulations described herein can be used in a variety of therapeutic methods, particularly for preventing or treating a disorder or disease or for preventing or treating a pathogen infection in an animal. The methods of the invention relate to delivering the complex lipid formulations or modified PMP formulations described herein to an animal (e.g., a human).

[0382] Provided herein are methods of administering the complex lipid formulations or modified PMP formulations disclosed herein to animals. The methods can be used to prevent or treat a condition or disease or to prevent pathogen infection in animals (eg, humans).

[0383] For example, a method for treating an animal suffering from a fungal infection is provided herein, wherein the method comprises administering to the animal an effective amount of a complex lipid formulation comprising a plurality of complex lipid particles or a modified PMP formulation comprising a plurality of modified PMPs, wherein the complex lipid formulation or the modified PMP formulation comprises an exogenous peptide, polypeptide or protein as a pathogen control agent, such as an antifungal agent. In some cases, the fungal infection is caused by Candida albicans. In some cases, the method reduces or substantially eliminates the fungal infection.

[0384] On the other hand, there is provided herein a method for treating an animal (e.g., a human) suffering from a bacterial infection, wherein the method comprises administering to the animal an effective amount of a composite lipid formulation comprising a plurality of composite lipid particles or a modified PMP formulation comprising a plurality of modified PMPs. In some cases, the method comprises administering to the animal an effective amount of a composite lipid formulation comprising a plurality of composite lipid particles or a modified PMP formulation comprising a plurality of modified PMPs, wherein the composite lipid formulation or the modified PMP formulation comprises an exogenous peptide, polypeptide or protein as a pathogen control agent, such as an antibacterial agent. In some cases, the bacterium is Streptococcus spp., Pneumococcus spp., Pseudamonas spp., Shigella spp., Salmonella spp., Campylobacter spp. or Escherichia spp. In some cases, the method reduces or substantially eliminates bacterial infection. In some cases, the animal is a human, a veterinary animal, or a livestock animal.

[0385] The inventive method can be used for treating (for example, such as caused by animal such as human pathogen) infection in animals, which refers to applying treatment to animals already suffering from disease to improve or stabilize the symptom of animals. This can be related to reducing the colonization of pathogens in, on or around animals relative to the starting amount (for example, reducing by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) and / or allowing individual benefits (for example, reducing colonization with an amount sufficient to resolve symptoms). In such cases, the treated infection can be manifested as a reduction in symptoms (for example, reducing by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). In some cases, the treated infection can be effective to increase the likelihood of survival of an individual (e.g., an increase in the likelihood of survival of about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or to increase the overall survival of a population (e.g., an increase in the likelihood of survival of about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, the compositions and methods can be effective to "substantially eliminate" an infection, which means reducing the infection by an amount sufficient to persistently resolve symptoms in the animal (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0386] The methods of the invention can be used to prevent infection (e.g., such as caused by an animal, such as a human pathogen), which refers to preventing an increase in colonization of one or more pathogens in, on, or around an animal (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% relative to an untreated animal) in an amount sufficient to maintain the initial pathogen population (e.g., about the amount found in a healthy individual), preventing the onset of infection and / or preventing symptoms or conditions associated with infection. For example, in immunocompromised individuals (e.g., individuals with cancer, individuals with HIV / AIDS, or individuals taking immunosuppressants), or in individuals undergoing long-term antibiotic therapy, individuals can receive prophylactic treatment to prevent fungal infection when preparing for an invasive medical procedure (e.g., preparing for surgery, such as receiving a transplant, stem cell therapy, a graft, a prosthesis, receiving long-term or frequent intravenous catheters, or receiving treatment in an intensive care unit).

[0387] The complex lipid formulation or modified PMP formulation can be formulated for administration or administered by any suitable method, including, for example, oral administration, enteral administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal administration, intraarterial administration, intraperitoneal administration, intralesional administration, intracranial administration, intraarticular administration, intraprostatic administration, intrapleural administration, intratracheal administration, intrathecal administration, intranasal administration, intravaginal administration, intrarectal administration (including intracolonic administration), topical administration, intratumoral administration, peritoneal administration, subconjunctival administration, intracapsular administration, intramucosal administration, intrapericardial administration, intraumbilical administration, intraocular administration, intraorbital administration, topical administration, transdermal administration, or intravitreal administration (e.g., by intravitreal injection), administration by eye drops, administration by inhalation (e.g., by a nebulizer), administration by injection, administration by implantation, administration by infusion, administration by continuous infusion, administration by direct local perfusion of target cells, administration by catheter, administration by lavage, administration in a cream, or administration in a lipid composition. The composition used in the methods described herein can also be systemically or locally administered. The method of administration can be varied according to various factors (e.g., the severity of the compound or composition administered and the condition, disease or illness treated). In some cases, the complex lipid formulation or the modified PMP formulation is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly or intranasally. Administration can be by any suitable route, such as oral or by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or chronic. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, bolus administration and pulse infusions at different time points.

[0388] Prevention or treatment of infections described herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity of the disease and the course. Whether the therapeutic agent is administered for preventive or therapeutic purposes will depend on previous therapy, the patient's clinical history and the response to the complex lipid formulation or modified PMP formulation. The complex lipid formulation or modified PMP formulation can be administered to the patient, for example, once or through a series of treatments. For repeated administration over a few days or longer, depending on the condition, treatment will generally continue until the desired disease symptoms are suppressed or the infection is no longer detectable. Such dosages can be administered intermittently, for example, weekly or biweekly (for example, so that the patient receives, for example, about two to about twenty doses of complex lipid formulations or modified PMP formulations). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens can be useful. The progress of this therapy can be easily monitored by conventional techniques and assays.

[0389] In some cases, the amount of the complex lipid formulation or modified PMP formulation administered to an individual (e.g., a human) can be in the range of about 0.01 mg / kg to about 5 g / kg (e.g., about 0.01 mg / kg-0.1 mg / kg, about 0.1 mg / kg-1 mg / kg, about 1 mg / kg-10 mg / kg, about 10 mg / kg-100 mg / kg, about 100 mg / kg-1 g / kg, or about 1 g / kg-5 g / kg) of the individual's body weight. In some cases, the amount of the complex lipid formulation or modified PMP formulation administered to an individual (e.g., a human) is at least 0.01 mg / kg (e.g., at least 0.01 mg / kg, at least 0.1 mg / kg, at least 1 mg / kg, at least 10 mg / kg, at least 100 mg / kg, at least 1 g / kg, or at least 5 g / kg) of the individual's body weight. The dosage can be administered as a single dose or as multiple doses (e.g., 2, 3, 4, 5, 6, 7 or more than 7 doses). In some cases, the complex lipid formulation or modified PMP formulation administered to the animal can be administered alone or in combination with another therapeutic agent or pathogen control agent. The dose of the antibody administered in the combination therapy can be reduced as compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.

[0390] In one aspect, the disclosure features a method for treating diabetes comprising administering to a subject in need thereof an effective amount of a composition comprising a plurality of composite lipid particles or a plurality of modified PMPs encapsulating one or more exogenous peptides, polypeptides or proteins. Administration of a plurality of composite lipid particles or a plurality of modified PMPs can reduce blood glucose in the subject. In some embodiments, the exogenous peptide, polypeptide or protein is insulin, exenatide, semaglutide or tesiparatide.

[0391] VI. Methods for treating pathogens or their vectors

[0392] The complex lipid formulations or modified PMP formulations and related methods described herein can be used to reduce the fitness of animal pathogens and thereby treat or prevent infection in animals, such as humans. Examples of animal pathogens or their vectors that can be treated with the compositions or related methods of the present invention are further described herein.

[0393] A. Fungi

[0394] The complex lipid formulations or modified PMP formulations and related methods can be used to reduce the adaptability of fungi, for example, to prevent or treat fungal infections in animals such as humans. Methods of delivering the complex lipid formulations or modified PMP formulations to fungi are included by contacting the fungi with the complex lipid formulations or modified PMP formulations. Additionally or alternatively, the methods include preventing or treating fungal infections (e.g., caused by the fungi described herein) in animals at risk or in need thereof by administering the complex lipid formulations or modified PMP formulations to the animals.

[0395] The complex lipid formulations or modified PMP formulations and related methods are suitable for treating or preventing fungal infections in animals Fungal infections in animals include those belonging to the phylum Ascomycota (Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis / posadasii, Candida albicans), the phylum Basidiomycota (Filobasidiella neoformans, Trichosporon), the phylum Microsporidia (Encephalitozoon cuniculi, Enterocytozoon bieneusi), the phylum Mucoromycotina (Mucor circinelloides), the phylum circinelloides), Rhizopus oryzae, and Lichtheimia corymbifera).

[0396] In some cases, the fungal infection is caused by a fungus belonging to the phylum Ascomycota, Basidiomycota, Chytridiomycota, Microsporum, or Zygomycota. Fungal infection or overgrowth may include one or more fungal species, such as Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. auris, C. krusei, Saccharomyces cerevisiae, Malassezia globose, M. restricta, or Debaryomyces hansenii, Gibberella moniliformis, Alternaria brassicicola, Cryptococcus neoformans, Pneumocystis carinii, carinii, P. jirovecii, P. murina, P. oryctolagi, P. wakefieldiae, and Aspergillus clavatus. Fungal species can be considered pathogens or opportunistic pathogens.

[0397] In some cases, the fungal infection is caused by a fungus in the genus Candida (i.e., a Candida infection). For example, a Candida infection can be caused by a fungus in the genus Candida, selected from the group consisting of: Candida albicans, Candida glabrata, Candida dubliniensis, Candida krusei, Candida auris, Candida parapsilosis, Candida tropicalis, Candida orthopsilosis, Candida guilliermondii, Candida rugose, and Candida lusitaniae. Candida infections that can be treated by the methods disclosed herein include, but are not limited to, candidemia, oropharyngeal candidiasis, esophageal candidiasis, mucosal candidiasis, genital candidiasis, vulvovaginal candidiasis, rectal candidiasis, hepatic candidiasis, renal candidiasis, pulmonary candidiasis, splenic candidiasis, otomycosis, osteomyelitis, septic arthritis, cardiovascular candidiasis (e.g., endocarditis), and invasive candidiasis.

[0398] B. Bacteria

[0399] The complex lipid formulations or modified PMP formulations and related methods can be used to reduce the fitness of bacteria, for example, to prevent or treat bacterial infections in animals such as humans. Methods of applying the complex lipid formulations or modified PMP formulations to bacteria by contacting the bacteria with the complex lipid formulations or modified PMP compositions are included. Additionally or alternatively, the method includes preventing or treating bacterial infections (e.g., caused by the bacteria described herein) in animals at risk or in need thereof by administering the complex lipid formulations or modified PMP formulations to the animals.

[0400] The complex lipid formulations or modified PMP formulations and related methods are suitable for preventing or treating bacterial infections in animals caused by any of the bacteria described further below. For example, the bacteria may be bacteria belonging to the following: Bacillales (Bacillales anthracis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes), Lactobacillales (S. pneumoniae, S. pyogenes), Clostridiales (C. botulinum, C. difficile, C. perfringens, C. tetani), Spirochaetales (Borrelia burgdorferi, Treponema pallidum), Chlamydiales (Chlamydia trachomatis, Chlamydophila psittaci), psittaci), Actinomycetales (C. diphtheriae, Mycobacterium tuberculosis, M. avium), Rickettsiales (R. prowazekii, R. rickettsii, R. typhi, A. phagocytophilum, E. chaffeensis), Rhizobiales (Brucella melitensis), Burkholderiales (Bordetella pertussis, Burkholderia mallei), Burkholderia pseudomallei), Neisseriales (Neisseria gonorrhoeae), Neisseria meningitidis (N.meningitidis), Campylobacterales (Campylobacter jejuni, Helicobacter pylori), Legionellales (Legionella pneumophila), Pseudomonadales (A. baumannii, Moraxella catarrhalis, P. aeruginosa), Aeromonadales (Aeromonas sp.), Vibrionales (Vibrio cholerae, V. parahaemolyticus), Thiotrichales, Pasteurellales (Haemophilus influenzae), influenzae), Enterobacteriales (Klebsiella pneumoniae, Proteus mirabilis, Yersinia pestis, Y. enterocolitica, Shigella flexneri, Salmonella enterica, Escherichia coli).

[0401] The present invention can be further represented by the following examples:

[0402] Example 1. A method for delivering a therapeutic peptide or protein to a human subject in need thereof, the method comprising orally or enterally administering to the human subject a pharmaceutical formulation comprising:

[0403] (a) a plurality of complex lipid particles characterized by: (i) comprising at least 10 plant lipids extracted from one or more plant sources; (ii) comprising sterols that are exogenous to the one or more plant sources; (iii) comprising polyethylene glycol (PEG) conjugated lipids; (iii) containing less than 10% w / w proteinaceous material that is endogenous to the one or more plant sources; and (iv) containing less than 10 mol% of exogenous ionizable lipids; and

[0404] (b) the therapeutic peptide or protein encapsulated in the composite lipid particle.

[0405] Embodiment 2. The method according to embodiment 1, wherein the therapeutic peptide or protein is a hormone or a glucagon-like peptide 1 (GLP-1) agonist.

[0406] Embodiment 3. The method of embodiment 2, wherein the therapeutic peptide or protein is insulin, exenatide, semaglutide or tesiparatide.

[0407] Embodiment 4. The method of embodiment 1, wherein the therapeutic peptide or protein is delivered to brain tissue of the human subject.

[0408] Example 5. A method according to Example 1, wherein the composite lipid particles contain ten or more lipids, and the ten or more lipids belong to one or more subclasses selected from the group consisting of: acyl steryl glycosides, ceramides, digalactosyl diacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyl diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyl diacylglycerols and sterols.

[0409] Embodiment 6. The method of embodiment 5, wherein the composite lipid particle contains lipids from at least five, at least six, at least seven, at least eight, at least nine or at least ten different subclasses.

[0410] Embodiment 7. The method of embodiment 1, wherein the complex lipid particles contain less than 5% w / w proteinaceous material endogenous to the one or more plant sources.

[0411] Embodiment 8. The method according to embodiment 1, wherein the composite lipid particles contain less than 5 mol % of exogenous ionizable lipids.

[0412] Embodiment 9. The method of embodiment 1, wherein at least one of the plant sources is grapefruit, lemon, pitaya, spinach, kale, strawberry, broccoli or soybean.

[0413] Embodiment 10. The method according to embodiment 1, wherein the composite lipid particle comprises:

[0414] about 85-95% w / w of said vegetable lipid,

[0415] about 5 to 8% w / w of said sterol,

[0416] About 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipid in the complex lipid formulation.

[0417] Embodiment 11. A complex lipid formulation comprising:

[0418] a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and

[0419] One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties:

[0420] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0421] ii) contains less than 50 mol % ionizable lipids.

[0422] Embodiment 12. The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide or protein is a therapeutic agent.

[0423] Embodiment 13. The complex lipid formulation according to embodiment 11, wherein the exogenous peptide, polypeptide or protein is an antibody or an antibody fragment.

[0424] Embodiment 14. The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide or protein is a hormone.

[0425] Embodiment 15. The complex lipid formulation according to embodiment 14, wherein the exogenous peptide, polypeptide or protein is insulin.

[0426] Embodiment 16. The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide or protein is a receptor agonist or a receptor antagonist.

[0427] Embodiment 17. The complex lipid formulation according to embodiment 16, wherein the exogenous peptide, polypeptide or protein is a glucagon-like peptide 1 (GLP-1) agonist.

[0428] Embodiment 18. The complex lipid formulation according to embodiment 17, wherein the exogenous peptide, polypeptide or protein is exenatide, semaglutide or tesiparatide.

[0429] Embodiment 19. The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide or protein is less than 100 kD in size.

[0430] Embodiment 20. The complex lipid formulation of embodiment 19, wherein the exogenous peptide, polypeptide or protein is less than 50 kD in size.

[0431] Embodiment 21. The complex lipid formulation of embodiment 19, wherein the exogenous peptide, polypeptide or protein has a size of at least 3 kD.

[0432] Embodiment 22. The complex lipid formulation of embodiment 19, wherein the exogenous peptide, polypeptide or protein comprises at least 30 amino acid residues.

[0433] Embodiment 23. The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain 5-1000 lipids extracted from one or more plant sources.

[0434] Embodiment 24. The complex lipid formulation according to embodiment 11, wherein the complex lipid particles contain at least 10 plant lipids, wherein the plant lipids belong to one or more of the categories selected from the group consisting of glycerolipids, sphingolipids and sterols.

[0435] Embodiment 25. The complex lipid formulation of embodiment 24, wherein the complex lipid particles contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG) and sulfatides (SL).

[0436] Embodiment 26. The complex lipid formulation of embodiment 24, wherein the complex lipid particles contain one or more sphingolipids selected from the group consisting of glycosylinositolphosphoceramide (GIPC), glucosylceramide (GCer), ceramide (Cer) and free long chain base (LCB).

[0437] Embodiment 27. The complex lipid formulation of embodiment 24, wherein the complex lipid particles contain one or more phytosterols selected from the group consisting of campesterol, stigmasterol, and sitosterol.

[0438] Embodiment 28. The complex lipid formulation of embodiment 24, wherein the complex lipid particle contains one or more lipids belonging to one or more of the subclasses selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesterol ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acids, fatty acid esters of hydroxy fatty acids, hemi-dimonoacylglycerophosphate, hexosylceramide, lyso Phosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidyl, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, sterol esters, stigmasterol, sulfatides, sulfonic acid lipids, sphingomyelin, sulfoisorhamnosyldiacylglycerol, sterols and triacylglycerols.

[0439] Embodiment 29. A complex lipid formulation according to embodiment 28, wherein the complex lipid particle contains ten or more lipids, and the ten or more lipids belong to one or more subclasses selected from the group consisting of: acyl steryl glycosides, ceramides, digalactosyl diacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyl diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyl diacylglycerols and sterols.

[0440] Embodiment 30. The complex lipid formulation of embodiment 28 or 29, wherein the complex lipid particle contains lipids from at least five, at least six, at least seven, at least eight, at least nine, or at least ten different subclasses.

[0441] Embodiment 31. The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain less than 30% w / w proteinaceous material endogenous to the one or more plant sources.

[0442] Embodiment 32. The complex lipid formulation of embodiment 31, wherein the complex lipid particles contain less than 5% w / w of proteinaceous material endogenous to the one or more plant sources.

[0443] Embodiment 33. The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain less than 20 mol% of exogenous ionizable lipids.

[0444] Embodiment 34. The complex lipid formulation of embodiment 33, wherein the complex lipid particles contain less than 5 mol% of exogenous ionizable lipids.

[0445] Embodiment 35. The complex lipid formulation of embodiment 11, wherein at least one of the plant sources is a citrus fruit.

[0446] Embodiment 36. The complex lipid formulation of embodiment 35, wherein the citrus fruit is grapefruit or lemon.

[0447] Embodiment 37. The complex lipid formulation of embodiment 11, wherein at least one of the plant sources is a non-citrus plant.

[0448] Embodiment 38. The complex lipid formulation of embodiment 37, wherein the non-citrus plant is pitaya, spinach, kale, strawberry, broccoli or soybean.

[0449] Embodiment 39. The complex lipid formulation of embodiment 11, wherein the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0450] Embodiment 40. The complex lipid formulation of embodiment 39, wherein the sterol is cholesterol or sitosterol.

[0451] Embodiment 41. The complex lipid formulation of embodiment 39, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

[0452] Embodiment 42. The complex lipid formulation according to embodiment 39, wherein the PEG-lipid conjugate is PEG2000-PE, PEG2000-DMG, PEG2000-DSPE or a derivative thereof.

[0453] Embodiment 43. The complex lipid formulation of embodiment 39, wherein the exogenous lipid further comprises a lipid selected from the group consisting of: a fatty acid, a glycerolipid, a glycerophospholipid, a sphingolipid, a second sterol, and an additive synthetic lipid.

[0454] Embodiment 44. The complex lipid formulation of embodiment 39, wherein the complex lipid particles comprise:

[0455] about 10-95% w / w of said vegetable lipid,

[0456] about 5 to 60% w / w of said sterol,

[0457] About 0.5-15% w / w of the polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipids in the complex lipid formulation.

[0458] Embodiment 45. The complex lipid formulation of embodiment 44, wherein the complex lipid particles comprise:

[0459] about 85-95% w / w of said vegetable lipid,

[0460] about 5 to 8% w / w of said sterol,

[0461] About 1-3.5% w / w of the polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipids in the complex lipid formulation.

[0462] Embodiment 46. The complex lipid formulation of embodiment 21, wherein the average size of the complex lipid particles is less than about 250 nm.

[0463] Embodiment 47. The complex lipid formulation of embodiment 46, wherein the average size of the complex lipid particles is about 100 to 180 nm.

[0464] Embodiment 48. The complex lipid formulation of embodiment 11, wherein the PDI of the complex lipid particles is about 0.1 to about 0.5.

[0465] Embodiment 49. The complex lipid formulation of embodiment 48, wherein the PDI of the complex lipid particles is about 0.2 to about 0.4.

[0466] Embodiment 50. The complex lipid formulation of embodiment 11, wherein the complex lipid particles further comprise one or more cryoprotectants or lyoprotectants.

[0467] Embodiment 51. The complex lipid formulation of embodiment 11, wherein the complex lipid formulation is a lyophilized composition.

[0468] Embodiment 52. The complex lipid formulation of embodiment 11, wherein the complex lipid formulation is a liquid composition.

[0469] Embodiment 53. The complex lipid formulation of embodiment 11, wherein the complex lipid formulation is stable at room temperature and / or at 4°C for at least two weeks without lyophilization.

[0470] Embodiment 54. A pharmaceutical composition comprising the complex lipid formulation according to any one of embodiments 1 to 53 and a pharmaceutically acceptable vehicle, carrier or excipient.

[0471] Embodiment 55. The pharmaceutical composition according to embodiment 54, wherein the pharmaceutical composition is in capsule form or tablet form.

[0472] Embodiment 56. A method for delivering a peptide, polypeptide or protein to a mammalian cell or a mammal, the method comprising:

[0473] contacting the mammalian cell with the complex lipid formulation or administering the complex lipid formulation to the mammal under conditions sufficient to allow the mammalian cell or the mammal to take up the complex lipid formulation,

[0474] Wherein the complex lipid formulation comprises:

[0475] a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and

[0476] One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties:

[0477] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0478] ii) contains less than 50 mol % ionizable lipids.

[0479] Embodiment 57. A method according to Embodiment 56, wherein the mammalian cell is a human cell, or the mammal is a human.

[0480] Embodiment 58. A method according to embodiment 56, wherein the uptake of the exogenous peptide, polypeptide or protein encapsulated by the composite lipid particle by the mammalian cell or the mammal is increased relative to the uptake of the exogenous peptide, polypeptide or protein not encapsulated by the composite lipid particle.

[0481] Embodiment 59. The method of embodiment 56, wherein the method is used to deliver a peptide, polypeptide or protein to a mammal, and the administration is performed via an oral, enteral, intranasal, intracolonic, intrarectal or intrajejunal route.

[0482] Embodiment 60. A method according to embodiment 56, wherein the mammalian cell is a brain cell.

[0483] Embodiment 61. A method for treating or preventing a disease or condition for which a therapeutic agent is indicated in a subject, the method comprising:

[0484] administering to the subject in need thereof an effective amount of a complex lipid formulation comprising:

[0485] a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and

[0486] One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties:

[0487] i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and

[0488] ii) contains less than 50 mol % ionizable lipids.

[0489] Embodiment 62. The method of embodiment 61, wherein said administration is via an oral, enteral, intranasal, intracolonic, intrarectal or intrajejunal route.

[0490] Embodiment 63. The method of embodiment 61, wherein the disease is diabetes and the exogenous peptide, polypeptide or protein is insulin, exenatide, semaglutide or tesiparatide.

[0491] Embodiment 64. A method of producing a complex lipid formulation comprising a plurality of complex lipid particles encapsulating an exogenous peptide, polypeptide or protein, the method comprising:

[0492] extracting at least five lipids from one or more plant sources;

[0493] mixing at least two exogenous lipids with the extracted plant lipids to form composite lipid particles; and

[0494] The complex lipid particles are loaded with the exogenous peptide, polypeptide or protein, wherein the loading causes the exogenous peptide, polypeptide or protein to be encapsulated by the complex lipid particles, thereby forming the complex lipid formulation.

[0495] Embodiment 65. A method according to embodiment 64, wherein the lipids are extracted from the plant sources by adding an extraction solvent to one or more plant sources, wherein the extraction solvent comprises methanol, ethanol, propanol, 1-butanol, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, methyl tert-butyl ether, chloroform, ethyl acetate or a mixture thereof.

[0496] Embodiment 66. A method according to embodiment 65, wherein the extraction solvent is dichloromethane:methanol; chloroform:methanol; methanol:methyl tert-butyl ether (MTBE); dimethylformamide:methanol; acetonitrile:methanol; acetone:methanol; tetrahydrofuran:methanol; dimethyl sulfoxide:methanol; acetonitrile:ethanol; or ethyl acetate:ethanol.

[0497] Embodiment 67. The method of embodiment 64, wherein the extracting step further comprises reducing or eliminating proteinaceous material endogenous to the one or more plant sources to less than 50% w / w.

[0498] Embodiment 68. The method of embodiment 64, wherein the mixing step is performed by thin film mixing or microfluidic mixing.

[0499] Embodiment 69. The method of embodiment 64, wherein the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0500] Embodiment 70. A method according to embodiment 64, wherein the exogenous lipids do not include ionizable lipids.

[0501] Embodiment 71. A modified plant messenger package (PMP) formulation comprising:

[0502] one or more PMPs modified with one or more sterols and one or more polyethylene glycol (PEG)-lipid conjugates,

[0503] wherein the modified PMP is formulated with one or more exogenous peptides, polypeptides or proteins, and wherein the one or more exogenous peptides, polypeptides or proteins are encapsulated by the modified PMP.

[0504] Embodiment 72. The modified PMP formulation of Embodiment 71, wherein the exogenous peptide, polypeptide or protein is a therapeutic agent.

[0505] Embodiment 73. The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide or protein is an enzyme.

[0506] Embodiment 74. The modified PMP formulation of embodiment 73, wherein the enzyme is a recombinase or an editing enzyme.

[0507] Embodiment 75. The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide or protein is an antibody or antibody fragment.

[0508] Embodiment 76. The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide or protein is an Fc fusion protein.

[0509] Embodiment 77. The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide or protein is a hormone.

[0510] Embodiment 78. The modified PMP formulation of embodiment 77, wherein the exogenous peptide, polypeptide or protein is insulin.

[0511] Embodiment 79. The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide or protein is a receptor agonist or a receptor antagonist.

[0512] Embodiment 80. The modified PMP formulation of any one of embodiments 71 to 79, wherein the exogenous peptide, polypeptide or protein is less than 100 kD in size.

[0513] Embodiment 81. The modified PMP formulation of embodiment 80, wherein the exogenous peptide, polypeptide or protein is less than 50 kD in size.

[0514] Embodiment 82. The modified PMP formulation of any one of embodiments 71 to 79, wherein the exogenous peptide, polypeptide or protein is at least 5 kD in size.

[0515] Embodiment 83. The modified PMP formulation of any one of embodiments 71 to 79, wherein the exogenous peptide, polypeptide or protein comprises at least 50 amino acid residues.

[0516] Embodiment 84. The modified PMP formulation of any one of embodiments 71 to 83, wherein the overall charge of the exogenous peptide, polypeptide or protein is neutral, or has been modified to be neutrally charged.

[0517] Embodiment 85. The modified PMP formulation of any one of embodiments 71 to 83, wherein the overall charge of the exogenous peptide, polypeptide or protein is positive or negative.

[0518] Embodiment 86. The modified PMP formulation of any one of embodiments 71 to 85, wherein the PMP comprises a purified plant extracellular vesicle (EV) or a fragment or extract thereof.

[0519] Embodiment 87. The modified PMP formulation of embodiment 86, wherein the PMP is obtained from citrus fruit.

[0520] Embodiment 88. The modified PMP formulation of embodiment 87, wherein the citrus fruit is grapefruit or lemon.

[0521] Embodiment 89. The modified PMP formulation of any one of embodiments 71 to 88, wherein the sterol is cholesterol or sitosterol.

[0522] Embodiment 90. The modified PMP formulation of any one of embodiments 71 to 88, wherein the PEG-lipid conjugate is C14-PEG2k or C18-PEG2k.

[0523] Embodiment 91. The modified PMP formulation of any one of embodiments 71 to 88, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

[0524] Embodiment 92. The modified PMP formulation of any one of embodiments 71 to 88, wherein the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof.

[0525] Embodiment 93. The modified PMP formulation of any one of embodiments 71 to 88, wherein the sterol is cholesterol and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof.

[0526] Embodiment 94. The modified PMP formulation of embodiment 86, wherein the concentration of the sterol ranges from about 0.5 to 15% w / w based on the amount of the total lipid extract.

[0527] Embodiment 95. The modified PMP formulation of embodiment 94, wherein the concentration of the sterol ranges from about 5 to 8% w / w based on the amount of the total lipid extract.

[0528] Embodiment 96. The modified PMP formulation of embodiment 86, wherein the concentration of the PEG-lipid conjugate ranges from about 0.5 to 5% w / w based on the amount of the total lipid extract.

[0529] Embodiment 97. The modified PMP formulation of embodiment 96, wherein the concentration of the PEG-lipid conjugate ranges from about 1 to 3.5% w / w based on the amount of the total lipid extract.

[0530] Embodiment 98. A modified PMP formulation according to embodiment 86, wherein the sterol is cholesterol in a concentration range of about 5 to 8% w / w based on the amount of total lipid extract; and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof in a concentration range of about 1 to 3.5% w / w based on the amount of total lipid extract.

[0531] Embodiment 99. The modified PMP formulation of any one of embodiments 71 to 98, wherein the modified PMP is a lipid nanoparticle.

[0532] Embodiment 100. The modified PMP formulation of any one of embodiments 71 to 98, wherein the modified PMP has a size of less than about 200 nm.

[0533] Embodiment 101. The modified PMP formulation of embodiment 100, wherein the modified PMP has a size of about 100 to 160 nm.

[0534] Embodiment 102. The modified PMP formulation of any one of embodiments 71 to 101, further comprising a phosphate, citrate, sodium bicarbonate, HEPES, TAE or TRIS buffer at a pH of about 3.0 to about 8.5.

[0535] Embodiment 103. The modified PMP formulation of any one of embodiments 71 to 102, further comprising one or more cryoprotectants.

[0536] Embodiment 104. The modified PMP formulation of embodiment 103, wherein the one or more cryoprotectants are selected from the group consisting of sucrose, glycerol, mannitol, and combinations thereof.

[0537] Embodiment 105. The modified PMP formulation of any one of embodiments 71 to 104, wherein the modified PMP formulation is a lyophilized composition.

[0538] Embodiment 106. The modified PMP formulation of any one of embodiments 71 to 105, wherein the modified PMP formulation is stable at room temperature and / or at 4°C.

[0539] Embodiment 107. A pharmaceutical composition comprising the modified PMP formulation according to any one of embodiments 71 to 106 and a pharmaceutically acceptable vehicle, carrier or excipient.

[0540] Embodiment 108. A method of producing a modified PMP formulation comprising an exogenous peptide, polypeptide or protein, the method comprising:

[0541] providing a solution comprising a modified PMP containing one or more PMPs, one or more sterols, and one or more polyethylene glycol (PEG)-lipid conjugates;

[0542] providing a solution comprising the exogenous peptide, polypeptide or protein; and

[0543] The modified PMP is loaded with the exogenous peptide, polypeptide or protein, wherein the loading causes the exogenous peptide, polypeptide or protein to be encapsulated by the modified PMP.

[0544] Embodiment 109. A method for delivering a peptide, polypeptide or protein to a mammalian cell or a mammal, the method comprising:

[0545] The modified PMP formulation according to any one of Embodiments 71-104 is contacted with the mammalian cell or administered to the mammal under conditions sufficient to allow uptake of the modified PMP formulation by the mammalian cell or the mammal.

[0546] Embodiment 110. The method of embodiment 109, wherein the method is used to deliver a peptide, polypeptide or protein to a mammalian cell, and the cell is a cell of a subject.

[0547] Embodiment 111. The method of Embodiment 109, wherein the exogenous peptide, polypeptide, or protein is released from the modified PMP formulation in a mammalian cell contacted with the modified PMP formulation.

[0548] Embodiment 112. The method of embodiment 111, wherein the exogenous peptide, polypeptide or protein is active in the cytoplasm or nucleus of the mammalian cell.

[0549] Embodiment 113. The method of any one of Embodiments 108 to 112, wherein said mammal is a human.

[0550] Embodiment 114. The method of any one of Embodiments 108 to 112, wherein the uptake of the exogenous peptide, polypeptide or protein encapsulated by the modified PMP formulation is increased by the mammalian cell or the mammal relative to the uptake of the exogenous peptide, polypeptide or protein not encapsulated by the modified PMP formulation.

[0551] Embodiment 115. The method of embodiment 109, wherein the method is for delivering a peptide, polypeptide or protein to a mammal, and the administration is via oral, intranasal or intrarectal route.

[0552] Embodiment 116. A method for treating or preventing a disease or condition for which a therapeutic agent is indicated in a subject, the method comprising administering to the subject in need thereof an effective amount of a modified PMP formulation according to any one of embodiments 71 to 106, wherein the therapeutic agent is an exogenous peptide, polypeptide or protein encapsulated by the modified PMP in the modified PMP formulation.

[0553] Embodiment 117. The method of Embodiment 116, wherein said administering is via oral, intranasal or intrarectal route.

[0554] Embodiment 118. The method of Embodiment 114, wherein the disease is diabetes and the exogenous peptide, polypeptide or protein is insulin.

[0555] Examples

[0556] The following are examples of various methods of the present invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0557] Example 1: Preparation of PMP and composite lipid particles

[0558] General procedure for preparing PMPs

[0559] Preparation of PMP and formulation of PMP can be achieved using methods similar to those disclosed in International Patent Application Publication No. WO 2023 / 069498, which is incorporated herein by reference in its entirety.

[0560] General procedure for preparing lipid extract fractions of CLP

[0561] The general preparation of complex lipid particles (CLP) begins with the isolation of lipid extracts from natural plant sources. Briefly, the method for extracting plant lipids in CLP is as follows:

[0562] 1) Collecting plant matter from natural plant sources (eg, juice, pulp, or blended parts of the plant).

[0563] 2) Filter and concentrate the plant matter.

[0564] 3) The plant concentrate was diafiltered with citrate-sodium chloride buffer to produce a diafiltered intermediate (DFI), the starting material for extraction.

[0565] 4) Extract lipids by adding extraction solvents such as dichloromethane (DCM) and methanol (MeOH). Alternative extraction solvents in this method can include chloroform:methanol and ethyl acetate:ethanol.

[0566] 5) Water was added to induce phase separation.

[0567] 6) Collect the resulting organic phase and dry it using a rotary evaporator (rotovap).

[0568] 7) The dried lipids are resuspended in an aqueous solution (eg, 90% DCM and 10% MeOH solution) and transferred to a vial for drying (eg, by Genevac or similar drying equipment).

[0569] 8) The obtained lipid extract was dried and stored at -20°C.

[0570] Exemplary modified extraction methods for preparing CLP

[0571] The above-mentioned modification of extracting lipid from natural source is described in detail below.Use two kinds of alternative extraction methods for preparing composite lipid particles: 1) ethanol extraction method, or 2) modified Matyash method using methanol.In this example, for ethanol extraction, 3:2 ethyl acetate: ethanol solvent is produced.25mL PBS is added to 2.5g powdered natural source, then vortexed for one minute with 1500RPM.41.5mL ethanol solvent is added to the aqueous sample of natural source.This is vortexed for one minute with 1500RPM on vertical oscillator.Add another 50mL ethyl acetate, and solution is vortexed for one minute with 1500RPM.Add 50mL liquid chromatography-mass spectrometry grade water, and solution is vortexed for another minute with 1500RPM, then centrifuged for 5 minutes at 4 ℃, 1500xg.Top organic layer is transferred in new flask, then evaporated using rotary evaporator, wherein water bath is set to 40 ℃, ensures to remove any residual solvent. The remaining extracted lipids were weighed, sparged with N2, and then capped and stored at -20°C.

[0572] In alternative examples, for modified methanol extraction, aqueous samples are prepared as described above. By 93.75mL of 1:2MeOH: methyl tert-butyl ether (MTBE) solution is added to the sample, with 1500RPM vortex stirring for one minute, then ultrasonic treatment 5 minutes at 100% power and 37Hz. Further add 31.25mL MTBE, and the solution is vortexed for another minute at 1500RPM, then centrifuged for 5 minutes at 1500x g. The top organic layer is transferred in a flask, and 60mL ethyl acetate is added. The new solution is vortexed for one minute at 1500RPM, vibrated for 30 seconds, then centrifuged for 5 minutes at 4 ℃, 1500x g. The top organic layer is transferred again. Then, as described for ethanol extraction, the solvent is evaporated using a rotary evaporator, weighed and stored.

[0573] The dried extracts were reconstituted in absolute ethanol to a given concentration of 10 mg / mL, vortexed at 2000 RPM for 30 seconds and sonicated at 80 Hz for 30 seconds. Once the lipids were reconstituted, the samples were filtered into pre-weighed vials and then dried using GeneVac and then stored at -20°C under nitrogen.

[0574] In addition to the lipid extraction methods described above using dichloromethane:methanol, chloroform:methanol, methanol:MTBE, and ethyl acetate:ethanol, alternative organic solvents that may be used include, but are not limited to, dimethylformamide:methanol, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, or dimethyl sulfoxide:methanol.

[0575] Example 2: Formulation and characterization of polypeptide-loaded complex lipid formulations

[0576] In this example, methods were developed to generate and load stable complex lipid particles containing complex lipids derived from various plant sources as well as peptides, polypeptides, and small proteins.

[0577] In summary, the method involves: 1) mixing the natural lipids obtained as described in Example 1 with components that facilitate increased stability (e.g., sterols such as cholesterol and polyethylene glycol (PEG) molecules) to maintain the particle size and amount of encapsulated cargo over time; 2) forming particles containing the above components (with or without lyophilization); 3) directly or passively loading the particles with the peptide cargo; and optionally 4) purifying the resulting peptide-loaded particles for further application / processing.

[0578] The various steps of processing, deployment, characterization and testing are set forth herein, and then described in detail below. The step for programming the particle may include deployment by microfluidics (e.g., using NanoAssemblr) or film, as described in each section below. In addition, the step may include direct loading or passive loading of goods. Purification steps and post-processing steps may include options such as dialysis, filtration, centrifugation, lyophilization and / or encapsulation. The testing of formulations may include measurements of stability and performance in vitro and in vivo (e.g., mice, etc.). At any stage in these given stages (e.g., programming, post-processing, testing), the characterization of a given formulation (e.g., size, API loading, lipidomics, etc.) may affect other formulations and the optional steps of producing exemplary complex lipid formulations.

[0579] General procedure for preparation and loading of empty composite lipid particles

[0580] In general, formulation of complex lipid particles involves the generation of empty particles followed by loading of the empty particles or loading of the particles by microfluidic mixing to directly generate loaded particles.

[0581] The generation of empty particles is performed by thin film or microfluidic mixing. Briefly, the generation of empty particles by thin film hydration involves the following steps: 1) lipid mixing, 2) thin film formation, 3) hydration, 4) homogenization, and 5) empty particle formation. Briefly, the generation of empty particles by microfluidic mixing involves the following steps: 1) lipid mixing, 2) microfluidic homogenization, and 3) empty particle formation.

[0582] After the empty particles are formulated, the particles can be optionally lyophilized by thin film or microfluidic mixing. If lyophilization is not performed, the empty particles are passively loaded by the following steps: 1) the empty particles are mixed with the active pharmaceutical ingredient (API) solution, 2) the subsequent formulation is homogenized, and optionally 3) the formulation is purified. If lyophilization is performed, the empty particles are passively loaded by the following steps: 1) the lyophilized particles are rehydrated in the API solution, then 2) homogenized, and then optionally 3) the formulation is purified. Alternatively, the generation of loaded particles can be performed directly by microfluidic mixing. In short, direct loading of particles by microfluidic mixing involves the following steps: 1) lipids are mixed with API, 2) microfluidic homogenization, 3) loading particle formation, and optionally 4) purification of the resulting formulation. After the particles produced are loaded by any of the given means, the particles can be tested. Testing can include in vitro characterization, in vivo functional testing, lyophilization and encapsulation, or lyophilization and rehydration.

[0583] Exemplary CLPs prepared by adding sterols and PEG to lipids extracted from plant sources

[0584] This example describes the use of lipids derived from plant sources to prepare stable particles (such as PMP or CLP). In this example, sterols (such as cholesterol) and PEGylated lipids are added to the lipid extracted from plant sources. Cholesterol serves as a molecule to promote the filling of residual lipids and to stabilize the formulation, but the cholesterol can increase the rigidity of lipid particles, which may be undesirable, particularly when lipid particles are designed to cross cell monolayers such as gastrointestinal epithelial layers. PEGylated lipids serve as molecules to prevent the fusion of the lipid particles prepared. In this example, the amount of the cholesterol and PEGylated lipids added is minimized for the following reasons. The sterols of natural plant sources are present in many natural lipid compositions. Therefore, adding a large amount of exogenous cholesterol can produce rigid lipid particles. In addition, adding a large amount of PEGylated lipids can increase the tendency of formulations to be recognized by immune cells, particularly in the case of repeated administration, so that the resulting lipid particles are not too effective.

[0585] In this example, the following assumptions were made to calculate the amount of cholesterol and PEG: i) the average molecular weight of the source lipid (natural lipid extract) was 720 Da, ii) 50% of the total lipid extract was structural lipid. Thus, for example, for 1 mg of lipid extract, there were 0.5 mg or 695 nanomoles (nmol) of structural lipid. Added cholesterol and PEGylation were calculated relative to the amount of structural lipid per total mass of lipid extract.

[0586] DSPE-PEG2000 (18:0PEG2000 PE, CAS#474922-77-5) with a molecular weight of 2800Da was used as PEGylated lipids. Four different concentrations were added to the lipid mixture; 0% w / w, 0.5% w / w (5 μg / mg total lipid extract), 1.25% w / w (12.5 μg / mg total lipid extract) and 2.5% w / w (25 μg / mg total lipid extract). For 1 mg total lipid extract or 695 nmol structural lipids, this translates to 0% mol, 0.26% mol, 0.64% mol and 1.28% mol PEGylated lipids.

[0587] Cholesterol (CAS#57-88-5) derived from plants with a molecular weight of 386.5Da was used as cholesterol. Three different concentrations were added to the lipid mixture; 0% w / w, 3.4% w / w (34 μg / mg total lipid extract) and 6.8% w / w (68 μg / mg total lipid extract). For 1 mg total lipid extract or 695 nmol structural lipids, this translates to 0% mol, 12.5% ​​mol and 25% mol cholesterol.

[0588] Exemplary generation of empty composite lipid particles

[0589] As briefly described above, two main formulation approaches for preparing complex lipid particles have been used: 1) thin film rehydration and 2) microfluidic enabling.

[0590] In this example, the formulation method for film rehydration involves the following steps, as shown in Figure 1A middle.

[0591] 1) Dissolve natural lipids (natural lipids extracted from plants such as lemon) and exogenous lipids (i.e., cholesterol and PEGylated lipids, as discussed above at various concentrations) in an organic solvent (such as chloroform or ethanol). Dissolve natural lipids at a given concentration (e.g., 5 mg / ml concentration).

[0592] 2) A thin film of mixed lipids was formed in the flask by evaporating the organic solvent at 42°C in a rotary evaporator and further dried using a stream of nitrogen gas.

[0593] 3) The film is hydrated in an aqueous buffer containing a cryoprotectant at 40°C for 30 minutes and then vortexed, with the lipid in the buffer being 5 mg / ml. This buffer is selected to promote downstream dissolution of the protein cargo (e.g., based on the isoelectric point of the protein cargo). In the case of insulin, the buffer is citrate at a pH of about 3, or sodium bicarbonate at a pH of about 8.2. In the case of GLP1 receptor agonists (e.g., semaglutide and exenatide), the buffer is 0.1 M sodium bicarbonate at a pH of about 8.2. The cryoprotectant is 2-5% sucrose or 2-5% mannitol. Alternatively, the cryoprotectant can range from 0% to 5%. The cryoprotectant can be about 0.5-2%.

[0594] 4) The lipid film was homogenized in an aqueous buffer in a sonicator to form small unilamellar vesicles (SUVs) containing native lipids and exogenous lipids. The sonication process involved two steps: a 20-minute sonication step at 42°C, brief mixing and vortexing, and a second 20-minute sonication step at about 42°C. If aggregates were observed, the particle solution was filtered through a cotton filter. The particle morphology properties were analyzed using a dynamic light scattering method.

[0595] 5) The lipid solution is frozen in liquid nitrogen for 10 minutes. Depending on the volume of the particle solution, the aqueous buffer containing the lipid solution and the cryoprotectant are lyophilized (freeze dried) at room temperature overnight or for 12-48 hours. The resulting complex lipid formulation containing natural source lipids and exogenous lipids is produced in the form of dry powder and stored at -20°C until use. The particles can be lyophilized in an alternative method called shelf lyophilization, in which the particles are controlled by the freezing process using a vacuum rate. Alternatively, the particles are not lyophilized.

[0596] The formulation method for microfluidics-enabled processing involves the following steps, as shown in Figure 1B middle.

[0597] 1) Natural lipids and exogenous lipids (ie, cholesterol and PEGylated lipids, as discussed above at various concentrations) are dissolved in an organic solvent at a given concentration (eg, 5 mg / ml concentration).

[0598] 2) The lipid mixture in organic solvent is co-injected with an aqueous buffer (eg, 0.1 M sodium bicarbonate, pH 8.2, supplemented with 5% mannitol as a cryoprotectant) in a microfluidic device (NanoAssemblr Ignite).

[0599] 3) The organic solvent is dialyzed overnight against an aqueous buffer (eg, 0.1 M sodium bicarbonate, pH 8.2, 5% mannitol as a cryoprotectant).

[0600] 4) Dynamic light scattering method was used to analyze the particle morphology characteristics.

[0601] 5) The lipid solution is frozen in liquid nitrogen for 10 minutes. The lipid solution is lyophilized (freeze dried) at room temperature overnight. The resulting composite lipid particles containing natural source lipids and exogenous lipids are produced in the form of dry powders and stored at -20°C until use. The particles can be lyophilized in an alternative method called shelf lyophilization, in which the particles are controlled by the freezing process using a vacuum rate. Alternatively, the particles are not lyophilized.

[0602] Other known methods, such as microfluidic T-junction generation, may be used instead.

[0603] Loading peptides into empty composite lipid particles

[0604] Composite lipid particles containing naturally derived lipids and exogenous lipids were taken out for formulation with exemplary active biomolecules (e.g., polypeptides such as insulin), which were produced in dry powder form and stored at -20°C and prepared according to the above formulation method.

[0605] In this example, the lyophilized particles were rehydrated with an aqueous buffer containing an active biomolecule (e.g., a polypeptide such as insulin) at a concentration of 5 mg / ml at a 1:1 w / w lipid-active biomolecule ratio for peptides less than or equal to 5 kD, or with 1 mg / mL at a 1:5 w / w lipid:peptide mass ratio for proteins greater than 5 kD. After rehydration, a brief homogenization was performed by sonication at 40°C for 20 minutes. This rehydration buffer is similar to the buffer used to hydrate the film (step 3 above) and is selected based on its properties for solubilizing active biomolecules. Again, in the case of insulin, the buffer is citrate at a pH of about 3, or sodium bicarbonate at a pH of about 8.2; in the case of GLP1 receptor agonists (e.g., semaglutide or exenatide), the buffer is 0.1 M sodium bicarbonate at a pH of about 8.2. The particle morphological properties were analyzed using a dynamic light scattering method. Alternatively, if the particles are not lyophilized, the empty particles are mixed with an aqueous buffer containing an active biomolecule (eg, a polypeptide such as insulin) in a manner similar to the methods described above.

[0606] Next, the mixture of rehydrated particles containing active biomolecules is purified and separated from free active biomolecules not loaded in the particles by a dialysis step overnight. As discussed above, the 100 kD dialysis membrane in the buffer used for the formulation method is dialyzed with a buffer volume of at least 2000x the volume of the solution to be purified (e.g., 0.1 M sodium bicarbonate, pH 8.2, buffer). The dialysate is concentrated to a preferred concentration in a centrifugal unit. Alternatively, the purification and separation steps can be performed with tangential flow filtration (TFF). The purification steps described are optional and are not performed for all formulations. The particle morphology characteristics are analyzed using a dynamic light scattering method. The particle loading efficiency is quantified using a bicinchoninic acid assay (BCA).

[0607] The above method for loading bioactive molecules into empty composite lipid particles containing lipids of natural origin and exogenous lipids is also described in Figure 2 Shown in.

[0608] An alternative method for loading bioactive molecules into composite lipid particles containing lipids of natural origin and exogenous lipids involves dissolving the lipid mixture in an organic solvent together with an aqueous buffer containing the active biomolecules before homogenizing the mixed solution by microfluidics to directly produce the loaded particle formulation. In another approach, the bioactive molecules can also be dissolved in the organic phase.

[0609] Characterization of peptide-loaded complex lipid formulations

[0610] The efficacy of the composite lipid formulations prepared according to the above-mentioned formulation and loading method is evaluated by a quantitative method for characterizing the size of the particles and the distribution of the size in the particle population. Particle size is measured by dynamic light scattering (DLS). The results are shown in Table 5. Table 5 lists the particle size (nm) of the composite lipid formulations measured by DLS by changing the amount of cholesterol (left column) and PEGylated lipid (top row) added in the composite lipid formulations as described above.

[0611] Table 5. Particle size of insulin loaded complex lipid formulations.

[0612]

[0613] As shown in Table 5, the addition of cholesterol alone resulted in a larger particle size, and when the amount of PEGylated lipid was 0.5% w / w, the addition of PEGylated lipid alone also slightly increased the particle size. When the complex lipid formulation contained 2.5% w / w of PEGylated lipid and 6.8% w / w of cholesterol based on the amount of total lemon lipid extract, the insulin-loaded complex lipid formulation had a smaller particle size.

[0614] When the above formulation and loading process is repeated multiple times (eg, hundreds of times), a complex lipid formulation containing 2.5% w / w PEGylated lipid and 6.8% w / w cholesterol can provide particle sizes ranging from 100-160 nm.

[0615] The insulin encapsulation efficiency (amount of insulin entrapped into the vesicles) of the complex lipid formulations was also characterized and the results were consistently in the range of 25-35%. Similar characterizations were measured for other formulations of complex lipid particles.

[0616] Stability of peptide-loaded complex lipid formulations

[0617] As shown in Table 5, as a control, the insulin-loaded complex lipid formulation (i.e., 0% w / w of PEGylated lipid and 0% w / w of cholesterol) that does not include exogenous lipids initially has a small particle size of about 120 nm. However, this formulation is unstable over time, and the particle size increases to 400-500 nm after one day of storage at 4°C. In addition, lyophilization is generally used for longer storage of formulations (e.g., more than two weeks), but after the second lyophilization, the protein-loaded complex lipid formulation that does not include exogenous lipids (i.e., 0% w / w of PEGylated lipid and 0% w / w of cholesterol) is unstable, and after the second lyophilization, the particle size increases to 400-500 nm. In addition, when the powder is rehydrated with a buffer solution, the protein-loaded complex lipid formulation that does not include exogenous lipids (i.e., 0% w / w of PEGylated lipid and 0% w / w of cholesterol) cannot be re-formulated.

[0618] On the other hand, the composite lipid formulation containing PEGylated lipid and cholesterol is stable at least two weeks at 4 ℃. For longer storage time, freeze drying is used. The composite lipid formulation containing PEGylated lipid and cholesterol is stable after freeze drying. The composite lipid formulation containing PEGylated lipid and cholesterol can also be redeployed when the powder is rehydrated with buffer solution. After redeployment, the particle size of the composite lipid formulation can reach about 90% of the original particle size before freeze drying, and the insulin encapsulation efficiency of the composite lipid formulation can reach about 30% of the initial encapsulation efficiency before freeze drying. For other formulations of composite lipid particles, similar stability measurements are used.

[0619] Lipidomics of plant lipid extracts and empty complex lipid particles

[0620] In this example, lipids were extracted from five plant sources (e.g., lemon, pitaya, spinach, kale, strawberry) as described in Example 1. These extracts were formulated into empty composite lipid particles or maintained as lipid extract compositions as described above in Example 2. Both the extracts and the empty particles were subjected to lipidomic analysis.

[0621] Briefly, the extracts or pellets were dissolved in a compatible solvent and analyzed by MS / MS. The prevalence of the determined lipid classes and subclasses identified in the extracts and pellets was analyzed. The 46 different lipid classes / subclasses annotated by lipidomics are as follows: acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycosides, bile acids, acylcarnitines, cholesterol esters, ceramides, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acids, fatty acid esters of hydroxy fatty acids, hemi-dimonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl- Lysophosphatidylethanolamine, lysophosphatidyl, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, sterol esters, stigmasterol, sulfatides, sulfonic acid lipids, sphingomyelin, sulforhamnosyldiacylglycerol, sterols and triacylglycerols.

[0622] The number of lipids annotated in lemon extract alone exceeded 800, and these lipids were classified into at least 20 of the above listed classes or subclasses. This highlights the complexity of the composition of lipid extracts from natural sources, which is evident in all five tested plant sources. The prevalence of certain lipid classes or subclasses was altered by the formulation process, as is evident when comparing the analyzed lipids present in the extract or in the empty composite lipid particles. Even if the prevalence of lipid classes or subclasses changed during the formulation process, the overall diversity of lipid classes / subclasses was not lost.

[0623] Among the five empty particle formulations tested, an average of 24 listed lipid classes were present in the positively charged particle analysis and an average of 31 annotated lipid classes were present in the negatively charged particle analysis. A minimum of 5 annotated lipid classes / subclasses overlapped within the particle analysis between the different sources, with some sources having a minimum of 20 overlapping lipid classes / subclasses within the particles.

[0624] Of note, non-lipid components found in the lipid extracts were not examined in the lipidomic analysis. That is, characterization of the lemon lipid extracts found that in multiple extractions, the total protein content was less than 10% w / w as measured by BCA. In some cases, the total protein content of the lipid extracts was less than 5% w / w of the extract composition. Additional characterization of the lemon lipid extracts found that the total dsDNA was less than 1% w / w of the extract composition as measured by the Picogreen assay. In some cases, the total dsDNA was less than 0.05% w / w of the extract composition.

[0625] Example 3: Production of oral dosage forms of complex lipid formulations

[0626] The following examples describe how capsule or tablet dosage forms can be produced from complex lipid formulations.

[0627] First, for capsule dosage forms, liquid complex lipid formulations are obtained according to the procedures described in Example 2. Additional excipients may be added, including but not limited to cryoprotectants, lyoprotectants, stabilizers, bulking agents, flow agents and / or anti-caking agents. The liquid formulation is then dried to a solid formulation using established pharmaceutical manufacturing procedures, such as freeze drying or spray drying. If the dried material is a free-flowing powder, it is filled in capsules using standard manufacturing procedures. If the dried material is not free-flowing, as in the case of a lyophilized cake, a powder is produced from the cake using procedures such as grinding. The powder is then filled in capsules using standard manufacturing procedures. The capsule shell may be uncoated or coated. The capsules may be packaged in bulk or individually.

[0628] First, for tablet dosage forms, a liquid composite lipid formulation is obtained according to the procedure described in Example 2. Additional excipients may be added, including but not limited to cryoprotectants, lyoprotectants, stabilizers, bulking agents, flow agents, binders, compression agents and / or anti-caking agents. The liquid formulation is then dried into a solid formulation using established pharmaceutical manufacturing procedures, such as freeze drying or spray drying. If the dried material is a free-flowing powder, it is compressed into tablets using standard manufacturing procedures. If the dried material is not free-flowing, as in the case of a lyophilized cake, a powder is produced from the cake using procedures such as grinding. The powder is then compressed into tablets using standard manufacturing procedures. The tablets may be uncoated or coated. The tablets may be packaged in bulk or individually.

[0629] Example 4: In vivo nasal and enteral delivery of protein-loaded complex lipid formulations

[0630] An insulin-loaded complex lipid formulation was obtained according to Example 2, wherein the complex lipid formulation contained 2.5% w / w of PEGylated lipid and 6.8% w / w of cholesterol based on the amount of total lemon lipid extract.

[0631] The animals used were C57 female 8-week-old mice (Jax#0664).

[0632] Colonic experiment

[0633] The mice were administered the insulin-loaded complex lipid formulations by 50 μL (50 U / kg) intrarectal doses according to the treatment schedule in Table 6. At various time points, as indicated in Table 6, blood was collected and plasma samples were obtained, and insulin levels were quantified by ELISA assay.

[0634] Table 6. Schedule of intracolonic administration

[0635]

[0636] The results are shown in Figure 3 middle. Figure 3 Shown is the insulin concentration in mouse plasma 1 hour after intrarectal administration of the insulin-loaded complex lipid formulation to mice, demonstrating the systemic absorption of the insulin-loaded complex lipid formulation. Compared to the baseline (10 U / kg, 12 minutes after injection) of insulin-containing fusogenic liposomes administered directly to the colon, the intrarectal administration of the insulin-loaded complex lipid formulation caused significantly higher plasma insulin levels than the baseline.

[0637] Intranasal experiment

[0638] The mice were administered the insulin-loaded complex lipid formulations by intranasal doses of 40 μL (20 μL per nostril, 50 U / kg) according to the treatment schedule in Table 7. The brains were weighed and proteins were extracted using acidified ethanol at various time points as shown in Table 7. Insulin levels were quantified by ELISA assay.

[0639] Table 7. Treatment schedule for intranasal administration

[0640]

[0641] The results are shown in Figure 4 middle. Figure 4 Shown is the insulin concentration in the mouse brain 2 hours after intranasal administration of the composite lipid particles loaded with insulin to the mice, indicating that the composite lipid particles loaded with insulin can effectively deliver insulin to the brain by intranasal administration. Compared with the benchmarks of intranasal administration of free insulin and intranasal administration of insulin with cell penetrating peptides, intranasal administration of composite lipid particles loaded with insulin causes significantly higher brain insulin levels compared with the two benchmarks.

[0642] Example 5: In vivo enteral delivery of protein-loaded complex lipid formulations

[0643] The insulin-loaded complex lipid formulation was obtained according to Example 2, wherein the complex lipid particles were formulated as described in Table 8.

[0644] Table 8. Complex lipid formulations loaded with insulin.

[0645]

[0646]

[0647] Colonic experiment

[0648] The animals used were C57BL6 female 8-week-old mice (Jax#0664).

[0649] C57BL6 female 8-week-old mice (Jax#0664) (n=4-5 mice / formulation) were used. The mice were weighed and pre-bleeding samples were collected (via tail vein, retro-orbital or submandibular means). The mice were fasted overnight and then administered by intracolonic administration of 50uL of a given insulin-loaded complex lipid formulation (Table 8). Non-terminal bleeding was collected at 30 minutes and 60 minutes after administration, alternating collection routes. Terminal bleeding was also collected 120 minutes after administration by cardiac puncture. Whole blood samples from pre-bleeding, 30 minutes, 60 minutes and 120 minutes of bleeding were processed into plasma and then stored at -80°C. Insulin levels were quantified by MSD determination. Particle characterization and insulin measurement are reported in Table 9.

[0650] Table 9. Particle characterization and insulin levels of insulin-loaded complex lipid formulations.

[0651]

[0652]

[0653] The exposure of the active biomolecule (e.g., insulin) was measured as the area under the curve (AUC) at four measurement time points, and the dose was measured as BCA*dose volume (0.05 mL). Therefore, the data for each mouse in Table 9 are shown as whole body insulin levels (or exposure normalized to dose). As shown in Table 9, 22 / 22 different complex lipid formulations showed positive whole body insulin levels. Unexpectedly, 20 of the 22 different complex lipid formulations (>90%) outperformed free IC insulin.

[0654] Example 6: In vivo jejunal delivery of protein-loaded complex lipid formulations

[0655] The exenatide loaded complex lipid formulation was obtained according to Example 2, wherein the complex lipid particles were formulated as described in Table 10. The mice were treated according to Table 11.

[0656] Table 10. Complex lipid formulations loaded with Exenatide.

[0657]

[0658] Table 11. Treatment schedule for intrajejunal administration.

[0659]

[0660] Jejunal experiment

[0661] The animals used were C57BL6 female 8-week-old mice (Jax#0664).

[0662] C57BL6 female 8-week-old mice (Jax#0664) (n=5 mice / formulation) were used. The mice were weighed and pre-bleeding samples were collected (via tail vein, retro-orbital or submandibular means). The mice were fasted overnight and then administered by intrajejunal administration of 50uL (50ug / mouse) of a given complex lipid formulation loaded with exenatide (Table 10). Non-terminal bleeding was collected at 30 minutes and 60 minutes after administration, alternating collection routes. Terminal bleeding was also collected 120 minutes after administration by cardiac puncture. Whole blood samples from pre-bleeding, 30 minutes, 60 minutes and 120 minutes of bleeding were processed into plasma and then stored at -80°C. Exenatide levels were quantified by ELISA assay. The formulations are reported in Table 10, and the treatment schedule is shown in Table 11. The results of particle characterization and exenatide measurement are shown in Table 12.

[0663] Table 12. Particle characterization and insulin levels of insulin-loaded complex lipid formulations.

[0664]

[0665] The exposure of the active biomolecule (e.g., exenatide) was measured as the area under the curve (AUC) at four measurement time points, and the dose was measured as BCA*dose volume (0.05 mL). Therefore, the data for each mouse in Table 12 is shown as whole body insulin levels (or exposure normalized to dose). As shown in Table 12, the results indicate that the complex lipid formulations incorporating plant lipid extracts from broccoli and strawberry perform well in delivering exenatide.

[0666] Other embodiments

[0667] Although for the purpose of clear understanding, the foregoing invention has been described in detail by way of illustration and example, these descriptions and examples should not be construed as limiting the scope of the present invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety. Other embodiments are within the claims.

Claims

1. A method for delivering a therapeutic peptide or protein to a human subject in need thereof, the method comprising orally or enterally administering to the human subject a pharmaceutical formulation comprising: (a) a plurality of complex lipid particles characterized by: (i) comprising at least 10 plant lipids extracted from one or more plant sources; (ii) comprising sterols that are exogenous to the one or more plant sources; (iii) comprising polyethylene glycol (PEG) conjugated lipids; (iii) containing less than 10% w / w proteinaceous material that is endogenous to the one or more plant sources; and (iv) containing less than 10 mol% of exogenous ionizable lipids, and (b) the therapeutic peptide or protein encapsulated in the composite lipid particle.

2. The method of claim 1, wherein the therapeutic peptide or protein is a hormone or a glucagon-like peptide 1 (GLP-1) agonist.

3. The method of claim 2, wherein the therapeutic peptide or protein is insulin, exenatide, semaglutide or tirzepatide.

4. The method of claim 1, wherein the therapeutic peptide or protein is delivered to brain tissue of the human subject.

5. The method of claim 1, wherein the complex lipid particle contains ten or more lipids belonging to one or more subclasses selected from the group consisting of acyl steryl glycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerols, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyldiacylglycerols, and sterols.

6. The method of claim 5, wherein the composite lipid particle contains lipids from at least five, at least six, at least seven, at least eight, at least nine or at least ten different subclasses.

7. The method of claim 1, wherein the complex lipid particles contain less than 5% w / w proteinaceous material endogenous to the one or more plant sources.

8. The method of claim 1, wherein the composite lipid particle contains less than 5 mol% of exogenous ionizable lipids.

9. The method of claim 1, wherein at least one of the plant sources is grapefruit, lemon, pitaya, spinach, kale, strawberry, broccoli, or soybean.

10. The method of claim 1, wherein the composite lipid particle comprises: about 85-95% w / w of said vegetable lipid, about 5 to 8% w / w of said sterol, and About 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipid in the complex lipid formulation.

11. A complex lipid formulation comprising: a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties: i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and ii) contains less than 50 mol % ionizable lipids.

12. The complex lipid formulation of claim 11, wherein the exogenous peptide, polypeptide or protein is a therapeutic agent.

13. The complex lipid formulation of claim 11, wherein the exogenous peptide, polypeptide or protein is an antibody or an antibody fragment.

14. The complex lipid formulation of claim 11, wherein the exogenous peptide, polypeptide or protein is a hormone.

15. The complex lipid formulation of claim 14, wherein the exogenous peptide, polypeptide or protein is insulin.

16. The complex lipid formulation of claim 11, wherein the exogenous peptide, polypeptide or protein is a receptor agonist or a receptor antagonist.

17. The complex lipid formulation of claim 16, wherein the exogenous peptide, polypeptide or protein is a glucagon-like peptide 1 (GLP-1) agonist.

18. The complex lipid formulation of claim 17, wherein the exogenous peptide, polypeptide or protein is exenatide, semaglutide or tesiparatide.

19. The complex lipid formulation of claim 11, wherein the exogenous peptide, polypeptide or protein is less than 100 kD in size.

20. The complex lipid formulation of claim 19, wherein the exogenous peptide, polypeptide or protein is less than 50 kD in size.

21. The complex lipid formulation of claim 19, wherein the exogenous peptide, polypeptide or protein has a size of at least 3 kD.

22. The complex lipid formulation of claim 19, wherein the exogenous peptide, polypeptide or protein comprises at least 30 amino acid residues.

23. The complex lipid formulation of claim 11, wherein the complex lipid particles contain 5-1000 lipids extracted from one or more plant sources.

24. The complex lipid formulation of claim 11, wherein the complex lipid particles contain at least 10 plant lipids belonging to one or more classes selected from the group consisting of glycerolipids, sphingolipids, and sterols.

25. The complex lipid formulation of claim 24, wherein the complex lipid particles contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG) and sulfatides (SL).

26. The complex lipid formulation of claim 24, wherein the complex lipid particles contain one or more sphingolipids selected from the group consisting of glycosylinositolphosphoceramide (GIPC), glucosylceramide (GCer), ceramide (Cer) and free long chain base (LCB).

27. The complex lipid formulation of claim 24, wherein the complex lipid particles contain one or more A phytosterol selected from the group consisting of campesterol, stigmasterol and sitosterol.

28. The complex lipid formulation of claim 24, wherein the complex lipid particle contains one or more lipids belonging to one or more subclasses selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesterol ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acids, fatty acid esters of hydroxy fatty acids, hemi-monoacylglycerophosphate, hexosylceramide, lysophospholipids, Lipoic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidyl, lysophosphatidylinositol, lysophosphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, sterol esters, stigmasterol, sulfatides, sulfonic acid lipids, sphingomyelin, sulfoisorhamnosyldiacylglycerol, sterols and triacylglycerols.

29. The complex lipid formulation of claim 28, wherein the complex lipid particle contains ten or more lipids belonging to one or more subclasses selected from the group consisting of acyl steryl glycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemi-dimonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerols, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoisorhamnosyldiacylglycerols, and sterols.

30. The complex lipid formulation of claim 28 or 29, wherein the complex lipid particle contains lipids from at least five, at least six, at least seven, at least eight, at least nine, or at least ten different subclasses.

31. The complex lipid formulation of claim 11, wherein the complex lipid particles contain less than 30% w / w proteinaceous material endogenous to the one or more plant sources.

32. The complex lipid formulation of claim 31, wherein the complex lipid particles contain less than 5% w / w proteinaceous material endogenous to the one or more plant sources.

33. The complex lipid formulation of claim 11, wherein the complex lipid particles contain less than 20 mol% of exogenous ionizable lipids.

34. The complex lipid formulation of claim 33, wherein the complex lipid particles contain less than 5 mol% of exogenous ionizable lipids.

35. The complex lipid formulation of claim 11, wherein at least one of the plant sources is a citrus fruit.

36. The complex lipid formulation of claim 35, wherein the citrus fruit is grapefruit or lemon.

37. The complex lipid formulation of claim 11, wherein at least one of the plant sources is a non-citrus plant.

38. The complex lipid formulation of claim 37, wherein the non-citrus plant is pitaya, spinach, kale, strawberry, broccoli, or soybean.

39. The complex lipid formulation of claim 11, wherein the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

40. The complex lipid formulation of claim 39, wherein the sterol is cholesterol or sitosterol.

41. The complex lipid formulation of claim 39, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

42. The complex lipid formulation of claim 39, wherein the PEG-lipid conjugate is PEG2000-PE, PEG2000-DMG, PEG2000-DSPE or a derivative thereof.

43. The complex lipid formulation of claim 39, wherein the exogenous lipids further comprise lipids selected from the group consisting of: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, a second sterol, and an additive synthetic lipid.

44. The complex lipid formulation of claim 39, wherein the complex lipid particle comprises: about 10-95% w / w of said vegetable lipid, about 5-60% w / w of said sterol, and About 0.5-15% w / w of the polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipids in the complex lipid formulation.

45. The complex lipid formulation of claim 44, wherein the complex lipid particle comprises: about 85-95% w / w of said vegetable lipid, about 5-8% w / w of said sterol, and About 1-3.5% w / w of the polyethylene glycol (PEG)-lipid conjugate based on the amount of total lipids in the complex lipid formulation.

46. ​​The complex lipid formulation of claim 21, wherein the average size of the complex lipid particles is less than about 250 nm.

47. The complex lipid formulation of claim 46, wherein the average size of the complex lipid particles is about 100 to 180 nm.

48. The complex lipid formulation of claim 11, wherein the PDI of the complex lipid particles is from about 0.1 to about 0.

5.

49. The complex lipid formulation of claim 48, wherein the PDI of the complex lipid particles is from about 0.2 to about 0.

4.

50. The complex lipid formulation of claim 11, wherein the complex lipid particle further comprises one or more cryoprotectants or lyoprotectants.

51. The complex lipid formulation of claim 11, wherein the complex lipid formulation is a lyophilized composition.

52. The complex lipid formulation of claim 11, wherein the complex lipid formulation is a liquid composition.

53. The complex lipid formulation of claim 11, wherein the complex lipid formulation is stable at room temperature and / or at 4°C for at least two weeks without lyophilization.

54. A pharmaceutical composition comprising the complex lipid formulation according to any one of claims 1 to 53 and a pharmaceutically acceptable vehicle, carrier or excipient.

55. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition is in capsule or tablet form.

56. A method for delivering a peptide, polypeptide or protein to a mammalian cell or a mammal, the method comprising: contacting the mammalian cell with the complex lipid formulation or administering the complex lipid formulation to the mammal under conditions sufficient to allow the mammalian cell or the mammal to take up the complex lipid formulation, Wherein the complex lipid formulation comprises: a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties: i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and ii) contains less than 50 mol % ionizable lipids.

57. The method of claim 56, wherein the mammalian cell is a human cell, or the mammal is a human.

58. The method of claim 56, wherein the uptake of the exogenous peptide, polypeptide or protein encapsulated by the composite lipid particle by the mammalian cell or the mammal is increased relative to the uptake of the exogenous peptide, polypeptide or protein not encapsulated by the composite lipid particle.

59. The method of claim 56, wherein the method is for delivering a peptide, polypeptide or protein to a mammal and the administration is via an oral, enteral, intranasal, intracolonic, intrarectal or intrajejunal route.

60. The method of claim 56, wherein the mammalian cell is a brain cell.

61. A method for treating or preventing a disease or condition for which a therapeutic agent is indicated in a subject, the method comprising: administering to the subject in need thereof an effective amount of a complex lipid formulation comprising: a plurality of complex lipid particles, each of the plurality of complex lipid particles comprising at least five lipids extracted from one or more plant sources and at least two exogenous lipids; and One or more exogenous peptides, polypeptides or proteins encapsulated in the composite lipid particle, wherein the composite lipid particle is characterized by one or more of the following properties: i) contains less than 50% w / w proteinaceous material endogenous to said one or more plant sources; and ii) contains less than 50 mol % ionizable lipids.

62. The method of claim 61, wherein the administration is via an oral, enteral, intranasal, intracolonic, intrarectal or intrajejunal route.

63. The method of claim 61, wherein the disease is diabetes and the exogenous peptide, polypeptide or protein is insulin, exenatide, semaglutide or tesiparatide.

64. A method for producing a complex lipid formulation comprising a plurality of complex lipid particles encapsulating an exogenous peptide, polypeptide or protein, the method comprising: extracting at least five lipids from one or more plant sources; mixing at least two exogenous lipids with the extracted plant lipids to form composite lipid particles; and The complex lipid particles are loaded with the exogenous peptide, polypeptide or protein, wherein the loading causes the exogenous peptide, polypeptide or protein to be encapsulated by the complex lipid particles, thereby forming the complex lipid formulation.

65. The method of claim 64, wherein the lipids are extracted from the plant source by adding an extraction solvent to one or more plant sources, the extraction solvent comprising methanol, ethanol, propanol, 1-butanol, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, methyl tert-butyl ether, chloroform, ethyl acetate, or a mixture thereof.

66. The method of claim 65, wherein the extraction solvent is dichloromethane:methanol; chloroform:methanol; methanol:methyl tert-butyl ether (MTBE); dimethylformamide:methanol; acetonitrile:methanol; acetone:methanol; tetrahydrofuran:methanol; dimethyl sulfoxide:methanol; acetonitrile:ethanol; or ethyl acetate:ethanol.

67. The method of claim 64, wherein the extracting step further comprises reducing or eliminating proteinaceous material endogenous to the one or more plant sources to less than 50% w / w.

68. The method of claim 64, wherein the mixing step is performed by thin film mixing or microfluidic mixing.

69. The method of claim 64, wherein the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

70. The method of claim 64, wherein the exogenous lipids do not include ionizable lipids.

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