Composite lipid nanoparticles encapsulating polypeptides and uses thereof - Patents.com

Abstract

A method for delivering a therapeutic peptide or protein to a human subject in need thereof is provided. [Solution] Described herein are complex lipid particles that encapsulate one or more exogenous peptides, polypeptides, or proteins, as well as methods for producing complex lipid formulations that include exogenous peptides, polypeptides, or proteins. Also described herein are modified plant messenger pack (PMP) formulations that encapsulate one or more exogenous peptides, polypeptides, or proteins, as well as methods for producing modified PMP formulations that include exogenous peptides, polypeptides, or proteins.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 339,043, filed May 6, 2022, which is incorporated by reference in its entirety.

[0002] Polypeptides (e.g., proteins or peptides) are used in therapy (e.g., for the treatment of disease or condition), for diagnostic purposes, and as pathogen control agents. However, current methods for delivering polypeptides to cells can be limited by the delivery mechanism, for example, the efficiency of delivering polypeptides to cells. Thus, there is a need in the art for methods and compositions for delivering polypeptides to cells. Summary of the Invention

[0003] One aspect of the present invention relates to a method of delivering a therapeutic peptide or protein to a human subject in need thereof, the method comprising administering to the human subject: (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 exogenous to the one or more plant sources; (iii) comprising polyethylene glycol (PEG) conjugated lipids; (iii) containing less than 10% w / w protein material endogenous to the one or more plant sources; and (iv) containing less than 10 mole % exogenous ionized lipids; (b) Oral or enteral administration of a pharmaceutical preparation comprising a therapeutic peptide or protein encapsulated in a complex lipid particle.

[0004] In some embodiments, the therapeutic peptide or protein is a hormone or a glucagon-like peptide 1 (GLP-1) agonist. In one embodiment, the therapeutic peptide or protein is insulin, exenatide, semaglutide, or tirzepatide.

[0005] In some embodiments, the therapeutic peptide or protein is delivered to brain tissue in a human subject.

[0006] In some embodiments, the composite lipid particles contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerol, diacylglyceryl glucuronide, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and sterols.

[0007] In some embodiments, composite lipid particles contain lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different subclasses.

[0008] In some embodiments, the complex lipid particles contain less than 5% w / w protein material endogenous to one or more plant sources.

[0009] In some embodiments, the composite lipid particles contain less than 5 mole percent exogenous ionizable lipids.

[0010] In some embodiments, at least one of the plant sources is grapefruit, lemon, dragon fruit, spinach, kale, strawberry, broccoli, or soybean.

[0011] In some embodiments, the complex lipid particles comprise, based on the amount of total lipid in the complex lipid formulation, Approximately 85-95% w / w of vegetable lipids, Approximately 5-8% w / w sterols, Contains approximately 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate.

[0012] Another aspect of the invention relates to a complex lipid formulation comprising a plurality of complex lipid particles, each complex lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids, and one or more exogenous peptides, polypeptides, or proteins encapsulated in the complex lipid particle. The complex lipid particles have the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mole % ionized lipids.

[0013] In some embodiments, the composite lipid particles contain 5 to 1000 lipids extracted from one or more plant sources. In some embodiments, the composite lipid particles contain at least 10 plant lipids belonging to one or more of the classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. For example, the composite lipid particles contain 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 of the classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. In some embodiments, the composite lipid particles contain lipids from at least two or at least three of these different classes.

[0014] In some embodiments, the complex lipid particles contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG), and sulfolipids (SL). In some embodiments, the complex lipid particles contain one or more sphingolipids selected from the group consisting of glycosyl inositol phosphoceramides (GIPC), glucosylceramides (GCer), ceramides (Cer), and free long chain bases (LCB). In some embodiments, the complex lipid particles contain one or more phytosterols selected from the group consisting of campesterol, stigmasterol, and sitosterol.

[0015] In some embodiments, the composite lipid particles are selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesteryl ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acid, fatty acid ester of hydroxyl fatty acid, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol ... The composite lipid particles contain one or more lipids belonging to one or more of the subclasses selected from the group consisting of sphatidylserine, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, steryl ester, stigmasterol, sulfatide, sulfonolipid, sphingomyelin, sulfoquinovosyldiacylglycerol, sterol, and triacylglycerol. In some embodiments, the composite lipid particles contain at least 10 plant lipids belonging to one or more of the subclasses selected from the group consisting of the subclasses listed above. For example, the composite lipid particles contain 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 of the subclasses selected from the group consisting of the subclasses listed above.

[0016] In some embodiments, the composite lipid particles contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerol, diacylglyceryl glucuronide, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and sterols. For example, the composite lipid particles contain 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 of the subclasses selected from the group consisting of the subclasses listed above.

[0017] In some embodiments, the composite lipid particles contain lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different of the above-listed subclasses.

[0018] The composite lipid particles contain less than 50% w / w of protein material endogenous to 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 of protein material endogenous to one or more plant sources, or essentially no protein material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 30% w / w of protein material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 20% w / w protein material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 10% w / w protein material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 5% w / w protein material endogenous to one or more plant sources.

[0019] The composite lipid particles may contain reduced or minimized residual dsDNA material endogenous to one or more plant sources. For example, the composite lipid particles may 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 of residual dsDNA material endogenous to one or more plant sources, or may be essentially free of residual dsDNA material endogenous to one or more plant sources. In some cases, the lipid bilayer of the composite lipid particles does not contain residual dsDNA. In some embodiments, the composite lipid particles contain less than 1% w / w of residual dsDNA material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 0.1% w / w of residual dsDNA material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 0.01% w / w residual dsDNA material endogenous to one or more plant sources.

[0020] The composite lipid particles contain less than 50 mol% of ionized lipids (e.g., ionized lipids exogenous to one or more plant sources). For example, the composite lipid particles contain less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, less than 9 mol%, less than 8 mol%, less than 7 mol%, less than 6 mol%, less than 5 mol%, less than 4 mol%, less than 3 mol%, less than 2 mol%, less than 1 mol%, less than 0.5 mol%, less than 0.1 mol% of ionized lipids (e.g., ionized lipids exogenous to one or more plant sources), or essentially no ionized lipids (e.g., ionized lipids exogenous to one or more plant sources). In some embodiments, the composite lipid particles contain less than 20 mol% of exogenous ionized lipids. In some embodiments, the composite lipid particles contain less than 5 mol% of exogenous ionized lipids. In some embodiments, the composite lipid particles are essentially free of exogenous ionizable lipids.

[0021] In some embodiments, the complex lipid formulation does not contain exogenous nucleic acid.

[0022] In some embodiments, at least one of the botanical sources is a citrus fruit. For example, the citrus fruit may be a grapefruit or a lemon.

[0023] In some embodiments, at least one of the plant sources is a non-citrus plant, for example, the non-citrus plant may be dragon fruit, spinach, kale, strawberry, broccoli, or soybean.

[0024] In some embodiments, the one or more plant sources may be citrus fruits, non-citrus plants, or combinations thereof. In some embodiments, the one or more plant sources may be grapefruit, lemon, dragon fruit, spinach, kale, strawberry, broccoli, soybean, or combinations thereof.

[0025] In some embodiments, the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0026] In some embodiments, the exogenous lipid further comprises a lipid selected from the group consisting of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, second sterols, and additional synthetic lipids. 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.

[0027] In some embodiments, the composite lipid particles comprise about 10-95% w / w plant lipid. For example, 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 plant lipid, based on the amount of total lipid in the composite lipid formulation.

[0028] In some embodiments, the complex lipid particles comprise, based on the amount of total lipid in the complex lipid formulation, Approximately 10-95% w / w of vegetable lipids, Approximately 5-60% w / w sterols, Contains about 0.5-15% w / w polyethylene glycol (PEG)-lipid conjugate.

[0029] In some embodiments, the complex lipid particles comprise, based on the amount of total lipid in the complex lipid formulation, Approximately 85-95% w / w of vegetable lipids, Approximately 5-8% w / w sterols, Contains approximately 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate.

[0030] Another aspect of the present invention relates to a modified plant messenger pack (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 the one or more exogenous peptides, polypeptides, or proteins are encapsulated by the modified PMPs.

[0031] In some embodiments, the PMP comprises purified plant extracellular vesicles (EVs), or segments or extracts thereof. In some embodiments, the EVs, or segments or extracts thereof, are obtained from citrus fruits, such as grapefruit or lemon.

[0032] In some embodiments, the PMP is obtained from a citrus fruit, such as grapefruit or lemon.

[0033] In some embodiments, the modified PMP is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric 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.

[0034] In any of the above aspects of the invention relating to complex lipid formulations or modified PMP formulations, the following embodiments may apply.

[0035] The exogenous peptide, polypeptide, or protein may be a therapeutic agent.

[0036] In some embodiments, the exogenous peptide, polypeptide, or protein is an enzyme, hi some embodiments, the enzyme is a recombinant enzyme or an editing enzyme.

[0037] In some embodiments, the exogenous peptide, polypeptide, or protein is an antibody or an antibody fragment.

[0038] In some embodiments, the exogenous peptide, polypeptide, or protein is an Fc fusion protein.

[0039] In some embodiments, the exogenous peptide, polypeptide, or protein is a hormone, hi some embodiments, the exogenous peptide, polypeptide, or protein is insulin.

[0040] In some embodiments, the exogenous peptide, polypeptide, or protein is a peptide.

[0041] In some embodiments, the exogenous peptide, polypeptide or protein is a receptor agonist or 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 tirzepatide.

[0042] 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.

[0043] In some embodiments, the exogenous peptide, polypeptide, or protein has a size of 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 exogenous peptide, polypeptide, or protein has a size of less than 50 kD. In some embodiments, the exogenous peptide, polypeptide, or protein is at least 3 kD, at least 4 kD, or at least 5 kD in size. In some embodiments, the exogenous peptide, polypeptide, or protein has a size of at least 3 kD. In some embodiments, the exogenous peptide, polypeptide, or protein is at least 5 kD in size.

[0044] 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.

[0045] In some embodiments, the exogenous peptide, polypeptide, or protein has an overall charge that is neutral. In some embodiments, the exogenous peptide, polypeptide, or protein has been modified to have a neutral charge. In some embodiments, the exogenous peptide, polypeptide, or protein has an overall charge that is positive. In some embodiments, the exogenous peptide, polypeptide, or protein has an overall charge that is negative.

[0046] In some embodiments, the exogenous peptide, polypeptide, or protein may be modified (e.g., a lipid modification, such as a lipid tail). In some embodiments, the exogenous peptide, polypeptide, or protein may be a lipopeptide. In some embodiments, the exogenous peptide, polypeptide, or protein may be synthetic or contain synthetic amino acids.

[0047] In some embodiments, the sterol is cholesterol or sitosterol.

[0048] 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, the 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.

[0049] In some embodiments, the sterol is cholesterol or sitosterol and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof. In some embodiments, the sterol is cholesterol or sitosterol and the PEG-lipid conjugate is DSPE-PEG2000.

[0050] In some embodiments, the concentration of sterol in the composite lipid particle or modified PMP is in the range of about 5-60% w / w, e.g., about 5-50% w / w, about 5-40% w / w, about 5-30% w / w, about 5-20% w / w, about 5-15% w / w, about 0.5-15% w / w, about 5-8% w / w, or about 6-7% w / w, based on the amount of total lipid in the composite lipid particle or modified PMP. In some embodiments, the sterol is in the range of about 15-20% w / w, about 20-30% w / w, about 30-40% w / w, about 40-50% w / w, or about 50-60% w / w, based on the amount of total lipid in the composite lipid particle or modified PMP.

[0051] In some embodiments, the concentration of the PEG-lipid conjugate is in the range of about 0.5-5% w / w, about 0.5-3.5% w / w, about 1-3.5% w / w, about 0.5-3% w / w, about 1-3% w / w, about 0.5-2.5% w / w, about 1-2.5% w / w, about 1.5-2.5% w / w, or about 2-2.5% w / w based on the amount of total lipid in the composite lipid particle or modified PMP. In some embodiments, the concentration of the PEG-lipid conjugate is in the range of about 0.5-15% w / w, about 1-15% w / w, about 2-5% w / w, about 5-8% w / w, about 8-12% w / w, or about 12-15% w / w based on the amount of total lipid in the composite lipid particle or modified PMP.

[0052] In some embodiments, the sterol is cholesterol or sitosterol having a concentration in the range of about 5-50% w / w based on the amount of total lipid in the complex lipid particle or modified PMP, and the PEG-lipid conjugate is PEG2000-PE, PEG2000-DMG, PEG2000-DSPE, or derivatives thereof having a concentration in the range of about 1-3.5% w / w based on the amount of total lipid in the complex lipid particle or modified PMP.

[0053] In some embodiments, the sterol is cholesterol or sitosterol having a concentration in the range of about 5-8% w / w based on the amount of total lipid in the complex lipid particle or modified PMP, and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof having a concentration in the range of about 1-3.5% w / w based on the amount of total lipid in the complex lipid particle or modified PMP.

[0054] In some embodiments, the sterol is cholesterol or sitosterol having a concentration in the range of about 20-25% w / w based on the amount of total lipid in the complex lipid particle or modified PMP, and the PEG-lipid conjugate is DSPE-PEG2000 or a derivative thereof having a concentration in the range of about 1-3.5% w / w based on the amount of total lipid in the complex lipid particle or modified PMP.

[0055] In some embodiments, the sterol is cholesterol or sitosterol having a concentration in the range of about 5-8% w / w based on the amount of total lipid in the complex lipid particle or modified PMP, and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof having a concentration in the range of about 1-3.5% w / w based on the amount of total lipid in the complex lipid particle or modified PMP.

[0056] The composite lipid particles or modified PMPs may have an average size of less than about 400 nm, less than about 350 nm, less than about 300 nm, less than about 250 nm, or less than about 200 nm. In one embodiment, the composite lipid particles or modified PMPs have an average size of less than about 200 nm. In one embodiment, the composite lipid particles or modified PMPs have an average size of about 100-180 nm. In one embodiment, the composite lipid particles or modified PMPs have an average size of about 100-160 nm.

[0057] The composite lipid particles or modified PMPs may have a polydispersity index (PDI) of less than about 0.7, less than about 0.6, less than about 0.5, or less than about 0.4. For example, the composite lipid particles or modified PMPs may have a PDI in the range of 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 composite lipid particles have a PDI of about 0.1 to about 0.5. In one embodiment, the composite lipid particles have a PDI of about 0.2 to about 0.4.

[0058] In some embodiments, the composite lipid particle formulation or modified PMP formulation, for example, the 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 molar concentration of 0.001M to 0.1M. The buffer solution may have a pH of about 3.0 to about 8.5. The HEPES or TRIS buffer may have a pH of about 7.0 to about 8.5. The HEPES or TRIS buffer may have a concentration of about 7 mg / mL to about 15 mg / mL. The aqueous phase may further comprise about 2.0 mg / mL to about 4.0 mg / mL NaCl. 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.

[0059] In some embodiments, the composite lipid particle formulation or modified PMP formulation further comprises one or more cryoprotectants. The one or more cryoprotectants may 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 formulation comprises 0.5-2% sucrose, 0.5-2% mannitol, or a combination thereof.

[0060] 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 frozen composition includes poloxamer, for example, poloxamer 188.

[0061] In some embodiments, the complex lipid particle formulation is not lyophilized, hi some embodiments, the complex lipid formulation is a liquid composition.

[0062] In some embodiments, the composite lipid particle formulation or modified PMP formulation is stable at room temperature for at least one day, with or without lyophilization, and / or stable at 4° C. for at least one day, at least one week, or at least one month. 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 particle formulation is stable at room temperature and / or 4° C. for at least two weeks, without lyophilization. In some embodiments, the composite lipid particle formulation or modified PMP formulation is stable at temperatures of at least 20° C., 24° C., or 37° C.

[0063] Some embodiments provide a composition comprising a plurality of modified PMP formulations of any of the above embodiments, in some embodiments, the modified PMP formulations in the composition are at a concentration effective to increase fitness of the mammal.

[0064] Some embodiments provide a composition comprising a plurality of composite lipid particle formulations of any of the above embodiments, in some embodiments, the composite lipid particle formulations in the composition are at a concentration effective to increase fitness of the mammal.

[0065] In some embodiments, the exogenous peptide, polypeptide, or protein in the composite lipid particle formulation is at a concentration of at least 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, or 1 μg / mL. In some embodiments, the exogenous peptide, polypeptide, or protein is at a concentration of at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 μg / mL.

[0066] In some embodiments, at least 15% of the composite lipid particles in the composite lipid particle formulations encapsulate an exogenous peptide, polypeptide, or protein. In some embodiments, at least 50% of the composite lipid particles in the composite lipid particle formulations encapsulate an exogenous peptide, polypeptide, or protein. In some embodiments, at least 95% of the composite lipid particles in the composite lipid particle formulations encapsulate an exogenous peptide, polypeptide, or protein.

[0067] In some embodiments, at least 15% of the modified PMPs in a plurality of modified PMP formulations encapsulate an exogenous peptide, polypeptide, or protein. In some embodiments, at least 50% of the modified PMPs in a plurality of modified PMP formulations encapsulate an exogenous peptide, polypeptide, or protein. In some embodiments, at least 95% of the modified PMPs in a plurality of modified PMP formulations encapsulate an exogenous peptide, polypeptide, or protein.

[0068] Another aspect of the present invention relates to a pharmaceutical composition or preparation comprising the complex lipid formulation described herein and a pharma- ceutically acceptable vehicle, carrier, or excipient.

[0069] In some embodiments, the pharmaceutical composition or preparation is in an oral dosage form, such as a capsule or tablet dosage form.

[0070] All the above explanations and all the embodiments discussed in the above aspects relating to complex lipid formulations or complex lipid particles are applicable to those aspects of the invention relating to pharmaceutical formulations comprising complex lipid formulations.

[0071] In another aspect, the disclosure features a pharmaceutical composition including a modified PMP formulation according to any one of the above embodiments and a pharma- ceutically acceptable vehicle, carrier, or excipient.

[0072] In some embodiments, the pharmaceutical composition or preparation is formulated for administration to a mammal, such as a human subject. In some embodiments, the pharmaceutical composition or preparation is formulated for administration to a mammalian cell.

[0073] In any of the above aspects of the invention relating to pharmaceutical compositions or preparations, the following embodiments may be applicable.

[0074] 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 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 and loading the complex lipid particles with an exogenous peptide, polypeptide, or protein, whereby the exogenous peptide, polypeptide, or protein is encapsulated by the complex lipid particles, thereby forming a complex lipid formulation.

[0075] 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.

[0076] In some embodiments, lipids are extracted from one or more plant sources by adding to the plant source an extraction solvent comprising methanol, ethanol, propanol, 1-butanol, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, dimethylsulfoxide, methyl tert-butyl ether, chloroform, ethyl acetate, or mixtures 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, dimethylsulfoxide:methanol, acetonitrile:ethanol, or ethyl acetate:ethanol.

[0077] In some embodiments, the extraction process further comprises reducing the protein 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 essentially completely removing the protein material endogenous to the one or more plant sources.

[0078] The composite lipid particles may contain reduced or minimized residual dsDNA material endogenous to one or more plant sources. For example, the composite lipid particles may 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 of residual dsDNA material endogenous to one or more plant sources, or may be essentially free of residual dsDNA material endogenous to one or more plant sources. In some cases, the lipid bilayer of the composite lipid particles does not contain residual dsDNA. In some embodiments, the composite lipid particles contain less than 1% w / w of residual dsDNA material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 0.1% w / w of residual dsDNA material endogenous to one or more plant sources. In some embodiments, the composite lipid particles contain less than 0.01% w / w residual dsDNA material endogenous to one or more plant sources.

[0079] In some embodiments, the mixing step is performed by thin film mixing or microfluidic mixing.

[0080] In some embodiments, the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate.

[0081] In some embodiments, the exogenous lipids do not include ionizable lipids. Thus, the complex lipid formulations are prepared without the addition of exogenous ionizable lipids.

[0082] In another aspect, the disclosure features a method of producing a modified PMP formulation containing an exogenous peptide, polypeptide, or protein, the method includes: (a) providing a solution containing a modified PMP that contains one or more PMPs, one or more sterols, and one or more polyethylene glycol (PEG)-lipid conjugates; providing a solution containing the exogenous peptide, polypeptide, or protein; and (b) loading the modified PMP with the exogenous peptide, polypeptide, or protein, whereby the exogenous peptide, polypeptide, or protein is encapsulated by the modified PMP.

[0083] In some embodiments, the exogenous peptide, polypeptide, or protein is soluble in solution.

[0084] In some embodiments, loading comprises one or more of sonication, electroporation, and lipid extrusion. In some embodiments, loading comprises sonication and lipid extrusion. In some embodiments, loading comprises lipid extrusion. In some embodiments, the PMP lipid is isolated prior to lipid extrusion. In some embodiments, the isolated PMP lipid comprises glycosyl inositol phosphorylceramide (GIPC).

[0085] Another aspect of the invention relates to a method of delivering a peptide, polypeptide, or protein to a mammalian cell or mammal, the method comprising contacting a mammalian cell with a complex lipid formulation or administering a complex lipid formulation to a mammal under conditions sufficient to allow uptake of the complex lipid formulation by the mammalian cell or by the mammal. a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five plant lipids and at least two exogenous lipids; and One or more exogenous peptides, polypeptides, or proteins encapsulated in a composite lipid particle, the composite lipid particle having the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mole % ionized lipids.

[0086] In some embodiments, the mammalian cell is a cell in a human or the mammal is a human. In some embodiments, uptake by the mammalian cell or by the mammal of an exogenous peptide, polypeptide, or protein encapsulated by a composite lipid particle is increased compared to uptake of an exogenous peptide, polypeptide, or protein that is not encapsulated by a composite lipid particle.

[0087] In some embodiments, the method is for delivering a peptide, polypeptide, or protein to a mammal, and administration is via oral, enteral, intranasal, rectal (including intracolonic), or intrajejunal routes.

[0088] In some embodiments, the mammalian cell is a brain cell.

[0089] Another aspect of the invention relates to a method of treating or preventing a disease or disorder in a subject in need of a therapeutic agent, the method comprising administering to a subject in need thereof: a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and The method comprises administering an effective amount of a complex lipid formulation comprising one or more exogenous peptides, polypeptides, or proteins encapsulated in complex lipid particles, the complex lipid particles having the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mole % ionized lipids.

[0090] In some embodiments, administration is via oral, enteral, intranasal, rectal (including intracolonic), or intrajejunal routes.

[0091] In some embodiments, the disease is diabetes and the exogenous peptide, polypeptide, or protein is insulin, exenatide, semaglutide, or tirzepatide.

[0092] In another aspect, the disclosure features a method of delivering a peptide, polypeptide, or protein to a mammalian cell, the method including contacting the cell with a modified PMP formulation of any of the above embodiments, in an amount and for a time sufficient to allow uptake of the modified PMP formulation by the cell. In some embodiments, the cell is a cell in a subject.

[0093] In some embodiments, the exogenous peptide, polypeptide, or protein is released from the modified PMP formulation in a mammalian cell with which the modified PMP formulation is contacted. In some embodiments, the exogenous peptide, polypeptide, or protein exerts its activity 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 exerts its activity in the nucleus of the mammalian cell.

[0094] In another aspect, the disclosure features a method of delivering a peptide, polypeptide, or protein to a mammal, the method including administering to the mammal a modified PMP formulation described in any of the above embodiments, wherein the administration occurs under conditions sufficient to allow uptake of the modified PMP formulation by the mammal.

[0095] In another aspect, the disclosure features a modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, where the mammal is a human.

[0096] 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 compared to uptake of an exogenous peptide, polypeptide, or protein that is not encapsulated by the modified PMP formulation.

[0097] In another aspect, the disclosure features a modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, wherein the efficacy of an exogenous peptide, polypeptide, or protein encapsulated by the modified PMP formulation is increased compared to the efficacy of an exogenous peptide, polypeptide, or protein that is not encapsulated by the modified PMP formulation.

[0098] In another aspect, the disclosure features a method of treating or preventing a disease or disorder in a subject in need of a therapeutic agent, the method including administering to a subject in need thereof an effective amount of a modified PMP formulation, as described in 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.

[0099] In some embodiments, the disease or disorder is diabetes. In some embodiments, administration of the modified PMP formulation reduces blood glucose in the subject. In some embodiments, the exogenous peptide, polypeptide, or protein is insulin.

[0100] In another aspect, the disclosure features a modified PMP formulation, composition, pharmaceutical composition, or method of any of the above embodiments, where the modified PMP formulation is not significantly degraded by gastric fluids, e.g., is not significantly degraded by fasting gastric fluids.

[0101] In another aspect, the disclosure features a method of any of the above embodiments, where administration is via oral, enteral, intranasal, or intrarectal (including intracolonic) routes. [Brief description of the drawings]

[0102] [Figure 1] FIG. 1 is a scheme illustrating an exemplary formulation process for preparing composite lipid particles containing natural source lipids and exogenous lipids, as described in Example 2. [Diagram 2] FIG. 2 is a scheme illustrating an exemplary process for loading bioactive molecules into composite lipid particles containing natural source lipids and exogenous lipids, as described in Example 2. [Diagram 3] Figure 3 is a graph showing insulin concentrations in mouse plasma one hour after rectal administration of insulin-loaded complex lipid formulations containing natural source lipids and exogenous lipids. (†) The dashed line reflects the benchmark of fused liposomes containing insulin administered directly to the colon (10 U / kg, 12 min after injection). [Figure 4] Figure 4 is a graph showing insulin concentrations in mouse brains two hours after intranasal administration of insulin-loaded complex lipid formulations containing natural source lipids and exogenous lipids. The dashed line reflects the benchmark for brain delivery of 10 U / kg insulin boosted by intranasal co-administration with (†) cell-penetrating peptide and (‡) free insulin 90 min after administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0103] I. Definition As used herein, the term "encapsulate" or "encapsulated" refers to the enclosure of a moiety (e.g., an exogenous peptide, polypeptide, or protein as defined herein) within an enclosed lipid membrane structure, e.g., a lipid bilayer. The lipid membrane structure may be, for example, a plant messenger pack (PMP) or a plant extracellular vesicle (EV), or may be obtained from or derived from a plant EV. The encapsulated moiety (e.g., an encapsulated exogenous peptide, polypeptide, or protein) is encapsulated by the lipid membrane structure, e.g., such an encapsulated moiety is located in the lumen of the enclosed lipid membrane structure (e.g., the lumen of a PMP). The encapsulated moiety (e.g., an encapsulated peptide, polypeptide, or protein) may, in some cases, interact or associate with the inner surface of the lipid membrane structure. The exogenous peptide, polypeptide, or protein may, in some cases, be intercalated with the lipid membrane structure. In some cases, the exogenous peptide, polypeptide, or protein has an extraluminal portion. Alternatively, the terms "encapsulate" or "encapsulated" may be used in the context of using a composite lipid particle to encapsulate a moiety (e.g., an exogenous peptide, polypeptide, or protein, as defined herein) within a composite lipid particle. In some cases, "encapsulate" may be used in the context of using a modified PMP to encapsulate a moiety (e.g., an exogenous peptide, polypeptide, or protein, as defined herein).

[0104] As used herein, the term "exogenous peptide, polypeptide, or protein" refers to a peptide, polypeptide, or protein (as defined herein) that is encapsulated in a complex lipid particle or modified PMP (e.g., a PMP derived from a plant extracellular vesicle and modified with one or more exogenous lipids) that does not naturally occur in a plant lipid vesicle (e.g., does not naturally occur in a plant extracellular vesicle), or is encapsulated in a complex lipid particle or modified PMP in an amount not found in naturally occurring plant extracellular vesicles. The exogenous peptide, polypeptide, or protein may, in some cases, be naturally present in the plant from which the plant lipid is extracted or the plant from which the PMP is derived. In other cases, the exogenous peptide, polypeptide, or protein is not naturally present in the plant from which the plant lipid is extracted or the plant from which the PMP is derived. The exogenous peptide, polypeptide, or protein may be artificially expressed in the plant from which the plant lipid is extracted or the plant from which the PMP is derived, e.g., a heterologous polypeptide. The exogenous peptide, polypeptide, or protein may be derived from another organism. In some embodiments, exogenous peptides, polypeptides, or proteins are loaded into composite lipid particles or modified PMP formulations, for example, using one or more of sonication, electroporation, lipid extraction, and lipid extrusion. The exogenous peptides, polypeptides, or proteins may be, for example, therapeutic agents, enzymes (e.g., recombinant or edited enzymes), hormones (e.g., insulin), receptor agonists or receptor antagonists (e.g., GLP-1 agonists such as exenatide, semaglutide, or tirzepatide), or pathogen control agents.

[0105] As used herein, "delivering" or "contacting" refers to providing or applying a complex lipid particle formulation or modified PMP formulation (e.g., a modified PMP formulation containing an exogenous protein or peptide) to an organism, e.g., an animal. Delivery to an animal may be, for example, oral or enteral delivery (e.g., delivery by feeding, or delivery into the GI tract, e.g., by tube feeding) or systemic delivery (e.g., delivery by injection). The modified PMP formulation may be delivered to the digestive tract, e.g., the stomach, small intestine, or large intestine. The complex lipid particle formulation or modified PMP formulation may be stable in the digestive tract.

[0106] 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.

[0107] 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 pharma- ceutically acceptable carrier.

[0108] As used herein, the term "infection" refers to the presence or colonization of a pathogen within an animal (e.g., within 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, for example, by causing disease, disease symptoms, or an immune (e.g., inflammatory) response.

[0109] As used herein, the term "pathogen" refers to an organism, such as a microorganism or invertebrate, that causes disease or symptoms of disease in an animal, for example, by (i) directly infecting the animal, (ii) producing an agent (e.g., a bacterium that produces a pathogenic toxin, etc.) that causes the disease or symptoms of disease in the animal, and / or (iii) by inducing an immune (e.g., an inflammatory response) in the animal (e.g., a biting insect, e.g., a bedbug). As used herein, pathogens include, but are not limited to, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids and viruses, or any combination thereof, each pathogen being capable of inducing disease or symptoms in humans, either by itself or in contact with another pathogen.

[0110] As used herein, the terms "polypeptide," "peptide," or "protein" encompass any chain of naturally or non-naturally occurring amino acids (either D- 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, including, for example, naturally occurring polypeptides, synthetic or recombinant polypeptides, hybrid molecules, peptoids, or peptidomimetics. A polypeptide may 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 in size. A polypeptide may be a full-length protein. Alternatively, a polypeptide may comprise one or more domains of a protein.

[0111] As used herein, the term "antibody" encompasses natural or partially or fully synthetically produced immunoglobulins, and fragments thereof capable of specifically binding to an antigen. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources or partially or fully synthetically produced. "Antibody" further includes polypeptides that include a framework region derived from an immunoglobulin gene or a fragment thereof that specifically binds and recognizes an antigen. The use of the term "antibody" is meant to include complete antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single chain antibodies (nanobodies), humanized antibodies, murine antibodies, chimeric antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. "Antibody" further includes bispecific and multispecific antibodies.

[0112] As used herein, the term "antigen-binding fragment" refers to a fragment of an intact immunoglobulin and any portion of a polypeptide that contains an antigen-binding region that has the ability to specifically bind to an antigen. For example, an antigen-binding fragment may be, but is not limited to, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, or a scFv fragment. A Fab fragment has one antigen-binding site and contains the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region CH1 of the heavy chain. A Fab' fragment differs from a Fab fragment in that the Fab' fragment additionally contains the hinge region of the heavy chain, which contains at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 region. A F(ab')2 fragment is generated, whereby the cysteine ​​residues of the Fab' fragment are linked by disulfide bonds at the hinge region. An Fv fragment is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region, and recombinant techniques for generating Fv fragments are well known in the art. The two-chain Fv fragment may have a structure in which the heavy chain variable region is non-covalently linked to the light chain variable region. The single-chain Fv (scFv) fragment may generally have a dimeric structure like the two-chain Fv fragment, in which the heavy chain variable region is covalently linked to the light chain variable region via a peptide linker, or the heavy chain variable region and the light chain variable region are directly linked to each other at their C-terminus. The antigen-binding fragment may be obtained using proteolytic enzymes (e.g., whole antibody is digested with papain to obtain Fab fragments and with pepsin to obtain F(ab')2 fragments) or may be prepared by recombinant gene technology. The dAb fragment consists of the VH domain.

[0113] A single chain antibody molecule may comprise a polymer having multiple individual molecules, eg, dimers, trimers or other polymers.

[0114] 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 that is not the plant or part of the plant in which the PMP is produced) (e.g., an agent added to the PMP using the loading approaches described herein) or (2) endogenous to the plant cell or tissue in which the PMP is produced, but present in the PMP at a concentration higher than that found in nature (e.g., higher than the concentration found in naturally occurring plant extracellular vesicles) (e.g., added to the PMP using the loading approaches described herein, genetic engineering, and in vitro or in vivo approaches).

[0115] As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, as described in Altschul et al., (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0116] As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and their progeny. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, ciliary regions, callus tissues, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant parts include, but are not limited to, the following: roots, stems, shoots, leaves, pollen, seeds, fruits, harvested products, tumor tissues, and various forms of cells and cultures (e.g., single cells, protists, embryos, and callus tissues). Plant tissues may be within a plant or within a plant organ, tissue, or cell culture. Additionally, plants may be genetically engineered to produce heterologous proteins or RNA.

[0117] As used herein, the term "complex lipid particle" refers to lipid particles having a complexity characterized by including a wide variety of lipids, including lipids extracted from one or more plant sources. The complex lipid particles may include 10% w / w to 99% w / w lipids derived from lipid structures derived from one or more plant sources, for example, may 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 lipids derived from lipid structures derived from one or more plant sources. The complex lipid particles may contain 5 to 1000 lipids extracted from one or more plant sources. The complex lipid particles may contain plant lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of lipids derived from plant sources. The complex lipid particles may contain all or a fraction of the lipid species present in the lipid structure derived from a plant source, for example, 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 substantially 100% of the lipid species present in the lipid structure derived from a plant source. The complex lipid particles may contain reduced or minimized protein material endogenous to one or more plant sources, for example, less than 0% w / w, less than 1% w / w, less than 5% w / w, less than 10% w / w, less than 15% w / w, less than 20% w / w, less than 30% w / w, less than 40% w / w, or less than 50% w / w of protein material endogenous to one or more plant sources. In some cases, the lipid bilayer of the complex lipid particle does not contain protein.

[0118] The composite lipid particles may further comprise at least two exogenous lipids. The composite lipid particles may comprise 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 of exogenous lipids. Exemplary exogenous lipids include sterols and PEG-lipid conjugates. The composite lipid particles may be used to encapsulate exogenous peptides, polypeptides, or proteins, allowing the delivery of exogenous peptides, polypeptides, or proteins to target cells or tissues.

[0119] As used herein, the term "plant extracellular vesicles", "plant EVs", or "EVs" refers to enclosed lipid bilayer structures naturally occurring in plants. Optionally, the plant EVs include one or more plant EV markers. As used herein, the term "plant EV marker" refers to a component that is 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 of the plant EV markers listed in the appendix disclosed in WO2021 / 041301, which is incorporated herein by reference in its entirety. In some cases, the plant EV marker is an identification marker for a plant EV but is not an insecticide. In some cases, the plant EV marker is both an identification marker for a plant EV and an insecticide (e.g., either associated with or encapsulated by multiple PMPs, or not directly associated with or encapsulated by multiple PMPs).

[0120] As used herein, the term "plant messenger pack" or "PMP" refers to lipid structures (e.g., lipid bilayer, monolayer, multilayer structures, e.g., vesicular lipid structures) that are about 5-2000 nm in diameter (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) that are derived from a plant source or segment, part, or extract thereof (e.g., enriched, isolated, or purified) and that contain lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated with the plant source or segment, part, or extract thereof, and that have been enriched, isolated, or purified from a plant, plant part, or plant cell, concentrate or isolate that has been stripped of one or more contaminants or undesirable components from the source plant. PMPs may be highly purified preparations of naturally occurring EVs. Preferably, at least 1% of contaminants or undesirable components are removed from the source plant (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% of one or more contaminants or undesirable components from the source plant, e.g., plant cell wall components, pectin, plant organelles (e.g., mitochondria, chloroplasts such as plastids, leucoplasts or amyloplasts, and nuclei), plant chromatin (e.g., plant chromosomes), or plant molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipid-protein structures). 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) compared to one or more contaminants or undesirable components from the source plant as measured by weight (w / w), spectroscopic imaging (% transmittance), or conductivity (S / m).

[0121] PMPs may be modified to include exogenous lipids, e.g., lipids, that are (1) exogenous to the plant (e.g., derived from a source that is not the plant or part of the plant in which the PMP is produced) (e.g., added to the PMP using methods described herein) or (2) endogenous to the plant cell or tissue in which the PMP is produced, but present in the PMP at a concentration higher than that found in nature (e.g., higher than that found in naturally occurring plant extracellular vesicles) (e.g., added to the PMP using methods described herein, genetic engineering, in vitro or in vivo approaches). The lipid composition of the PMP may 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, ionized lipids, zwitterionic lipids, and lipidoids.

[0122] PMPs may be modified to optionally include additional agents, such as polypeptides (e.g., peptides or proteins), therapeutic agents, polynucleotides, or small molecules. PMPs can carry or associate with additional agents (e.g., polypeptides) in a variety of ways to enable delivery of the agent to the target plant, for example, by encapsulating the agent, incorporating the agent into the lipid bilayer structure, or associating the agent with the surface of the lipid bilayer structure (e.g., by conjugation). Heterologous functional agents can be incorporated into PMPs either in vivo (e.g., in plants) or in vitro (e.g., in tissue culture, cell culture, or synthetically incorporated).

[0123] As used herein, the term "pure" refers to a PMP preparation in 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 compared to the initial sample, or portion thereof, isolated from the plant.

[0124] As used herein, the term "exogenous lipid" refers to a lipid that is exogenous to a plant, i.e., the lipid is derived from a source that is not the plant source from which the lipid is extracted (e.g., lipid added to a composite lipid particle formulation using the methods described herein). The term "exogenous lipid" does not exclude plant-derived lipids (such as plant-derived sterols). That is, the exogenous lipid may be a plant-derived lipid (such as a plant-derived sterol that is exogenous to the plant source from which the lipid is extracted, e.g., the exogenous lipid may be a plant-derived sterol that is added to a composite lipid particle formulation). The exogenous lipid may be a cell-penetrating agent, may increase the delivery of peptides, polypeptides, or proteins to cells by the composite lipid formulation, and / or may increase the loading (e.g., loading efficiency or loading capacity) of peptides, polypeptides, or proteins. In some embodiments, the exogenous lipid may be a stabilizing lipid. In some embodiments, the exogenous lipid may be a structural lipid. Exemplary exogenous lipids include sterols and PEGylated lipids.

[0125] As used herein, the term "treatment" refers to administering a pharmaceutical composition to an animal for prophylactic and / or therapeutic purposes. To "prevent an infection" refers to prophylactic treatment of an animal that does not already have a disease or condition, but is susceptible to or otherwise at risk for a particular disease or condition. To treat an infectious disease refers to administering treatment to an animal that already has a disease to improve or stabilize the animal's condition.

[0126] As used herein, the term "treating an infection" refers to administering a treatment to an individual (e.g., an animal) that already has a disease to improve or stabilize the individual's condition. This can involve reducing the colonization of one or more pathogens in, on, or around the animal compared to the starting amount (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) and / or allowing a benefit to the individual (e.g., reducing colonization by an amount sufficient to resolve symptoms). In such cases, a treated infection can be manifested as a reduction in symptoms (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some cases, the treated infection is effective to increase the chances of survival of an individual (e.g., increase the chances of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or increase the overall survival of a population (e.g., increase the chances of survival by 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" the infection, which refers to a reduction in infection in an amount sufficient to provide a sustained resolution of symptoms in the animal (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0127] 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 in an amount sufficient to maintain the initial pathogen population (e.g., approximately the amount found in a healthy individual), prevent the onset of infection, and / or prevent symptoms or conditions associated with infection (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to an untreated animal). For example, an individual (e.g., an animal, e.g., a human) may be receiving prophylactic treatment to prevent fungal infection in preparation for an invasive medical procedure (e.g., undergoing transplantation, stem cell therapy, grafts, prosthetics, long-term or frequent intravenous catheterization, or intensive care unit care), an immunocompromised individual (e.g., having cancer, HIV / AIDS, or taking immunosuppressants), or an individual receiving long-term antibiotic therapy.

[0128] As used herein, a complex lipid formulation or modified PMP formulation is "stable" means that the complex lipid formulation or modified PMP formulation is stable for 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), and, optionally, within a defined temperature range (e.g., 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 -2°C, "complex lipid formulation" refers to a complex lipid formulation or modified PMP composition that 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 starting number of complex lipid particles or modified PMPs (e.g., complex lipid particles or modified PMPs per mL of solution) compared to the number of complex lipid particles or modified PMPs in the complex lipid formulation or modified PMP composition (e.g., at the time of production or formulation) at temperatures of 0°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).

[0129] Alternatively, the expression may refer to 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), and, optionally, a defined temperature range (e.g., 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 − ... For example, it refers to a complex lipid formulation or modified PMP composition that 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 compared to the starting activity of the complex lipid formulation or modified PMP formulation (e.g., at the time of production or formulation) at a temperature of 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).

[0130] Alternatively, the expression may refer to 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), and, optionally, a defined temperature range (e.g., 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., −25° C., −30° C., −35° C., −40° C., −45° C., −50° C., −60° C., −70° C., −80° C., −90° C., −100° C., −150° C., −250° C., −250° C., −300° C., −35 ... "complex lipid particles" refers to complex lipid or modified PMP formulations that retain their particle size, i.e., have no more than 5% (e.g., 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 or less) increase in particle size compared to the starting size of the complex lipid particles or modified PMPs (e.g., at the time of production or formulation) at temperatures below 30°C (e.g., at temperatures below 30°C, -5°C, or 0°C), or at -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C, or -30°C).

[0131] In some embodiments, a stable complex lipid formulation or modified PMP continues to encapsulate or remain associated with an exogenous peptide, polypeptide, or protein with which it is loaded, e.g., 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 for more than 90 days.

[0132] 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 insects with piercing and sucking mouthparts found in Hemiptera and some Hymenoptera and Diptera, such as mosquitoes, bees, wasps, midges, ticks, tsetse flies, flies, fleas and ants.

[0133] As used herein, the term "juice vesicle" or "juice vesicle" refers to the juice-containing membrane-bound components of the endocarp (carpel) of a mandarin fruit, e.g., a citrus fruit. In some embodiments, the vesicle is separated from other parts of the fruit, e.g., the peel (exocarp or flavedo), endocarp (mesocarp, albedo, or mesocarp), stele (placenta), segment walls, atta, or seeds. In some embodiments, the vesicle is a grapefruit, lemon, lime, or orange vesicle.

[0134] II. Complex lipid particles or modified PMPs encapsulating polypeptides One aspect of the invention relates to a complex lipid formulation comprising a plurality of complex lipid particles, each complex lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids, and one or more exogenous peptides, polypeptides, or proteins encapsulated in the complex lipid particle. The complex lipid particles have the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mole % ionized lipids.

[0135] Another aspect of the present invention relates to plant messenger packs (PMPs) modified with one or more exogenous lipids.

[0136] The complex lipid formulations or modified PMP formulations described herein include an exogenous peptide, polypeptide, or protein, such as an exogenous peptide, polypeptide, or protein described in Section III herein. A plurality of complex lipid particles or modified PMPs may be loaded with an exogenous peptide, polypeptide, or protein 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 the exogenous peptide, polypeptide, or protein.

[0137] The exogenous peptide, polypeptide, or protein may be, for example, a therapeutic agent, a pathogen control agent (e.g., an agent having anti-pathogen activity (e.g., antibacterial, antifungal, insecticidal, antiparasitic, or antiviral activity)), or an enzyme (e.g., a recombinant enzyme or an editing enzyme).

[0138] A.CLP The composite lipid particles (CLPs) described herein comprise a wide variety of lipids extracted from one or more plant sources. The composite lipid particles may comprise between 10% w / w and 99% w / w lipid derived from lipid structures derived from one or more plant sources, for example, may 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 lipid derived from lipid structures derived from one or more plant sources.

[0139] In some embodiments, the composite lipid particles comprise about 10-95% w / w plant lipid. For example, 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 plant lipid, based on the amount of total lipid in the composite lipid formulation.

[0140] The composite lipid particles may contain 5 to 1000 lipids extracted from one or more plant sources. In some embodiments, the composite lipid particles contain at least 10 plant lipids belonging to one or more of the classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. For example, the composite lipid particles contain 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 of the classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. In some embodiments, the composite lipid particles contain lipids from at least two or at least three of these different classes.

[0141] The complex lipid particles may contain one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG), and sulfolipids (SL). In some embodiments, the complex lipid particles contain one or more sphingolipids selected from the group consisting of glycosyl inositol phosphoceramides (GIPC), glucosylceramides (GCer), ceramides (Cer), and free long chain bases (LCB). In some embodiments, the complex lipid particles contain one or more phytosterols selected from the group consisting of campesterol, stigmasterol, and sitosterol.

[0142] The complex lipid particles are composed of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesteryl ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acids, fatty acid esters of hydroxyl fatty acids, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylceryl. The composite lipid particles may contain one or more lipids belonging to one or more of the subclasses selected from the group consisting of plant lipids, monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, steryl ester, stigmasterol, sulfatide, sulfonolipid, sphingomyelin, sulfoquinovosyldiacylglycerol, sterol, and triacylglycerol. In some embodiments, the composite lipid particles contain at least 10 plant lipids belonging to one or more of the subclasses selected from the group consisting of the subclasses listed above. For example, the composite lipid particles contain 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 of the subclasses selected from the group consisting of the subclasses listed above.

[0143] The composite lipid particles may contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerol, diacylglyceryl glucuronide, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and sterols. For example, the composite lipid particles contain 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 of the subclasses selected from the group consisting of the subclasses listed above.

[0144] The composite lipid particles may contain plant lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of lipids derived from plant sources.

[0145] The identity (as well as class and subclass) and amount of lipids extracted from plant sources can be analyzed by lipidomic analysis by solubilizing the lipid extract or complex lipid particles in a compatible solvent and analyzing by mass spectrometry (e.g., MS / MS). An example of MS / MS-based lipidomic analysis of complex lipid particles is shown in Example 2.

[0146] The composite lipid particles may comprise all or a fraction of the lipid species present in the lipid structures derived from a plant source, for example, 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 substantially 100% of the lipid species present in the lipid structures derived from a plant source.

[0147] The complex lipid particles may contain reduced or minimized proteinaceous material endogenous to one or more plant sources. For example, the complex lipid particles may 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 of proteinaceous material endogenous to one or more plant sources, or may be essentially free of proteinaceous material endogenous to one or more plant sources. In some cases, the lipid bilayer of the complex lipid particles does not contain protein. To calculate the % w / w of the remaining protein material endogenous to the plant source(s), 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 the percentage of the mass of the total protein endogenous to the plant source(s) based on the mass of the total lipid extract.

[0148] The complex lipid particles may contain reduced or minimized residual dsDNA material endogenous to one or more plant sources.For example, the complex lipid particles may 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 of residual dsDNA material endogenous to one or more plant sources, or may be essentially free of residual dsDNA material endogenous to one or more plant sources.In some cases, the lipid bilayer of the complex lipid particles does not contain residual dsDNA.To calculate the % w / w of residual dsDNA material endogenous to one or more plant sources, divide the total adjusted dsDNA by the concentration of the plant lipid extract and then multiply by 100. Alternatively, % w / w is calculated as the percentage of the mass of total residual dsDNA endogenous to one or more plant sources, based on the mass of the total lipid extract.

[0149] The composite lipid particles may further comprise at least two exogenous lipids. The composite lipid particles may comprise 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 of exogenous lipids. For example, the composite lipid particles may contain sterol and PEG-lipid conjugates. Additional exogenous lipids suitable for inclusion in composite lipid particles are described herein below.

[0150] B.PMP The PMPs can include plant EVs, or segments, parts, or extracts thereof, and the plant EVs are about 5-2000 nm in diameter. For example, the PMPs 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 600-650 nm, about 650-700 nm, about The plant EVs may have an average diameter of 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1000 nm, about 1000-1250 nm, about 1250-1500 nm, about 1500-1750 nm, or about 1750-2000 nm, or a segment, part, or extract thereof. In some cases, the PMP comprises a plant EV, or a segment, part, or extract 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 certain cases, the plant EV, or a segment, part, or extract thereof, has an average diameter of about 50-200 nm. In certain cases, the plant EV, or a segment, part, or extract thereof, has an average diameter of about 50-300 nm. In certain cases, the plant EVs, or segments, parts, or extracts thereof, have an average diameter of about 200-500 nm, In certain cases, the plant EVs, or segments, parts, or extracts thereof, have an average diameter of about 30-150 nm.

[0151] In some cases, the PMP may comprise a plant EV, or a segment, portion, or extract 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, 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. Various methods standard in the art (e.g., dynamic light scattering) can be used to measure the particle diameter of plant EVs, or segments, parts, or extracts thereof.

[0152] In one case, the PMP is 77 nm 2 ~3.2×10 6 nm 2 (For example, 77 to 100 nm 2 , 100~1000nm 2 , 1000~1×10 4 nm 2 , 1×10 4 ~1×10 5 nm 2 , 1×10 5 ~1×10 6 nm 2 , or 1 × 10 6 ~3.2×10 6 nm 2 In some cases, the PMP may comprise a plant EV, or a segment, part, or extract thereof, having an average surface area of ​​65 nm 3~5.3×10 8 nm 3 (For example, 65 to 100 nm 3 , 100~1000nm 3 , 1000~1×10 4 nm 3 , 1×10 4 ~1×10 5 nm 3 , 1×10 5 ~1×10 6 nm 3 , 1×10 6 ~1×10 7 nm 3 , 1×10 7 ~1×10 8 nm 3 , 1×10 8 ~5.3×10 8 nm 3 In some cases, the PMP may comprise a plant EV, or a segment, part, or extract thereof, having an average volume of at least 77 nm 2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1 × 10 4 nm 2 , at least 1 × 10 5 nm 2 , at least 1 × 10 6 nm 2 , or at least 2 × 10 6 nm 2 In some cases, the PMP may comprise a plant EV, or a segment, part, or extract thereof, having an average surface area of ​​at least 65 nm 3 (e.g., at least 65 nm 3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1 × 10 4 nm 3 , at least 1 × 10 5 nm 3 , at least 1 × 10 6 nm 3 , at least 1 × 10 7 nm 3, at least 1 × 10 8 nm 3 , at least 2 × 10 8 nm 3 , at least 3 × 10 8 nm 3 , at least 4 × 10 8 nm 3 , or at least 5 × 10 8 nm 3 The plant EVs, or segments, parts, or extracts thereof, may comprise a plant EV having an average volume of about 1000 mg / kg or more.

[0153] In some cases, PMPs may have the same size as plant EVs or segments, extracts, or portions thereof. Alternatively, PMPs may have a different size than the initial plant EVs from which they are generated. For example, PMPs may have a diameter of about 5-2000 nm in diameter. For example, PMPs may have a 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 550-600 nm, about 600-650 nm, about 650 The nanoparticles may have an average diameter of about 700 nm, about 700 to 750 nm, about 750 to 800 nm, about 800 to 850 nm, about 850 to 900 nm, about 900 to 950 nm, about 950 to 1000 nm, about 1000 to 1200 nm, about 1200 to 1400 nm, about 1400 to 1600 nm, about 1600 to 1800 nm, or about 1800 to 2000 nm. In some cases, PMPs may have 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, at least 1000 nm, at least 1200 nm, at least 1400 nm, at least 1600 nm, at least 1800 nm, or about 2000 nm. Various methods (e.g., dynamic light scattering) that are standard in the art can be used to measure the particle diameter of PMPs. In some cases, the size of PMPs is determined after loading with a different functional agent or other modifications to PMPs.

[0154] In one case, the PMP is 77 nm 2 ~1.3×10 7 nm 2 (For example, 77 to 100 nm 2 , 100~1000nm 2 , 1000~1×104 nm 2 , 1×10 4 ~1×10 5 nm 2 , 1×10 5 ~1×10 6 nm 2 , or 1 × 10 6 ~1.3×10 7 nm 2 In some cases, the PMP may have an average surface area of ​​65 nm 3 ~4.2×10 9 nm 3 (For example, 65 to 100 nm 3 , 100~1000nm 3 , 1000~1×10 4 nm 3 , 1×10 4 ~1×10 5 nm 3 , 1×10 5 ~1×10 6 nm 3 , 1×10 6 ~1×10 7 nm 3 , 1×10 7 ~1×10 8 nm 3 , 1×10 8 ~1×10 9 nm 3 , or 1 × 10 9 ~4.2×10 9 nm 3 In some cases, the PMP may have an average volume of at least 77 nm 2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1 × 10 4 nm 2 , at least 1 × 10 5 nm 2 , at least 1 × 10 6 nm 2 , or at least 1 × 10 7 nm 2 In some cases, the PMP has an average surface area of ​​at least 65 nm 3 (e.g., at least 65 nm3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1 × 10 4 nm 3 , at least 1 × 10 5 nm 3 , at least 1 × 10 6 nm 3 , at least 1 × 10 7 nm 3 , at least 1 × 10 8 nm 3 , at least 1 × 10 9 nm 3 , at least 2 × 10 9 nm 3 , at least 3 × 10 9 nm 3 , or at least 4 × 10 9 nm 3 ) average volume.

[0155] In some cases, the PMP may include an intact plant EV. Alternatively, the PMP may include a segment, portion, or extract of the total surface area of ​​a vesicle of a plant EV (e.g., a segment, portion, or extract that includes less than 100% (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 10%, 5%, or 1%) of the total surface area of ​​the vesicle). The segment, portion, or extract may be of any shape, such as a circumferential segment, a spherical segment (e.g., a hemisphere), a curved segment, a straight segment, or a flat segment. When the segment is a spherical segment of a vesicle, the spherical segment may represent one resulting from the division of a spherical vesicle along a pair of parallel lines or one resulting from the division of a spherical vesicle along a pair of non-parallel lines. Thus, the PMPs may contain intact plant EVs, plant EV segments, parts, or extracts, or a mixture of intact plant EVs and plant EV segments. Those skilled in the art will understand that the ratio of intact plant EVs to segmented plant EVs will depend on the particular isolation method used. For example, grinding or blending a plant, or parts thereof, may produce a PMP that contains a higher percentage of plant EV segments, parts, or extracts than non-destructive extraction methods such as vacuum infiltration.

[0156] When the PMP comprises a segment, part, or extract of a plant EV, the EV segment, part, or extract has an average surface area smaller than the average surface area of ​​an intact vesicle, e.g., 77 nm 2 , 100 nm 2 , 1000nm 2 , 1×10 4 nm 2 , 1×10 5 nm 2 , 1×10 6 nm 2 , or 3.2 × 10 6 nm 2 In some cases, the EV segments, portions, or extracts may have an average surface area of ​​less than 70 nm 2 , 60nm 2 , 50 nm2 , 40nm 2 , 30nm 2 , 20nm 2 , or 10 nm 2 In some cases, the PMPs have an average volume smaller than the average volume of an intact vesicle, e.g., 65 nm 3 , 100 nm 3 , 1000nm 3 , 1×10 4 nm3, 1×10 5 nm 3 , 1×10 6 nm 3 , 1×10 7 nm 3 , 1×10 8 nm 3 , or 5.3 × 10 8 nm 3 The plant EV, or a segment, part or extract thereof, may comprise:

[0157] Where a PMP comprises an extract of a plant EV, for example where a PMP comprises lipids extracted from a plant EV (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 the plant EV (e.g., with chloroform). A plurality of PMPs may comprise plant EV segments and / or plant EV extracted lipids or mixtures thereof.

[0158] C. How to generate PMP PMPs may be produced from plant EVs, or segments, parts or extracts thereof (e.g., lipid extracts) that occur naturally in a plant, or part thereof, including plant tissues or plant cells. An exemplary method for producing PMPs includes (a) providing an initial sample from a plant or part thereof, the plant or part thereof including EVs, and (b) isolating a crude PMP fraction from the initial sample, the crude PMP fraction having a reduced level of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the initial sample. The method may further include an additional step (c) including purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, the plurality of pure PMPs having a reduced level of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the crude EV fraction. Each production step is discussed in further detail below. Exemplary methods for isolating and purifying PMPs can be found, for example, in 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.

[0159] For example, a plurality of PMPs can be prepared by (a) providing an initial sample from a plant, or part thereof, the plant, or part thereof, comprising EVs; (b) isolating a crude PMP fraction from the initial sample, the crude PMP fraction comprising a reduced level (e.g., a level that is at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced) of at least one contaminant or undesirable component from the plant, or part thereof, as compared to the level in the initial sample. and (c) purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, the plurality of pure PMPs having a reduced level (e.g., at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced) of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the crude EV fraction.

[0160] The PMP provided herein can comprise plant EVs isolated from various plants, or segments, parts, or extracts thereof.PMP can be isolated from any genus of plant (vascular or non-vascular), including but not limited to angiosperms (monocotyledonous and dicotyledonous), gymnosperms, ferns, Selaginellaceae, horsetail, archaic scoparium, lycopodidae, algae (e.g., unicellular or multicellular, e.g., archaic plastids), or mosses.In certain cases, PMP can be produced from vascular plants, such as monocotyledonous or dicotyledonous plants or gymnosperms. For example, PMPs may be used to treat plant crops such as alfalfa, apple, Arabidopsis, banana, barley, canola, castor bean seed, chicory, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, yam, eucalyptus, fescue, flax, gladiolus, lilac, linseed, millet, muskmelon, mustard, oats, oil palm, rapeseed, papaya, peanut, pineapple, ornamental plants, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugar beet, sugarcane, sunflower, strawberry, tobacco, tomato, turfgrass, wheat, or lettuce. PMPs may be produced from fruits and nut trees such as celery, broccoli, cauliflower, cucurbits, apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel, vines such as grapes, kiwi, hops, shrubs such as raspberry, blackberry, gooseberry, and forest trees such as bramble, ash, pine, fir, maple, oak, chestnut, poplar, alfalfa, canola, castor seed, corn, cotton, crambe, flax, linseed, mustard, oil palm, rapeseed, peanut, potato, rice, safflower, sesame, soybean, sugar beet, sunflower, tobacco, tomato, or wheat. In some embodiments, PMPs may be produced from dragon fruit, kale, spinach, or strawberry.

[0161] PMPs may be produced using the entire plant (e.g., the entire rosette or the entire seedling), or alternatively from one or more plant parts (e.g., leaves, seeds, roots, fruits, vegetables, pollen, phloem sap, or xylem sap). For example, PMPs may be produced from shoot vegetative organs / structures (e.g., leaves, stems, or tubers), roots, floral and floral organs / structures (e.g., pollen, bracts, bracts, sepals, petals, stamens, carpels, anthers, or ovules), seeds (including embryos, endosperm, or seed coats), fruits (mature ovaries), sap (e.g., phloem or xylem sap), plant tissues (e.g., vascular tissue, crushed tissue, tumor tissue, etc.), and cells (e.g., single cells, protists, embryos, callus tissue, guard cells, egg cells, etc.), or their progeny. For example, the isolation step may include (a) providing a plant, or a part thereof, where the plant part is an Arabidopsis thaliana leaf. The plant may be at any stage of development. For example, the PMPs may be produced from seedlings, e.g., 1-week-old, 2-week-old, 3-week-old, 4-week-old, 5-week-old, 6-week-old, 7-week-old, or 8-week-old seedlings (e.g., Arabidopsis thaliana seedlings). Other exemplary PMPs may include PMPs produced from roots (e.g., ginger root), fruit juice (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem sap (e.g., Arabidopsis thaliana phloem sap), or xylem sap (e.g., tomato plant xylem sap). In some embodiments, the PMPs are produced from citrus fruits, e.g., grapefruit or lemon.

[0162] PMPs can be produced from plants, or parts thereof, by various methods. Any method that allows the release of the EV-containing apoplastic fraction of the plant, or the extracellular fraction (e.g., cell culture medium) that contains PMPs, including otherwise secreted EVs, is suitable for the present method. EVs can be separated from plants or parts of plants by either disruptive (e.g., grinding or mixing the plant, or any part of the plant) or non-disruptive (washing or vacuum infiltration of the plant, or any part of the plant) methods. For example, the plant, or parts thereof, can be vacuum infiltrated, crushed, mixed, or a combination thereof, to isolate EVs from the plant or part of the plant, thereby producing PMPs. For example, the isolating step may include (b) isolating a crude PMP fraction from an initial sample (e.g., a plant, a part of a plant, or a sample derived from a plant or a part of a plant), the crude PMP fraction having a reduced level of at least one contaminant or undesirable component from the plant or part thereof compared to the level in the initial sample, and the isolating step may include vacuum infiltrating the plant (e.g., with a vesicle isolation buffer) to release and collect the apoplastic fraction. Alternatively, the isolating step may include (b) grinding or blending the plant to release the EVs and thereby generate PMPs.

[0163] When isolating plant EVs and thereby generating PMPs, the PMPs can be separated or collected into a crude PMP fraction (e.g., an apoplastic fraction). For example, the separation step can include separating a plurality of PMPs into a crude PMP fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the PMP-containing fraction from bulky contaminants, including plant tissue fragments, plant cells, or plant cell organelles (e.g., nuclei or chloroplasts). Thus, the crude PMP fraction will have a reduced number of bulky contaminants, including, for example, plant tissue fragments, plant cells, or plant cell organelles (e.g., nuclei, mitochondria, or chloroplasts), compared to the initial sample from the source plant or plant part.

[0164] 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 density gradients (iodixanol or sucrose), molecular sieves, and / or other approaches to remove aggregated components (e.g., precipitation or size exclusion chromatography). The resulting pure PMP may 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), nuclei, cell wall components, organelles, or combinations thereof) compared to one or more fractions produced during a previous separation step, or compared to a pre-established threshold level, for example, a commercially available shipping specification. For example, pure PMPs may have reduced levels (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) of plant organelles or cell wall components compared to the levels in the initial sample. In some cases, pure PMPs are substantially free (e.g., have 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. Further examples of release and separation processes can be found in WO2021 / 041301, which is incorporated herein by reference in its entirety. PMP, for example, 1 x 10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 5×10 10 , 1×10 11 , 2×10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11, 7×10 11 , 8×10 11 , 9×10 11 , 1×10 12 , 2×10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1×10 13 , or 1 × 10 13 It may be at a concentration of PMP / mL.

[0165] For example, protein aggregates may be removed from the isolated PMPs. For example, the isolated PMP solution may be taken through a range of pH (e.g., measured using a pH probe) to precipitate protein aggregates in the solution. The pH may be adjusted, for example, to pH 3, pH 5, pH 7, pH 9, or pH 11, for example, by the addition of sodium hydroxide or hydrochloric acid. Once the solution is at the specified pH, it may be filtered to remove particles. Alternatively, the isolated PMP solution may be aggregated using the addition of a charged polymer, such as Polymin-P or Plastol 2640. Briefly, Polymin-P or Plastol 2640 is added to the solution and mixed with an impeller. The solution may then be filtered to remove particles. Alternatively, the aggregates may be solubilized by increasing the salt concentration. For example, NaCl may be added to the isolated PMP solution, for example, until 1 mol / L. The solution may then be filtered to isolate the PMPs. Alternatively, the aggregates may be solubilized by increasing the temperature. For example, the isolated PMPs can be heated under mixing for 5 minutes until the solution reaches a uniform temperature, for example, 50°C. The PMP mixture can then be filtered to isolate the PMPs. Alternatively, soluble contaminants from the PMP solution can be separated by a size-exclusion chromatography column according to standard procedures, with the PMPs eluting in the first fraction, while proteins and ribonucleoproteins and some lipoproteins are eluted later. The efficiency of protein aggregate removal can be determined by measuring and comparing the protein concentration before and after removal of protein aggregates via BCA / Bradford protein quantification. In some embodiments, protein aggregates are removed before the exogenous peptide, polypeptide, or protein is encapsulated by the PMP. In other embodiments, protein aggregates are removed after the exogenous peptide, polypeptide, or protein is encapsulated by the PMP.

[0166] 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 at any step of the production process. PMPs can be characterized by various analytical methods to estimate PMP yield, PMP concentration, PMP purity, PMP composition, or PMP size. PMPs can be evaluated by a number of methods known in the art that allow visualization, quantification, or qualitative characterization (e.g., composition identification) of PMPs, such as microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectroscopy (e.g., Fourier transform infrared analysis), or mass spectrometry (protein and lipid analysis). In certain cases, methods (e.g., mass spectrometry) may be used to identify plant EV markers present on PMPs, such as the markers disclosed in the appendix disclosed in WO2021 / 041301, the entirety of which is incorporated herein by reference. To aid in the analysis and characterization of PMP fractions, PMPs can be further labeled or stained. For example, PMPs can be stained using 3,3'-dihexyloxacarbocyanine iodide (DIOC6), a fluorescent lipophilic dye, PKH67 (Sigma Aldrich), Alexa Fluor® 488 (Thermo Fisher Scientific), or DyLight™ 800 (Thermo Fisher). In the absence of sophisticated forms of nanoparticle tracking, this relatively simple approach can be used to quantify total membrane content and indirectly measure the concentration of PMPs (Rutter and Innes, Plant Physiol. 173(1):728-741, 2017; Rutter et al, Bio. Protoc. 7(17):e2533, 2017). For more precise measurements and to assess the size distribution of PMPs, nanoparticle tracking, nanoflow cytometry, or tunable resistive pulse sensing can be used.

[0167] During the production process, the PMPs can be optionally prepared such that the PMPs are at an increased concentration (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or greater than 100-fold) compared to the EV levels in the control or initial sample. Isolated PMPs can constitute from about 0.1% to about 100% of the PMP composition, such as any one 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 comprises at least 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more PMP, for example, as measured by weight / volume, percentage of PMP protein composition, and / or percentage of lipid composition (e.g., by measuring fluorescently labeled lipids). In some cases, the concentrated agent is used as a commercial product, e.g., the end user may use a diluted agent having a substantially lower concentration of active ingredient. In some embodiments, the composition is formulated as a PMP concentrated formulation, e.g., an ultra-low volume concentrated formulation. In some embodiments, the PMP in the composition is at a concentration effective to increase the fitness of an organism, e.g., a plant, an animal, an insect, a bacterium, or a fungus. In other embodiments, the PMP in the composition is at a concentration effective to decrease the fitness of an organism, e.g., a plant, an animal, an insect, a bacterium, or a fungus.

[0168] PMPs can be produced from various plants, or parts thereof (e.g., leaf apoplast, seed apoplast, roots, fruits, vegetables, pollen, flax, or wood kernels). For example, PMPs can be released from the apoplast of a plant, such as the leaf apoplast (e.g., the leaf apoplast of Arabidopsis thaliana) or the seed apoplast (e.g., the seed apoplast of sunflower). Other exemplary PMPs are produced from roots (e.g., ginger root), fruit juice (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem sap (e.g., phloem sap of Arabidopsis thaliana), xylem sap (e.g., xylem sap of tomato plants), or cell culture supernatant (e.g., BY2 tobacco cell culture supernatant). WO2021 / 041301, which is incorporated by reference in its entirety, further demonstrates the production of PMPs from these various plant sources.

[0169] PMPs can be produced and purified by a variety of methods, for example, by ultracentrifugation and / or methods to remove aggregated contaminants, for example, by using density gradients (iodixanol or sucrose) in conjunction with precipitation or molecular sieve chromatography. Further description of the production, purification, and characterization of PMPs can be found in WO2021 / 041301, which is incorporated by reference in its entirety.

[0170] In some cases, the PMPs of the compositions and methods can be isolated from a plant, or part thereof, and used without further modification to the PMP. In other cases, the PMPs can be modified prior to use, as further outlined herein.

[0171] D. Plant EV markers PMPs may have a wide range of markers that identify plant EVs and / or PMPs generated from plant EVs, including segments, portions, or extracts thereof. As used herein, the term "plant EV marker" refers to a component that is naturally associated with a plant and is incorporated into or on a plant EV in a plant body, 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 Physiol. 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, J of Exp. Biol. 68(20): 5485-5496, 2017, each of which is incorporated herein by reference. Further examples of plant EV markers are listed in the appendix disclosed in WO2021 / 041301, which is incorporated herein by reference in its entirety, and are further outlined herein.

[0172] Plant EV markers may include plant lipids. Examples of plant lipid markers that may be found in PMPs include phytosterol, campesterol, β-sitosterol, stigmasterol, avenasterol, glycosyl inositol phosphorylceramide (GIPC), glycolipids (e.g., monogalactosyl diacylglycerol (MGDG) or digalactosyl diacylglycerol (DGDG)), or combinations thereof. For example, PMPs may include GIPC, which represents a 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 infection), such as phosphatidic acid (PA) or phosphatidylinositol-4-phosphate (PI4P).

[0173] Alternatively, the plant EV marker may comprise a plant protein. In some cases, the protein plant EV marker may be an antimicrobial protein that is naturally produced by plants, including defense proteins that plants secrete in response to abiotic or biotic stress factors (e.g., bacterial or fungal infection). Plant pathogen defense proteins include soluble N-ethylmalemide sensitive factor-associated protein receptor protein (SNARE) proteins (e.g., syntaxin-121 (SYP121, GenBank Accession Number: NP_187788.1 or NP_974288.1), penitration 1 (PEN1, GenBank Accession Number: NP_567462.1)) or ABC transporter penitration 3 (PEN3, GenBank Accession Number: 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 Number: NP_193719.1), calcium-dependent lipid-binding proteins, or lectins (e.g., jacalin-related lectins, e.g., sunflower jacalin (Helja, GenBank: AHZ86978.1). For example, an RNA-binding protein may be glycine-rich RNA-binding protein-7 (GRP7, GenBank Accession Number: NP_179760.1). Additionally, proteins that regulate plasmodesmata function can, in some cases, be found in plant EVs, such as Synapse-Totgamin. A (GenBank Accession Number: NP_565495.1). In some cases, the plant EV marker may include a protein involved in lipid metabolism, such as phospholipase C or phospholipase D. In some cases, the plant protein EV marker is a cellular transport protein in plants. In certain instances where the plant EV marker is a protein, the protein marker may lack a signal peptide typically associated with a secreted protein.Non-conventional secreted proteins appear to share several common features, such as (i) the lack of a leader sequence, (ii) the absence of ER or Golgi apparatus-specific PTMs, and / or (iii) secretion unaffected by Brefeldin A, which blocks the classical ER / Golgi-dependent secretory pathway. Those skilled in the art can use various tools freely accessible to the public (e.g., CresignomeP database, SUBA3 (SUB subcellular localization database of Arabidopsis proteins)) to evaluate proteins for signal sequences, or lack thereof.

[0174] When 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 to a plant EV marker, such as any of the plant EV markers listed in the appendix disclosed in WO2021 / 041301, which is incorporated 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 to PEN1 from Arabidopsis thaliana (GenBank Accession Number: NP_567462.1).

[0175] In some cases, the plant EV marker comprises a nucleic acid encoded in the plant, such as a plant RNA, a plant DNA, or a plant PNA. For example, the PMP may comprise a dsRNA, an mRNA, a viral RNA, a microRNA (miRNA), or a small interfering RNA (siRNA) encoded by the plant. In some cases, the nucleic acid may be associated with a protein that facilitates long-term transport of RNA in the plant, as discussed herein. In some cases, the nucleic acid plant EV marker may be involved in host-induced gene silencing (HIGS), which is a process by which a plant silences a foreign transcript of a plant pest (e.g., a pathogen such as a fungus). For example, the nucleic acid may be a nucleic acid that silences a bacterial or fungal gene. In some cases, the nucleic acid may be a microRNA, such as miR159 or miR166, that targets a gene of a fungal pathogen (e.g., Verticillium dahliae). In some cases, the protein may be one involved in carrying plant defense compounds, such as proteins involved in glucosinolate (GSL) transport and metabolism, including glucosinolate transporter-1-1 (GTR1, GenBank Accession Number: NP_566896.2), glucosinolate transporter-2 (GTR2, NP_201074.1), or epithio-specific regulator 1 (ESM1, NP_188037.1).

[0176] When 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 to a plant EV marker, such as, for example, those encoding the plant EV markers listed in the appendix disclosed in WO2021 / 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 to miR159 or miR166.

[0177] In some cases, plant EV markers include compounds produced by plants. For example, the compounds may be defense compounds produced in response to abiotic or biotic stress factors, such as secondary metabolites. One such secondary metabolite found in PMPs is glucosinolates (GSLs), which are nitrogen- and sulfur-containing secondary metabolites found primarily in cruciferous plants. Other secondary metabolites may include allelochemicals.

[0178] In some cases, PMPs may also be identified as being produced from plant EVs based on the lack of certain markers (e.g., lipids, polypeptides, or polynucleotides) that are not typically produced by plants but are generally associated with other organisms (e.g., markers of animal EVs, bacterial EVs, or fungal EVs). For example, in some cases, PMPs lack lipids typically found in animal EVs, bacterial EVs, or fungal EVs. In some cases, PMPs lack lipids typical of animal EVs (e.g., sphingomyelin). In some cases, PMPs do not contain lipids typical of bacterial EVs or bacterial membranes (e.g., LPS). In some cases, PMPs lack lipids typical of fungal membranes (e.g., ergosterol).

[0179] Plant EV markers can be identified using any approach known in the art that allows for the identification of 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 comprise a detectable amount, e.g., a predetermined threshold amount, of a plant EV marker described herein.

[0180] E. Exogenous lipids In some embodiments, composite lipid particles not only include lipids extracted from one or more plant sources, but also contain two or more exogenous lipids.

[0181] In some embodiments, the PMP is modified to contain two or more exogenous lipids.

[0182] The exogenous lipid may be a cell-penetrating agent, may increase the delivery of peptide, polypeptide or protein by complex lipid formulations to cells, and / or may increase the loading of peptide, polypeptide or protein (e.g., loading efficiency or loading capacity). In some embodiments, the exogenous lipid may be a stabilizing lipid. In some embodiments, the exogenous lipid may be a structured lipid. Exemplary exogenous lipids include sterols and PEGylated lipids.

[0183] In some embodiments, the complex lipid particles include other components (eg, lipids, eg, sterols, eg, cholesterol, or small molecules).

[0184] In some embodiments, the PMP may be modified with other components (e.g., lipids, e.g., sterols, e.g., cholesterol, or small molecules) to further alter the functional and structural characteristics of the PMP. For example, the PMP may be further modified with a stabilizing molecule that increases the stability of the PMP (e.g., stable at room temperature for at least one day and / or stable at 4°C for at least one week).

[0185] In some embodiments, the complex lipid particles further comprise a sterol, such as sitosterol, sitostanol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., ovine cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or an analog of any sterol (e.g., a glycoside, ester, or peptide).

[0186] In some embodiments, the PMP is modified with a sterol, such as sitosterol, sitostanol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., ovine cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or an analog of any sterol (e.g., a glycoside, ester, or peptide).

[0187] In some cases, the exogenous sterol is added to the preparation prior to a mixing step, such as step (b), e.g., mixed with the extracted bacterial lipids prior to step (b). The exogenous sterol may be added in an amount of, e.g., 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 preparation.

[0188] In some embodiments, the concentration of sterol in the composite lipid particle is in the range of about 5-60% w / w, e.g., about 5-50% w / w, about 5-40% w / w, about 5-30% w / w, about 5-20% w / w, about 5-15% w / w, about 0.5-15% w / w, about 5-8% w / w, or about 6-7% w / w, based on the amount of total lipid in the composite lipid particle. In some embodiments, the sterol is in the range of about 15-20% w / w, about 20-30% w / w, about 30-40% w / w, about 40-50% w / w, or about 50-60% w / w, based on the amount of total lipid in the composite lipid particle.

[0189] In some embodiments, the concentration of the PEG-lipid conjugate is in the range of about 0.5-5% w / w, about 0.5-3.5% w / w, about 1-3.5% w / w, about 0.5-3% w / w, about 1-3% w / w, about 0.5-2.5% w / w, about 1-2.5% w / w, about 1.5-2.5% w / w, or about 2-2.5% w / w based on the amount of total lipid in the composite lipid particle. In some embodiments, the concentration of the PEG-lipid conjugate is in the range of about 0.5-15% w / w, about 1-15% w / w, about 2-5% w / w, about 5-8% w / w, about 8-12% w / w, or about 12-15% w / w based on the amount of total lipid in the composite lipid particle.

[0190] In some embodiments, the sterol is cholesterol or sitosterol. In some cases, the composite lipid particle or modified PMP comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% sterol (e.g., cholesterol or sitosterol), for example, 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, or 50%-60% molar ratio of sterol. In some embodiments, the composite lipid particle or modified PMP comprises about 35%-50% sterol (e.g., cholesterol or sitosterol), for example, about 36%, 38.5%, 42.5%, or 46.5% molar ratio of sterol. In some embodiments, the composite lipid particles or modified PMPs comprise a molar ratio of sterol of about 20% to 40%.

[0191] In some embodiments, a PMP that is modified with a sterol has altered stability (e.g., increased stability) compared to a PMP that is not modified with a sterol, hi some embodiments, a PMP that is modified with a sterol has a faster fusion rate with the membrane of a target cell compared to a PMP that is not modified with a sterol.

[0192] In some cases, the composite lipid particle or modified PMP comprises an exogenous lipid and an exogenous sterol.

[0193] In some embodiments, the composite lipid particles comprise PEGylated lipids.

[0194] In some embodiments, the PMP is modified with a PEGylated lipid.

[0195] The length of the polyethylene glycol (PEG) can vary from 1 kDa to 10 kDa, and in some aspects, PEGs with a length of 2 kDa are used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k.

[0196] In some cases, the composite lipid particle or modified PMP is 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%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 10%, 20%, 30%, 40%, 50%, or greater than 50% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., 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% molar ratio of PEGylated lipid. In some embodiments, the composite lipid particle or modified PMP comprises about 0.1% to 10% molar ratio of PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., about 1% to 3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid.

[0197] In some embodiments, PMPs modified with PEGylated lipids have altered stability (e.g., increased stability) compared to PMPs not modified with PEGylated lipids, hi some embodiments, PMPs modified with PEGylated lipids have altered particle size compared to PMPs not modified with PEGylated lipids.

[0198] In some embodiments, composite lipid particles or modified PMPs containing PEGylated lipids are less likely to be phagocytosed than those that do not contain PEGylated lipids.

[0199] The addition of PEGylated lipids may also affect stability in the GI tract and enhance particle translocation through mucus. PEG may be used as a method of attaching targeting moieties.

[0200] The cellular uptake of complex lipid particles or modified PMP can be measured by various methods known in the art.For example, complex lipid particles or modified PMP, or its components, can be labeled with a marker (e.g., fluorescent marker) that can be detected in isolated cells to confirm uptake.

[0201] The composite lipid particles contain less than 50 mol% of ionized lipids (e.g., ionized lipids exogenous to one or more plant sources). For example, the composite lipid particles contain less than 45 mol%, less than 40 mol%, less than 35 mol%, less than 30 mol%, less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, less than 9 mol%, less than 8 mol%, less than 7 mol%, less than 6 mol%, less than 5 mol%, less than 4 mol%, less than 3 mol%, less than 2 mol%, less than 1 mol%, less than 0.5 mol%, less than 0.1 mol% of ionized lipids (e.g., ionized lipids exogenous to one or more plant sources), or essentially no ionized lipids (e.g., ionized lipids exogenous to one or more plant sources). In some embodiments, the composite lipid particles contain less than 20 mol% of exogenous ionized lipids. In some embodiments, the composite lipid particles contain less than 5 mol% of exogenous ionized lipids.

[0202] In some embodiments, the complex lipid formulations provided herein comprise two or more different types of complex lipid particles, e.g., complex lipid particles derived from two or more different plant sources, and / or complex lipid particles comprising different species and / or different ratios of exogenous lipids, such as sterols and / or PEGylated lipids.

[0203] In some embodiments, the modified PMP formulations provided herein include two or more different modified PMPs, e.g., modified PMPs derived from different unmodified PMPs (e.g., unmodified PMPs from two or more different plant sources), and / or modified PMPs containing different species and / or different ratios of exogenous lipids, such as sterols and / or PEGylated lipids.

[0204] In some cases, the organic solvent in which lipid membrane is dissolved is chloroform, ethanol, or dimethylformamide:methanol (DMF:MeOH).Alternatively, the organic solvent or solvent combination may be, for example, acetonitrile, acetone, chloroform, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethylsulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethylsulfoxide:methanol, or dimethylformamide:methanol.

[0205] F. Pharmaceutical Preparations For example, the present invention includes a complex lipid formulation or modified PMP formulation that can be formulated into a pharmaceutical composition for administration to animals, such as 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 the dosage, the pharmaceutical composition of the method described herein is formulated into a suitable pharmaceutical composition to allow easy delivery.A single dose can be in a unit dosage form as needed.

[0206] The complex lipid formulation or modified PMP formulation may be formulated, for example, for oral, enteral, intravenous (e.g., injection or infusion), or subcutaneous administration to an animal (e.g., a human). For injectable formulations, a variety of effective pharmaceutical carriers are known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 22 nd ed.,(2012) and ASHP Handbook on Injectable Drugs,18 th ed., (2014).

[0207] The pharma- ceutically acceptable carriers and excipients in the compositions are non-toxic to recipients at the dosages 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, octadecyldimethylbenzylammonium 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 compositions may be formulated according to conventional pharmaceutical practice.The concentration of the compound in the formulation will vary depending on a number of factors, including the dosage of the effective agent to be administered (e.g., the exogenous peptide, polypeptide or protein encapsulated by the complex lipid formulation or modified PMP) and the route of administration.

[0208] For oral administration to animals, the complex lipid formulation or modified PMP formulation can be prepared in the form of oral preparation.The preparation for oral use can include tablets, caplets, capsules, syrups, or oral liquid dosage forms that contain active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may 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 and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginates, or alginic acid), binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), as well as lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharma- ceutically acceptable excipients may 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 oil medium). The compositions disclosed herein may also further include immediate release, extended release, or sustained release formulations.

[0209] For parenteral administration to animals, the complex lipid formulation or modified PMP composition may be formulated in the form of a liquid solution or suspension and administered by parenteral routes of administration (e.g., topical, subcutaneous, intravenous, or intramuscular). The pharmaceutical composition may be formulated for injection or infusion. The pharmaceutical composition for parenteral administration may be formulated using a sterile solution or any pharma- ceutical acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, or cell culture media (e.g., Dulbecco's modified Eagle's medium (DMEM), alpha modified Eagle's medium (alpha-MEM), and F-12 medium). Formulation methods are known in the art, see, for example, Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd ed.) Taylor&Francis Group, CRC Press (2009).

[0210] III. Exogenous Peptides, Polypeptides, or Proteins The present invention includes a composite lipid formulation or modified PMP formulation in which the composite lipid particle or modified PMP encapsulates an exogenous peptide, polypeptide, or protein. The exogenous peptide, polypeptide, or protein may be enclosed within the composite lipid particle or modified PMP, e.g., may be located within a lipid membrane structure, and may be separated from the surrounding material or solution by both leaves of the lipid bilayer, e.g.,. In some embodiments, the encapsulated exogenous peptide, polypeptide, or protein may interact or associate with the lipid membrane inside the composite lipid particle or modified PMP. In some embodiments, the encapsulated exogenous peptide, polypeptide, or protein may interact or associate with the lipid membrane outside the composite lipid particle or modified PMP. The exogenous peptide, polypeptide, or protein may, in some cases, be intercalated with the lipid membrane structure. In some cases, the exogenous peptide, polypeptide, or protein has an extraluminal portion. In some cases, the exogenous peptide, polypeptide, or protein is conjugated to the outer surface of the lipid membrane structure, e.g., using click chemistry.

[0211] The exogenous peptide, polypeptide, or protein may be a peptide, polypeptide, or protein that is not naturally present in plant EVs. Alternatively, the exogenous peptide, polypeptide, or protein may be naturally present in plant EVs, but is encapsulated in the complex lipid particle or modified PMP in an amount not found in naturally occurring plant extracellular vesicles. The exogenous peptide, polypeptide, or protein may, in some cases, be naturally present in the plant from which the plant lipids are extracted or the plant from which the PMP is derived. In other cases, the exogenous peptide, polypeptide, or protein is not naturally present in the plant from which the plant lipids are extracted or the plant from which the PMP is derived. The exogenous polypeptide may be artificially expressed in the plant from which the plant lipids are extracted or the plant from which the PMP is derived, for example, a heterologous polypeptide. The exogenous peptide, polypeptide, or protein may be derived from another organism. In some embodiments, the exogenous peptide, polypeptide, or protein is loaded into the complex lipid particle or modified PMP using, for example, one or more of sonication, electroporation, lipid extraction, and lipid extrusion.

[0212] The peptides, polypeptides, or proteins included herein may include naturally occurring or recombinantly produced variants. In some cases, they may be functional fragments or variants thereof (e.g., enzymatically active fragments or variants thereof). For example, the peptides, polypeptides, or proteins may be functionally active variants of any of the peptides, polypeptides, or proteins described herein that 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% identity to the sequences described herein or the sequences of naturally occurring peptides, polypeptides, or proteins, for example, over a specific region or over the entire sequence. In some cases, a peptide, polypeptide, or protein may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a subject peptide, polypeptide, or protein.

[0213] The peptides, polypeptides, or proteins described herein may be formulated in a composition for any of the uses described herein. The compositions disclosed herein may include any number or type (e.g., class) of peptides, polypeptides, or proteins, such as at least about one of 1, 2, 3, 4, 5, 10, 15, 20, or more. The appropriate concentration of each peptide, polypeptide, or protein in the composition depends on factors such as the efficacy, stability of the peptide, polypeptide, or protein, the number of distinct species in the composition, and the method of application of the formulation. In some cases, each peptide, polypeptide, or protein in a liquid composition is about 0.1 ng / mL to about 100 mg / mL. In some cases, each peptide, polypeptide, or protein in a solid composition is about 0.1 ng / g to about 100 mg / g.

[0214] Methods for making peptides, polypeptides, or proteins are routine in the art. See generally, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0215] The method of producing peptides, polypeptides, or proteins involves expression in plant cells, but recombinant proteins can also be produced using insect cells, yeast, bacteria, mammalian cells, or other cells under the control of a suitable promoter. Mammalian expression vectors can include non-transcribed elements such as origin of replication, suitable promoters and enhancers, and other 5' or 3' adjacent non-transcribed sequences, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 virus genome, such as SV40 origin, early promoter, enhancer, splice, and polyadenylation sites, can be used to provide other genetic elements required for the expression of heterologous DNA sequences. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0216] A variety of mammalian cell culture systems can be used to express and produce 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 the production of protein therapeutics is described, for example, in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances 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 may be chemically synthesized.

[0217] In some cases, the complex lipid formulation or modified PMP comprises an antibody or its antigen-binding fragment. For example, the agent described herein may be an antibody that blocks or enhances the activity and / or function of a pathogen component. The antibody may act as an antagonist or agonist of a polypeptide (e.g., an enzyme or a cellular receptor) in a pathogen. The production and use of antibodies against target antigens in a pathogen are known in the art. For antibody engineering, the use of degenerate oligonucleotides, recombinant antibody production methods including 5'-RACE, phage display, and mutagenesis, antibody testing and characterization, antibody pharmacokinetics and pharmacodynamics, antibody purification and storage, and screening and labeling techniques, 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.

[0218] Exogenous peptide, polypeptide or protein can be released from complex lipid formulation or modified PMP in target cell.In some embodiments, exogenous peptide, polypeptide or protein exerts activity in the cytoplasm of target cell or the nucleus of target cell.Exogenous peptide, polypeptide or protein can be translocated to the nucleus of target cell.

[0219] In some embodiments, cellular uptake of an exogenous peptide, polypeptide, or protein encapsulated by a complex lipid particle or modified PMP is increased compared to uptake of an exogenous peptide, polypeptide, or protein that is not encapsulated by a complex lipid particle or modified PMP.

[0220] In some embodiments, the efficacy of an exogenous polypeptide or protein encapsulated by a complex lipid particle or modified PMP is increased compared to the efficacy of an exogenous peptide, polypeptide, or protein that is not encapsulated by a complex lipid particle or modified PMP.

[0221] A. Therapeutic Agents The exogenous peptide, polypeptide, or protein may be a therapeutic agent, e.g., an agent used to prevent or treat a condition or disease. In some embodiments, the disease is cancer, an autoimmune condition, or a metabolic disorder.

[0222] In some examples, the therapeutic agent is a peptide (e.g., a naturally occurring, recombinant, or synthetic peptide) or a protein (e.g., a naturally occurring, recombinant, or synthetic protein). In some examples, the protein is a fusion protein.

[0223] In some cases, the peptide, polypeptide, or protein is endogenous to the organism (e.g., mammal) to which the complex lipid formulation or modified PMP is delivered. In other cases, the peptide, polypeptide, or protein is not endogenous to the organism.

[0224] In some examples, the therapeutic agent is an antibody (e.g., a monoclonal antibody, e.g., 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 bispecific antibodies), a nanobody, a conjugated antibody, or an antibody-related polypeptide.

[0225] In some examples, the therapeutic agent is an antibacterial, antimicrobial, antifungal, analgesic, antiparasitic, or antiviral polypeptide.

[0226] In some examples, the therapeutic agent is an allergenic, allergen, or antigen.

[0227] In some examples, the therapeutic agent is a vaccine (eg, a conjugate vaccine, an inactivated vaccine, or a live attenuated vaccine).

[0228] In some examples, the therapeutic agent is an enzyme, such as a metabolic recombinase, a helicase, an integrase, an RNAse, a DNAse, a ubiquitinating protein, hi some examples, the enzyme is a recombinase.

[0229] 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.

[0230] In some examples, the therapeutic agent is any one of a cytokine, a hormone, a signal transduction ligand, a transcription factor, a receptor, a receptor antagonist, a receptor agonist, a blocking or neutralizing polypeptide, a riboprotein, or a chaperone.

[0231] 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).

[0232] In some examples, the therapeutic agent is one of an aptamer, a blood derivative, a cellular therapy, or an immunotherapy (eg, a cellular immunotherapy).

[0233] In some embodiments, the therapeutic agent is a protein or peptide therapeutic agent with enzymatic, regulatory, or targeting activity, such as a protein or peptide therapeutic agent with activity affecting one or more of endocrine and growth control, metabolic enzyme deficiencies, hematopoiesis, hemostasis and thrombosis, gastrointestinal disorders, pulmonary disorders, immune deficiencies and / or immune regulation, fertility, aging (e.g., anti-aging activity), autophagy regulation, epigenetic regulation, oncology, or infectious disease (e.g., an antimicrobial peptide, an antifungal agent, or an antiviral agent).

[0234] In some embodiments, the therapeutic agent is a protein vaccine, eg, a vaccine for use in protecting against harmful foreign agents, treating autoimmune diseases, or treating cancer (eg, neoantigens).

[0235] In some instances, the peptides, polypeptides, or proteins are globular, fibrous, or disordered.

[0236] In some examples, the peptide, polypeptide, or protein has a size of less than 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD, for example, 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).

[0237] In some examples, a peptide, polypeptide, or protein has an overall charge that is positive, negative, or neutral. A peptide, polypeptide, or protein may be modified such that the overall charge is altered, for example, by adding one or more 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, e.g., one or more (e.g., 1-10 or 5-10) positively or negatively charged amino acids.

[0238] 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 a patient an effective amount of a composition comprising a plurality of composite lipid particles or modified PMPs, and 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 the blood glucose of the subject. In some embodiments, the therapeutic agent is insulin. In some embodiments, the therapeutic agent is exenatide, semaglutide, or tirzepatide.

[0239] 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. [Table 1] TIFF2025515189000002.tif230169 TIFF2025515189000003.tif225169 TIFF2025515189000004.tif230169 TIFF2025515189000005.tif214169 TIFF2025515189000006.tif227169 TIFF2025515189000007.tif230169 TIFF2025515189000008.tif220169 TIFF2025515189000009.tif230169 TIFF2025515189000010.tif230169 TIFF2025515189000011.tif231169 TIFF2025515189000012.tif230169 TIFF2025515189000013.tif231169 TIFF2025515189000014.tif230169 TIFF2025515189000015.tif230169 TIFF2025515189000016.tif210169 TIFF2025515189000017.tif219169 TIFF2025515189000018.tif230169 TIFF2025515189000019.tif230169 TIFF2025515189000020.tif229169 TIFF2025515189000021.tif229169 TIFF2025515189000022.tif235169 TIFF2025515189000023.tif229169 TIFF2025515189000024.tif235169 TIFF2025515189000025.tif231169 TIFF2025515189000026.tif81169

Table 2

Table 3

Table 4

[0240] B. Enzymes The exogenous peptide, polypeptide, or protein may be an enzyme, for example, an enzyme that catalyzes a biological reaction used in the prevention or treatment of a condition or disease, the prevention or treatment of a pathogen infection, the diagnosis of a disease, or the diagnosis of a disease or condition.

[0241] The enzyme may be a recombinase enzyme, such as a Cre recombinase enzyme. In some embodiments, the Cre recombinase enzyme is delivered to cells containing a Cre reporter construct by a complex lipid formulation or modified PMP.

[0242] The enzyme may be an editing enzyme, for example, a gene editing enzyme. In some embodiments, the gene editing enzyme is, for example, a component of a CRISPR-Cas system (e.g., Cas9 enzyme), a TALEN, or a zinc finger nuclease.

[0243] C. Pathogen Control Agents The exogenous peptide, polypeptide, or protein may be a peptide, polypeptide, or protein that is a pathogen control agent, for example, an antibacterial, antifungal, insecticidal, pesticide, antiparasitic, or virucidal agent used in human health. In some cases, the complex lipid formulation or modified PMP formulation described herein includes a peptide, polypeptide, or protein, or a functional fragment or derivative thereof, that targets a pathogen pathway. The complex lipid formulation or modified PMP formulation that includes the peptide, polypeptide, or protein described herein can be administered to a pathogen in an amount and for a time sufficient to (a) reach a target level (e.g., a predetermined level or threshold level) of peptide, polypeptide, or protein concentration, and (b) reduce or eliminate the pathogen. In some cases, complex lipid formulations or modified PMP formulations comprising the peptides, polypeptides, or proteins described herein can be administered to an animal having or at risk of infection by a pathogen in an amount and for a 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 the pathogen. The peptides, polypeptides, or proteins described herein can be formulated in complex lipid formulations or modified PMP formulations, or in some cases associated with complex lipid formulations or modified PMPs thereof, for any of the methods described herein.

[0244] Examples of peptides, polypeptides, or proteins that may be used herein include enzymes (e.g., metabolic recombinases, helicases, integrases, RNAses, DNAses, or ubiquitinating 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.

[0245] The complex lipid formulations or modified PMP formulations described herein may include a bacteriocin. In some cases, the bacteriocin is 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 lantibiotics), colicins, microcins, and pyocins.

[0246] The complex lipid formulation or modified PMP formulation described herein may contain antimicrobial peptides (AMPs). Any AMP suitable for inhibiting microorganisms may be used. AMPs are a diverse group of molecules, which are divided into subgroups based on their amino acid composition and structure. AMPs may be derived from or produced by any organism that naturally produces AMPs, including the AMPs derived from plants (e.g., copsin), insects (e.g., mastrene, poneratoxin, cecropin, moricin, melittin), frogs (e.g., magain, dermaseptin, aurein), and mammals (e.g., cathelicidin, defensin and protegrin).

[0247] IV. METHODS FOR PRODUCING COMPLEX LIPID FORMULATIONS OR MODIFIED PMPS COMPRISING EXOGENOUS POLYPEPTIDES 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 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

[0248] and loading the complex lipid particles with an exogenous peptide, polypeptide, or protein, whereby the exogenous peptide, polypeptide, or protein is encapsulated by the complex lipid particles, thereby forming a complex lipid formulation.

[0249] Further description of general procedures and exemplary methods for producing composite lipid particles and encapsulating the composite lipid particles with exogenous peptides, polypeptides, or proteins can be found in Examples 1-2.

[0250] In another aspect, the disclosure generally features a method of producing a modified PMP that includes an exogenous peptide, polypeptide, or protein. Thus, the method includes (a) providing a solution that includes the exogenous peptide, polypeptide, or protein, and (b) loading the modified PMP with the exogenous peptide, polypeptide, or protein, whereby the exogenous peptide, polypeptide, or protein is encapsulated by the modified PMP.

[0251] Exogenous peptide, polypeptide or protein may be placed in a solution, for example, a phosphate buffered saline (PBS) solution. Exogenous peptide, polypeptide or protein may or may not be soluble in the solution. If peptide, polypeptide or protein is not soluble in the solution, the pH of the solution can be adjusted until the polypeptide is soluble in the solution. Insoluble peptide, polypeptide or protein is also useful for loading.

[0252] Loading of composite lipid particles or modified PMPs with exogenous peptides, polypeptides, or proteins may include or consist of sonication of a solution containing the exogenous peptide, polypeptide, or protein (e.g., a soluble or insoluble exogenous polypeptide) and a plurality of composite lipid particles or modified PMPs, for example, sonication according to the protocol described in Wang et al., Nature Comm., 4:1867, 2013, to induce dispersion of portions of the composite lipid particles or modified PMPs and the peptide, polypeptide, or protein into the composite lipid particles or modified PMPs.

[0253] Alternatively, loading of the composite lipid particles or modified PMPs with exogenous peptides, polypeptides, or proteins may comprise or consist of electroporation of a solution containing the exogenous peptide, polypeptide, or protein (e.g., a soluble or insoluble exogenous polypeptide) and a plurality of the composite lipid particles or modified PMPs, e.g., sonication according to the protocol described in Wahlgren et al., Nucl. Acids. Res., 40(17), e130, 2012.

[0254] Alternatively, small amounts of surfactants (e.g., saponin) can be added to increase loading of exogenous peptides, polypeptides, or proteins into composite lipid particles or modified PMPs, for example, as described in Fuhrmann et al., J Control Release., 205:35-44, 2015.

[0255] Loading of composite lipid particles or modified PMPs with exogenous peptides, polypeptides, or proteins may include or consist of lipid extraction and lipid extrusion. Briefly, plant lipids can be isolated by adding MeOH:CHCl3 (e.g., 3.75 mL of 2:1 (v / v) MeOH:CHCl3) to PMPs in PBS solution (e.g., 1 mL of PMPs in PBS) and vortexing the mixture. Then, CHCl3 (e.g., 1.25 mL) and ddH2O (e.g., 1.25 mL) are added sequentially and vortexed. The mixture is then centrifuged at 2,000 rpm for 10 minutes in a glass tube at 22°C to separate the mixture into two layers (aqueous and organic). The organic layer sample containing plant lipids is dried by heating under nitrogen (2 psi). To load peptides, isolated plant lipids are mixed with a peptide, polypeptide, or protein solution and passed through a lipid extruder, for example following the protocol of Haney et al., J Control Release, 207:18-30, 2015.

[0256] Plant lipids may also be isolated using methods to isolate additional plant lipid classes, such as glycosyl inositol phosphorylceramides (GIPCs), as described in Casas et al., Plant Physiology, 170:367-384, 2016. Briefly, to extract plant lipids, including GIPCs, chloroform:methanol:HCl (e.g., 3.5 mL of chloroform:methanol:HCl (200:100:1, v / v / v)) and butylated hydroxytoluene (e.g., 0.01% (w / v) butylated hydroxytoluene) are added to PMPs and incubated with PMPs. Next, NaCl (e.g., 2 mL of 0.9% (w / v) NaCl) is added and vortexed for 5 minutes. The sample is then centrifuged to induce the organic layer to aggregate at the bottom of the glass tube, and the organic layer is collected. The upper layer may be re-extracted with chloroform (e.g., 4 mL of pure chloroform) to isolate lipids. The organic phases are combined and dried. After drying, the aqueous phase is resuspended in water (e.g., 1 mL of pure water) and the GIPCs are back-extracted twice using butanol-1 (e.g., 1 mL of butanol-1). To load exogenous peptides, polypeptides, or proteins, the isolated plant lipid layer is mixed with a peptide, polypeptide, or protein solution and passed through a lipid extruder according to the protocol of Haney et al., J Control Release, 207:18-30, 2015. Alternatively, lipids may be extracted with methyl tertiary butyl ether (MTBE):methanol:water + butylated hydroxytoluene (BHT) or propan-2-ol:hexane:water.

[0257] In some embodiments, isolated GIPCs may be added to isolated plant lipids.

[0258] In some embodiments, loading of composite lipid particles or modified PMPs with exogenous peptides, polypeptides, or proteins involves sonication and lipid extrusion, as described above.

[0259] In some embodiments, exogenous peptides, polypeptides, or proteins may be pre-complexed (e.g., using protamine sulfate) or cationic lipids (e.g., DOTAP) may be added to facilitate encapsulation of negatively charged proteins.

[0260] Prior to use, the loaded composite lipid particles or loaded modified PMPs may be purified to remove peptides, polypeptides, or proteins that are not bound to or encapsulated in the composite lipid particles or modified PMPs. The loaded composite lipid particles or loaded modified PMPs may be characterized and their stability may be tested. The loading of exogenous peptides, polypeptides, or proteins may be quantified by methods known in the art for quantifying proteins. For example, the Pierce quantitative colorimetric peptide assay may be used on a small sample of loaded or unloaded composite lipid particles or modified PMPs, or Western blots using specific antibodies may be used to detect exogenous peptides, polypeptides, or proteins. Alternatively, the peptides, polypeptides, or proteins may be fluorescently labeled and fluorescence may be used to determine the concentration of the labeled exogenous peptides, polypeptides, or proteins in loaded or unloaded composite lipid particles or modified PMPs. Further description of the purification, characterization, stability, and loading of PMPs can be found in WO2021 / 041301, which is incorporated by reference in its entirety.

[0261] V. Treatment method The complex lipid formulations or modified PMP formulations described herein are useful in various therapeutic methods, particularly for preventing or treating a condition or disease, or for preventing or treating a pathogen infection in an animal. The method includes delivering the complex lipid formulations or modified PMP formulations described herein to an animal (e.g., a human).

[0262] Provided herein is a method for administering the complex lipid formulation or modified PMP formulation disclosed herein to animals.The method can be useful for preventing or treating a condition or disease, or for preventing pathogen infection in animals (e.g., humans).

[0263] For example, provided herein are methods of treating an animal having a fungal infection, the methods comprising 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, comprising an exogenous peptide, polypeptide, or protein that is a pathogen control agent, e.g., an antifungal agent. In some cases, the fungal infection is caused by Candida albicans. In some cases, the methods reduce or substantially eliminate the fungal infection.

[0264] In another aspect, provided herein is a method of treating an animal (e.g., a human) with a bacterial infection, the method comprising 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. In some cases, 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, the ... exogenous peptides, polypeptides, or proteins that are pathogen control agents, e.g., antimicrobial agents. In some cases, the bacteria is Streptococcus, Pneumococcus, Pseudomonas, Shigella, Salmonella, Campylobacter, or Escherichia. In some cases, the method reduces or substantially eliminates the bacterial infection. In some cases, the animal is a human, a veterinary animal, or a livestock animal.

[0265] The method is useful for treating infections in animals (e.g., caused by animal, e.g., human pathogens), which refers to administering treatment to an animal already suffering from a disease to improve or stabilize the animal's condition. This may involve reducing the colonization of pathogens in, on, or around the animal by one or more pathogens (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) compared to the starting amount, and / or may involve allowing a benefit to the individual (e.g., reducing colonization by an amount sufficient to resolve symptoms). In such cases, the treated infection may be manifested as a reduction in symptoms (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some cases, the treated infection is effective to increase the chances of survival of an individual (e.g., increase the chances of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or increase the overall survival of a population (e.g., increase the chances of survival by 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" the infection, which refers to a reduction in infection in an amount sufficient to provide a sustained resolution of symptoms in the animal (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0266] The method is useful for preventing infection (e.g., caused by an animal, e.g., human pathogen), which is useful for preventing an increase in colonization of one or more pathogens in, on, or around an animal in an amount sufficient to maintain the initial pathogen population (e.g., approximately the amount found in a healthy individual), prevent the onset of infection, and / or prevent symptoms or conditions associated with infection (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to an untreated animal). For example, prophylactic treatment may be administered to prevent fungal infection in individuals preparing for an invasive medical procedure (e.g., transplant, stem cell therapy, graft, prosthetics, undergoing long-term or frequent intravenous catheterization, or undergoing intensive care unit treatment), immunocompromised individuals (e.g., those with cancer, HIV / AIDS, or taking immunosuppressants), or individuals undergoing long-term antibiotic therapy.

[0267] The complex lipid formulation or modified PMP formulation may be formulated for administration or may be administered by any suitable method, including, for example, oral, enteral, intravenous, intramuscular, subcutaneous, intradermal, transdermal, intra-arterial, intraperitoneal, intracerebral, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal (including intracolonic), topical, intratumoral, peritoneal, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, topical, transdermal, intravitreal (e.g., by intravitreal injection), eye drop, inhalation (e.g., by nebulizer), injection, implantation, infusion, continuous infusion, localized perfusion directly bathing the target cells, catheter, lavage, in creams, lipid compositions. The compositions utilized in the methods described herein may also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).In some cases, the complex lipid formulation or modified PMP formulation is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally.Administration can be by any suitable route, for example, orally or by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or chronic.Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0268] The prevention or treatment of infections described herein (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the severity and course of the disease. Whether a therapeutic agent is administered for prophylactic or therapeutic purposes depends 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 over a series of treatments. For repeated administration over several days or more, depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs or the infection is no longer detectable. Such doses may be administered intermittently, for example, weekly or biweekly (e.g., such that the patient receives, for example, about 2 to about 20 doses of the complex lipid formulation or modified PMP formulation). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0269] In some cases, the amount of the complex lipid formulation or modified PMP formulation administered to an individual (e.g., a human) may be within the range of about 0.01 mg / kg to about 5 g / kg of the individual's body weight (e.g., about 0.01 mg / kg to 0.1 mg / kg, about 0.1 mg / kg to 1 mg / kg, about 1 mg / kg to 10 mg / kg, about 10 mg / kg to 100 mg / kg, about 100 mg / kg to 1 g / kg, or about 1 g / kg to 5 g / kg). 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 of the individual's body weight (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). The dose 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 additional therapeutic or pathogen control agents.The dose of the antibody administered in combination therapy can be reduced compared to single therapy.The progress of this therapy can be easily monitored by conventional techniques.

[0270] In one aspect, the disclosure features a method for treating diabetes, comprising administering to a subject in need thereof a composition comprising a plurality of composite lipid particles or a plurality of modified PMPs that encapsulate an effective amount of one or more exogenous peptides, polypeptides, or proteins.Administering the plurality of composite lipid particles or the plurality of modified PMPs reduces the blood glucose of the subject.In some embodiments, the exogenous peptide, polypeptide, or protein is insulin, exenatide, semaglutide, or tirzepatide.

[0271] VI. METHODS FOR TREATING PATHOGENS OR THEIR VECTORS The complex lipid formulation or modified PMP formulation and related methods described herein are useful for reducing the fitness of animal pathogens, thereby treating or preventing infection in animals, such as humans.Examples of animal pathogens, or vectors thereof, that can be treated using the composition or related methods are further described herein.

[0272] A. Fungi Complex lipid formulations or modified PMP formulations and related methods can be useful for reducing the fitness of fungi, for example, to prevent or treat fungal infection in animals, for example, humans.Included are methods of delivering complex lipid formulations or modified PMP formulations to fungi by contacting fungi with complex lipid formulations or modified PMP formulations.In addition, or alternatively, the methods include administering complex lipid formulations or modified PMP formulations to animals to prevent or treat fungal infection (e.g., caused by fungi as described herein) in animals at risk or in need thereof.

[0273] The complex lipid formulations or modified PMP formulations and related methods have been demonstrated to be effective against a wide variety of fungal organisms, including Ascomycota (Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp., Coccidioides immitis / Coccidioides posadasii, Candida albicans, Basidiomycota (Filobasidiella neoformans, Trichosporon spp., Microsporidia (Encephalitozoon cuniculi, Enterocytozoon bieneusi), Mucor (Mucor circinelloides, Rhizopus oryzae), and fungi (Fucorrhizobia cerevisiae, Fucoidans spp. ... The present invention is suitable for the treatment or prevention of fungal infections in animals, including infections caused by fungi belonging to the family Lichtheimia oryzae, Lichtheimia corymbifera, and the like.

[0274] In some cases, the fungal infection is caused by fungi belonging to the phylum Ascomycota, Basidiomycota, Chytridiomycota, Microsporidia, or Zygomycota. The fungal infection or overgrowth may be caused by one or more fungal species, such as Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida auris, Candida krusei, Saccharomyces cerevisiae, Malassezia globosa, Malassezia restricta, or Debaryomyces hansenii, Gibberella moniliformis, Alternaria brassicicola, Cryptococcus neoformans, or any of a number of other fungal species. Neoformans, Pneumocystis carinii, Pneumocystis jiroveci, P. murina, Pneumocystis oryctolagi, P. wakefieldiae, and Aspergillus clavatus. Fungal species may be considered pathogens or opportunistic pathogens.

[0275] In some cases, the fungal infection is caused by a fungus of the Candida genus (i.e., a Candida infection). For example, the Candida infection can be caused by a fungus of the Candida genus 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 rugosa, and Candida lusitaniae. Candida infections that may be treated by the methods disclosed herein include, but are not limited to, candidemia, oropharyngeal candidiasis, esophageal candidiasis, mucosal candidiasis, genital candidiasis, vulvar candidiasis, rectal candidiasis, hepatic candidiasis, renal candidiasis, pulmonary candidiasis, splenic candidiasis, external otitis mycosis, osteomyelitis, septic arthritis, cardiovascular candidiasis (e.g., endocarditis), and invasive candidiasis.

[0276] B. Bacteria The complex lipid formulation or modified PMP formulation and related methods can be useful for reducing the fitness of bacteria, for example, to prevent or treat bacterial infection in animals, for example, humans.Included are methods of administering complex lipid formulations or modified PMP formulations to bacteria by contacting bacteria with complex lipid formulations or modified PMP compositions.In addition, or alternatively, the methods include administering complex lipid formulations or modified PMP formulations to animals to prevent or treat bacterial infection (e.g., caused by bacteria described herein) in animals at risk or in need thereof.

[0277] 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 further described below. For example, bacteria may be classified into the following order: Bacillales (B. anthracis, B. cereus, Staphylococcus aureus, L. monocytogenes), Lactobacillales (Streptococcus pneumoniae, S. pyogenes), Clostridiales (C. botulinum, C. difficile, C. perfringens, C. tetani), Spirochaetales (Borrelia burgdorferi, Treponema pallidum), and the following order: pallidum), Chlamydiales (Chlamydia trachomatis, Chlamydophila psittaci), Actinomycetales (Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium avium), Rickettsiales (R. prowazekii, R. rickettsii, R. typhi, Anaplasma phagocytophilum, Ehrlichia chaffeensis), Rhizobium (Brucella melitensis), melitensis), Burkholderiales (Bordetella pertussis, Burkholderia mallei, B. pseudomallei), Neisseriales (Neisseria gonorrhoeae, N.meningitidis), Campylobacterales (Campylobacter jejuni, Helicobacter pylori), Legionellale (Legionella pneumophila), Pseudomonadales (Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa), Aeromonadales (Aeromonas species), Vibriales (Vibrio cholerae, V. parahaemolyticus), Thiothrixales, Pasteurellale (Haemophilus influenzae), Enterobacteriaceae (Klebsiella pneumoniae), pneumoniae, Proteus mirabilis, Yersinia pestis, Yersinia enterocolitica, Shigella flexneri, Salmonella enterica, Escherichia coli.

[0278] The present invention can be further illustrated by the following embodiments.

[0279] Embodiment 1. A method of delivering a therapeutic peptide or protein to a human subject in need thereof, comprising administering to the human subject: (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 exogenous to the one or more plant sources; (iii) comprising polyethylene glycol (PEG) conjugated lipids; (iii) containing less than 10% w / w protein material endogenous to the one or more plant sources; and (iv) containing less than 10 mole % exogenous ionized lipids; (b) Oral or enteral administration of a pharmaceutical preparation comprising a therapeutic peptide or protein encapsulated in a complex lipid particle. Embodiment 2. The method of embodiment 1, wherein the therapeutic peptide or protein is a hormone or a glucagon-like peptide 1 (GLP-1) agonist. Embodiment 3. The method of embodiment 2, wherein the therapeutic peptide or protein is insulin, exenatide, semaglutide, or tirzepatide. Embodiment 4 The method of embodiment 1, wherein the therapeutic peptide or protein is delivered to brain tissue in a human subject. Embodiment 5. The method of embodiment 1, wherein the composite lipid particles contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemibismonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholines, lysophosphatidylethanolamines, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylethanol, phosphatidylglycerols, phosphatidylinositols, sulfoquinovosyldiacylglycerols, and sterols. Embodiment 6 The method of embodiment 5, wherein the composite lipid particles contain lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different subclasses. Embodiment 7. The method of embodiment 1, wherein the complex lipid particles contain less than 5% w / w protein material endogenous to one or more plant sources. Embodiment 8 The method of embodiment 1, wherein the composite lipid particles contain less than 5 mole % exogenous ionizable lipid. Embodiment 9 The method of embodiment 1, wherein at least one of the plant sources is grapefruit, lemon, dragon fruit, spinach, kale, strawberry, broccoli, or soybean. Embodiment 10. The complex lipid particles comprise, based on the amount of total lipid in the complex lipid formulation: Approximately 85-95% w / w of vegetable lipids, Approximately 5-8% w / w sterols, 2. The method of embodiment 1, comprising about 1-3.5% w / w polyethylene glycol (PEG)-lipid conjugate. Embodiment 11. A complex lipid formulation comprising: a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and One or more exogenous peptides, polypeptides, or proteins encapsulated in a composite lipid particle, the composite lipid particle having the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mol % ionizable lipids. Embodiment 12 The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide, or protein is a therapeutic agent. Embodiment 13 The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide, or protein is an antibody or an antibody fragment. Embodiment 14 The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide, or protein is a hormone. Embodiment 15 The combined lipid formulation of embodiment 14, wherein the exogenous peptide, polypeptide, or protein is insulin. Embodiment 16 The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide, or protein is a receptor agonist or a receptor antagonist. Embodiment 17 The complex lipid formulation of embodiment 16, wherein the exogenous peptide, polypeptide, or protein is a glucagon-like peptide 1 (GLP-1) agonist. Embodiment 18 The complex lipid formulation of embodiment 17, wherein the exogenous peptide, polypeptide, or protein is exenatide, semaglutide, or tirzepatide. Embodiment 19 The complex lipid formulation of embodiment 11, wherein the exogenous peptide, polypeptide, or protein has a size of less than 100 kD. Embodiment 20 The complex lipid formulation of embodiment 19, wherein the exogenous peptide, polypeptide, or protein has a size of less than 50 kD. Embodiment 21 The complex lipid formulation of embodiment 19, wherein the exogenous peptide, polypeptide, or protein has a size of at least 3 kD. Embodiment 22 The complex lipid formulation of embodiment 19, wherein the exogenous peptide, polypeptide, or protein comprises at least 30 amino acid residues. Embodiment 23. The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain from 5 to 1000 lipids extracted from one or more plant sources. Embodiment 24. The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain at least 10 plant lipids belonging to one or more of the classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. Embodiment 25. The complex lipid formulation of embodiment 24, wherein the complex lipid particle contains one or more glycerolipids selected from the group consisting of phospholipids (PL), galactolipids (GL), triacylglycerols (TG), and sulfolipids (SL). 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 glycosyl inositol phosphoceramides (GIPCs), glucosylceramides (GCer), ceramides (Cer), and free long chain bases (LCBs). 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. Embodiment 28. The composite lipid particles are selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesteryl ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acid, fatty acid ester of hydroxyl fatty acid, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylserine, 25. The complex lipid formulation of embodiment 24, comprising one or more lipids belonging to one or more of the subclasses selected from the group consisting of monogalactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, steryl esters, stigmasterol, sulfatides, sulfonolipids, sphingomyelin, sulfoquinovosyldiacylglycerol, sterols, and triacylglycerols. Embodiment 29. The complex lipid formulation of embodiment 28, wherein the complex lipid particles contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerols, diacylglyceryl glucuronides, hemibismonoacylglycerophosphates, hexosylceramides, lysophosphatidylcholines, lysophosphatidylethanolamines, monogalactosyldiacylglycerols, phosphatidylcholines, phosphatidylethanolamines, phosphatidylethanol, phosphatidylglycerols, phosphatidylinositols, sulfoquinovosyldiacylglycerols, and sterols. Embodiment 30. The complex lipid formulation of embodiment 28 or 29, wherein the complex lipid particles contain lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different subclasses. Embodiment 31 The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain less than 30% w / w protein material endogenous to one or more plant sources. Embodiment 32. The complex lipid formulation of embodiment 31, wherein the complex lipid particles contain less than 5% w / w protein material endogenous to one or more plant sources. Embodiment 33 The complex lipid formulation of embodiment 11, wherein the complex lipid particles contain less than 20 mol % exogenous ionizable lipid. Embodiment 34 The complex lipid formulation of embodiment 33, wherein the complex lipid particles contain less than 5 mole % exogenous ionizable lipid. Embodiment 35 The complex lipid formulation of embodiment 11, wherein at least one of the plant sources is a citrus fruit. Embodiment 36 The complex lipid formulation of embodiment 35, wherein the citrus fruit is grapefruit or lemon. Embodiment 37 The complex lipid formulation of embodiment 11, wherein at least one of the plant sources is a non-citrus plant. Embodiment 38. The complex lipid formulation of embodiment 37, wherein the non-citrus plant is dragon fruit, spinach, kale, strawberry, broccoli, or soybean. Embodiment 39 The complex lipid formulation of embodiment 11, wherein the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate. Embodiment 40 The complex lipid formulation of embodiment 39, wherein the sterol is cholesterol or sitosterol. Embodiment 41 The complex lipid formulation of embodiment 39, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE. Embodiment 42. The complex lipid formulation of embodiment 39, wherein the PEG-lipid conjugate is PEG2000-PE, PEG2000-DMG, PEG2000-DSPE, or a derivative thereof. 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 additional synthetic lipid. Embodiment 44. The complex lipid particles comprise, based on the amount of total lipid in the complex lipid formulation: Approximately 10-95% w / w of vegetable lipids, Approximately 5-60% w / w sterols, 40. The complex lipid formulation of embodiment 39, comprising about 0.5-15% w / w of a polyethylene glycol (PEG)-lipid conjugate. Embodiment 45. The complex lipid particles comprise, based on the amount of total lipid in the complex lipid formulation: Approximately 85-95% w / w of vegetable lipids, Approximately 5-8% w / w sterols, The complex lipid formulation of embodiment 44, comprising about 1-3.5% w / w of a polyethylene glycol (PEG)-lipid conjugate. Embodiment 46 The complex lipid formulation of embodiment 21, wherein the complex lipid particles have an average size of less than about 250 nm. Embodiment 47. The complex lipid formulation of embodiment 46, wherein the complex lipid particles have an average size of about 100-180 nm. Embodiment 48. The complex lipid formulation of embodiment 11, wherein the complex lipid particles have a PDI of about 0.1 to about 0.5. Embodiment 49. The complex lipid formulation of embodiment 48, wherein the complex lipid particles have a PDI of about 0.2 to about 0.4. Embodiment 50 The complex lipid formulation of embodiment 11, wherein the complex lipid particles further comprise one or more cryoprotectants or lyoprotectants. Embodiment 51 The complex lipid formulation of embodiment 11, wherein the complex lipid formulation is a lyophilized composition. Embodiment 52 The complex lipid formulation of embodiment 11, wherein the complex lipid formulation is a liquid composition. Embodiment 53. The complex lipid formulation of embodiment 11, wherein the complex lipid formulation is stable at room temperature and / or 4° C. for at least two weeks without lyophilization. Embodiment 54. A pharmaceutical composition comprising a complex lipid formulation according to any one of embodiments 1 to 53, and a pharma- ceutically acceptable vehicle, carrier, or excipient. Embodiment 55. The pharmaceutical composition of embodiment 54, wherein the pharmaceutical composition is in capsule or tablet form. Embodiment 56. A method for delivering a peptide, polypeptide, or protein to a mammalian cell or mammal, comprising: contacting a mammalian cell with the complex lipid formulation or administering the complex lipid formulation to a mammal under conditions sufficient to permit uptake of the complex lipid formulation by the mammalian cell or by the mammal; The complex lipid formulation is a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and One or more exogenous peptides, polypeptides, or proteins encapsulated in a composite lipid particle, the composite lipid particle having the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mol % ionized lipids. Embodiment 57 The method of embodiment 56, wherein the mammalian cell is a cell in a human or the mammal is a human. Embodiment 58. The method of embodiment 56, wherein uptake by a mammalian cell or by a mammal of an exogenous peptide, polypeptide, or protein encapsulated by a composite lipid particle is increased compared to uptake of an exogenous peptide, polypeptide, or protein that is not encapsulated by a composite lipid particle. Embodiment 59. The method of embodiment 56, for delivering a peptide, polypeptide, or protein to a mammal, wherein administration is via oral, enteral, intranasal, intracolonic, intrarectal, or intrajejunal routes. Embodiment 60 The method of embodiment 56, wherein the mammalian cell is a brain cell. Embodiment 61. A method of treating or preventing a disease or disorder in a subject in need of a therapeutic agent, comprising: For those who need it, a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and The method comprises administering an effective amount of a complex lipid formulation comprising one or more exogenous peptides, polypeptides, or proteins encapsulated in complex lipid particles, the complex lipid particles having the following characteristics: i) containing less than 50% w / w protein material endogenous to one or more plant sources; and ii) containing less than 50 mol % ionized lipids. Embodiment 62. The method of embodiment 61, wherein administration is via oral, enteral, intranasal, intracolonic, intrarectal, or intrajejunal routes. Embodiment 63. The method of embodiment 61, wherein the disease is diabetes and the exogenous peptide, polypeptide, or protein is insulin, exenatide, semaglutide, or tirzepatide. Embodiment 64. A method of producing a complex lipid formulation comprising a plurality of complex lipid particles encapsulating an exogenous peptide, polypeptide, or protein, 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 loading a complex lipid particle with an exogenous peptide, polypeptide, or protein, whereby the exogenous peptide, polypeptide, or protein is encapsulated by the complex lipid particle, thereby forming a complex lipid formulation. Embodiment 65. The method of embodiment 64, wherein the lipids are extracted from one or more plant sources by adding to the plant source an extraction solvent comprising methanol, ethanol, propanol, 1-butanol, acetonitrile, acetone, dimethylformamide, tetrahydrofuran, dimethylsulfoxide, methyl tert-butyl ether, chloroform, ethyl acetate, or a mixture thereof. Embodiment 66. The method of 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, dimethylsulfoxide:methanol, acetonitrile:ethanol, or ethyl acetate:ethanol. Embodiment 67. The method of embodiment 64, wherein the extraction step further comprises reducing to less than 50% w / w or removing protein material endogenous to the one or more plant sources. Embodiment 68. The method of embodiment 64, wherein the mixing step is performed by thin film mixing or microfluidic mixing. Embodiment 69 The method of embodiment 64, wherein the exogenous lipid comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate. Embodiment 70 The method of embodiment 64, wherein the exogenous lipid does not include an ionizable lipid. Embodiment 71. A modified plant messenger pack (PMP) formulation comprising: one or more PMPs modified with one or more sterols and one or more polyethylene glycol (PEG)-lipid conjugates; A modified plant messenger pack (PMP) formulation in which a modified PMP is formulated with one or more exogenous peptides, polypeptides, or proteins, and the one or more exogenous peptides, polypeptides, or proteins are encapsulated by the modified PMP. Embodiment 72 The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide, or protein is a therapeutic agent. Embodiment 73 The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide, or protein is an enzyme. Embodiment 74 The modified PMP formulation of embodiment 73, wherein the enzyme is a recombinant enzyme or an edited enzyme. Embodiment 75 The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide, or protein is an antibody or antibody fragment. Embodiment 76 The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide, or protein is an Fc fusion protein. Embodiment 77 The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide, or protein is a hormone. Embodiment 78 The modified PMP formulation of embodiment 77, wherein the exogenous peptide, polypeptide, or protein is insulin. Embodiment 79 The modified PMP formulation of embodiment 71, wherein the exogenous peptide, polypeptide, or protein is a receptor agonist or a receptor antagonist. Embodiment 80 The modified PMP formulation of any one of embodiments 71-79, wherein the exogenous peptide, polypeptide, or protein has a size of less than 100 kD. Embodiment 81 The modified PMP formulation of embodiment 80, wherein the exogenous peptide, polypeptide, or protein has a size of less than 50 kD. Embodiment 82 The modified PMP formulation of any one of embodiments 71-79, wherein the exogenous peptide, polypeptide, or protein has a size of at least 5 kD. Embodiment 83 The modified PMP formulation of any one of embodiments 71-79, wherein the exogenous peptide, polypeptide, or protein comprises at least 50 amino acid residues. Embodiment 84. The modified PMP formulation of any one of embodiments 71-83, wherein the exogenous peptide, polypeptide, or protein has an overall charge that is neutral or has been modified to have a neutral charge. Embodiment 85 The modified PMP formulation of any one of embodiments 71-83, wherein the exogenous peptide, polypeptide, or protein has an overall charge that is positive or negative. Embodiment 86. The modified PMP formulation of any one of embodiments 71-85, wherein the PMP comprises purified plant extracellular vesicles (EVs), or segments or extracts thereof. Embodiment 87. The modified PMP formulation of embodiment 86, wherein the PMP is obtained from a citrus fruit. Embodiment 88 The modified PMP formulation of embodiment 87, wherein the citrus fruit is grapefruit or lemon. Embodiment 89. The modified PMP formulation of any one of embodiments 71-88, wherein the sterol is cholesterol or sitosterol. Embodiment 90. The modified PMP formulation of any one of embodiments 71-88, wherein the PEG-lipid conjugate is C14-PEG2k or C18-PEG2k. Embodiment 91. A modified PMP formulation according to any one of embodiments 71-88, wherein the PEG lipid conjugate is PEG-DMG or PEG-PE. Embodiment 92. The modified PMP formulation of any one of embodiments 71-88, wherein the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof. Embodiment 93. The modified PMP formulation of any one of embodiments 71-88, wherein the sterol is cholesterol and the PEG-lipid conjugate is C18-PEG2000 PE or a derivative thereof. Embodiment 94. The modified PMP formulation of embodiment 86, wherein the concentration of sterol is in the range of about 0.5-15% w / w based on the amount of total lipid extract. Embodiment 95. The modified PMP formulation of embodiment 94, wherein the concentration of sterol is in the range of about 5-8% w / w based on the amount of total lipid extract. Embodiment 96. The modified PMP formulation of embodiment 86, wherein the concentration of the PEG-lipid conjugate is in the range of about 0.5-5% w / w based on the amount of total lipid extract. Embodiment 97. The modified PMP formulation of embodiment 96, wherein the concentration of the PEG-lipid conjugate is in the range of about 1-3.5% w / w, based on the amount of total lipid extract. Embodiment 98. The modified PMP formulation of embodiment 86, wherein the sterol is cholesterol having a concentration ranging from 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 having a concentration ranging from about 1 to 3.5% w / w based on the amount of total lipid extract. Embodiment 99. The modified PMP formulation of any one of embodiments 71-98, wherein the modified PMP is a lipid nanoparticle. Embodiment 100. The modified PMP formulation of any one of embodiments 71-98, wherein the modified PMP has a size of less than about 200 nm. Embodiment 101. The modified PMP formulation of embodiment 100, wherein the modified PMP has a size of about 100-160 nm. Embodiment 102. The modified PMP formulation of any one of embodiments 71-101, further comprising a phosphate, citrate, sodium bicarbonate, HEPES, TAE, or TRIS buffer at a pH of about 3.0 to about 8.5. Embodiment 103. The modified PMP formulation of any one of embodiments 71-102, further comprising one or more cryoprotectants. 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. Embodiment 105 The modified PMP formulation of any one of embodiments 71-104, wherein the modified PMP formulation is a lyophilized composition. Embodiment 106 The modified PMP formulation of any one of embodiments 71-105, wherein the modified PMP formulation is stable at room temperature and / or at 4°C. Embodiment 107. A pharmaceutical composition comprising a modified PMP formulation according to any one of embodiments 71-106, and a pharma- ceutically acceptable vehicle, carrier, or excipient. Embodiment 108. A method for producing a modified PMP formulation comprising an exogenous peptide, polypeptide, or protein, comprising: Providing a solution comprising one or more PMPs, one or more sterols, and a modified PMP comprising one or more polyethylene glycol (PEG)-lipid conjugates; Providing a solution containing an exogenous peptide, polypeptide, or protein; and A method comprising loading a modified PMP with an exogenous peptide, polypeptide or protein, whereby the exogenous peptide, polypeptide or protein is encapsulated by the modified PMP. Embodiment 109. A method for delivering a peptide, polypeptide, or protein to a mammalian cell or mammal, comprising: A method comprising contacting a mammalian cell with a modified PMP formulation described in any one of embodiments 71-104 or administering to a mammal a modified PMP formulation described in any one of embodiments 71-104 under conditions sufficient to allow uptake of the modified PMP formulation by the mammalian cell or by the mammal. Embodiment 110. The method of embodiment 109, wherein the method is for delivering a peptide, polypeptide, or protein to a mammalian cell, and the cell is a cell in a subject. 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 with which the modified PMP formulation is contacted. Embodiment 112 The method of embodiment 111, wherein the exogenous peptide, polypeptide, or protein exerts activity in the cytoplasm or nucleus of a mammalian cell. Embodiment 113 The method of any one of embodiments 108 to 112, wherein the mammal is a human. Embodiment 114. The method of any one of embodiments 108-112, wherein uptake by a mammalian cell or by a mammal of an exogenous peptide, polypeptide, or protein encapsulated by the modified PMP formulation is increased compared to uptake of an exogenous peptide, polypeptide, or protein that is not encapsulated by the modified PMP formulation. Embodiment 115. The method of embodiment 109, wherein the method is for delivering a peptide, polypeptide, or protein to a mammal, and administration is via oral, intranasal, or intrarectal routes. Embodiment 116. A method for treating or preventing a disease or disorder in a subject in need of a therapeutic agent, comprising administering to a subject in need thereof an effective amount of a modified PMP formulation described in 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. Embodiment 117. The method of embodiment 116, wherein administration is via oral, intranasal, or rectal routes. Embodiment 118 The method of embodiment 114, wherein the disease is diabetes and the exogenous peptide, polypeptide, or protein is insulin. EXAMPLES

[0280] Below are examples of various methods of the present invention: From the general description above, various other embodiments may be implemented.

[0281] Example 1: Preparation of PMPs and composite lipid particles General procedure for preparing PMPs Preparation of PMPs and formulation of PMPs may be accomplished utilizing methods similar to those disclosed in International Patent Application Publication No. WO2023 / 069498, which is incorporated herein by reference in its entirety.

[0282] General procedure for preparing the lipid extract components of CLP The typical preparation of complex lipid particles (CLPs) begins with the isolation of lipid extracts from natural plant sources. Briefly, the method of extraction of plant lipids in CLPs is as follows. 1) Plant material (e.g., juice, pulp, or mixed parts of the plant) is collected from a natural plant source. 2) Filter and concentrate the plant material. 3) The plant concentrate is diafiltered with a citrate-sodium chloride buffer to obtain the diafiltration intermediate (DFI), which is the starting material for extraction. 4) The lipids are extracted by adding an extraction solvent such as dichloromethane (DCM) and methanol (MeOH). Alternative extraction solvents in this process can include chloroform:methanol and ethyl acetate:ethanol. 5) Water is added to induce phase separation. 6) The resulting organic phase is collected and dried using a rotary evaporator (rotovap). 7) The dried lipids are resuspended in an aqueous solution (e.g., 90% DCM and 10% MeOH solution), transferred to a vial, and dried (e.g., via Genevac or similar drying apparatus). 8) The resulting lipid extract is dried and stored at -20°C.

[0283] Exemplary Modified Extraction Methods for Preparing CLPs Modifications to the above extraction of lipids from natural sources are detailed below. Two alternative extraction methods for the preparation of composite lipid particles were used: 1) ethanol extraction method or 2) modified Matthias method using methanol. In this example, a 3:2 ethyl acetate:ethanol solvent was made for ethanol extraction. 25 mL of PBS was added to 2.5 g of powdered natural source, then vortexed at 1500 RPM for 1 minute. 41.5 mL of ethanol solvent was added to the aqueous sample of natural source. This was vortexed on a vertical shaker at 1500 RPM for 1 minute. An additional 50 mL of ethyl acetate was added and the solution was vortexed at 1500 RPM for 1 minute. 50 mL of liquid chromatography-mass spectrometry grade water was added and the solution was vortexed at 1500 RPM for an additional minute, then centrifuged at 1500×g for 5 minutes at 4° C. The top organic layer was transferred to a new flask and then evaporated using a rotovap with a water bath set at 40° C. to ensure removal of residual solvent. The remaining extracted lipids were weighed and sparged with N2, then capped and stored at -20°C.

[0284] In an alternative example, an aqueous sample was prepared as described above for modified methanol extraction. 93.75 mL of a 1:2 MeOH:methyl tert-butyl ether (MTBE) solution was added to the sample, vortexed at 1500 RPM for 1 minute, then sonicated at 100% power and 37 Hz for 5 minutes. An additional 31.25 mL of MTBE was added, and the solution was vortexed at 1500 RPM for an additional minute, then centrifuged at 1500×g for 5 minutes. The upper organic layer was transferred to a flask, and 60 mL of ethyl acetate was added. The new solution was vortexed at 1500 RPM for 1 minute, shaken for 30 seconds, then centrifuged at 1500×g for 5 minutes at 4° C. The upper organic layer was transferred again. The solvent was then evaporated using a rotovap, weighed, and stored as described for the ethanol extraction.

[0285] The dried extracts were reconstituted in absolute ethanol to a given concentration of 10 mg / mL, vortexed at 2000 RPM for 30 second intervals, and sonicated at 80 Hz for 30 second intervals. Upon lipid reconstitution, samples were filtered into pre-weighed vials, then dried using a GeneVac and stored at -20°C under nitrogen.

[0286] In addition to the above dichloromethane:methanol, chloroform:methanol, methanol:MTBE, and ethyl acetate:ethanol lipid extraction methods, alternative organic solvents that can be used include, but are not limited to, dimethylformamide:methanol, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethylsulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, or dimethylsulfoxide:methanol.

[0287] Example 2: Formulation and characterization of polypeptide-loaded complex lipid formulations In this example, a method was developed to create and load stable complex lipid particles containing complex lipids derived from various plant sources with peptides, polypeptides, and small proteins.

[0288] In summary, the method involved: 1) mixing natural lipids obtained as described in Example 1 with components that promote increased stability (e.g., sterols such as cholesterol and poly-ethylene glycol (PEG) molecules) to preserve particle size and amount of encapsulated cargo over time; 2) forming particles containing the above components (with or without lyophilization); 3) loading the particles with peptidic cargo, either directly or passively; and, optionally, 4) purifying the resulting peptide-loaded particles for further applications / processing.

[0289] The various steps of processing, formulation, characterization and testing are described here and then in detail below. The programming step of the particle can include formulation via microfluidics (e.g., using NanoAssemblr) or membrane, as described in the following section. In addition, the process can include direct or passive loading of cargo. Purification and post-processing steps can include options such as dialysis, filtration, centrifugation, lyophilization, and / or encapsulation. Testing of the formulation can include stability and performance measurements both in vitro and in vivo (e.g., in mice, etc.). At any of these given stages (e.g., programming, post-processing, testing), characterization of a given formulation (size, API loading, lipidomics, etc.) can influence further formulation and optional steps to generate an exemplary complex lipid formulation.

[0290] General procedure for preparing and loading empty composite lipid particles In general, the formulation of composite lipid particles involves the generation of empty particles followed by loading of the empty particles or loading of particles via microfluidic mixing to directly generate loaded particles.

[0291] The generation of empty particles occurs via either thin film or microfluidic mixing. Briefly, the generation of empty particles via thin film hydration includes the steps of 1) lipid mixing, 2) thin film formation, 3) hydration, 4) homogenization, and 5) empty particle formation. Briefly, the generation of empty particles via microfluidic mixing includes the following steps: 1) lipid mixing, 2) microfluidic homogenization, and 3) empty particle formation.

[0292] After empty particle formulation, either using thin film or microfluidic mixing, the particles can be optionally freeze-dried. If freeze-drying does not occur, the empty particles are passively loaded via the following steps: 1) mixing the empty particles with an active pharmaceutical ingredient (API) solution, 2) homogenizing the subsequent formulation, and optionally, 3) purifying the formulation. If freeze-drying occurs, the empty particles are passively loaded via the following steps: 1) rehydrating the freeze-dried particles in an API solution, then 2) homogenizing, then optionally, 3) purifying the formulation. Alternatively, the generation of loaded particles can occur directly via microfluidic mixing. Briefly, the direct loading of particles via microfluidic mixing includes the following steps: 1) lipid mixing with the API, 2) microfluidic homogenization, 3) forming loaded particles, and optionally, 4) purifying the resulting formulation. After loading the particles generated through any of the given means, the particles can be tested. Testing may include in vitro characterization, in vivo functional testing, lyophilization and capsule filling, or lyophilization and rehydration.

[0293] Exemplary CLPs prepared by adding sterols and PEG to lipids extracted from plant sources This example describes the formulation of stable particles using lipids derived from plant sources (such as PMP or CLP). In this example, sterols (e.g., cholesterol) and PEGylated lipids were added to lipids extracted from plant sources. Cholesterol acts as a molecule that promotes the packing of residual lipids and the stabilization of the formulation, but can increase the rigidity of the lipid particles, which may not be desirable, especially when the lipid particles are designed to cross a cell monolayer, such as the gastrointestinal epithelial layer. PEGylated lipids act as molecules to prevent fusion of formulated lipid particles. In this example, the amount of cholesterol and PEGylated lipid added was minimized for the following reasons: Natural plant-derived sterols are present in many natural lipid compositions. Thus, adding large amounts of exogenous cholesterol may result in rigid lipid particles. Furthermore, adding large amounts of PEGylated lipids may increase the tendency of the formulation to be identified by immune cells, reducing the efficacy of the resulting lipid particles, especially in the case of repeated administration.

[0294] 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) is 720 Da, ii) 50% of the total lipid extract is structured lipid. Thus, for example, for 1 mg of lipid extract, there was 0.5 mg or 695 nmol of structured lipid. Added cholesterol and PEGylation were calculated relative to the amount of structured lipid per total mass of lipid extract.

[0295] DSPE-PEG2000 (18:0 PEG2000 PE, CAS number 474922-77-5) with a molecular weight of 2800 Da was used as the PEGylated lipid. Four different concentrations were added to the lipid mixture; 0% w / w, 0.5% w / w (5 μg per mg of total lipid extract), 1.25% w / w (12.5 μg per mg of total lipid extract), and 2.5% w / w (25 μg per mg of total lipid extract). For 1 mg of total lipid extract or 695 nmol of structured lipid, this translates to 0% mol, 0.26% mol, 0.64% mol, and 1.28% mol of PEGylated lipid.

[0296] Plant-derived cholesterol (CAS number 57-88-5) with a molecular weight of 386.5 Da was used as cholesterol. Three different concentrations were added to the lipid mixture; 0% w / w, 3.4% w / w (34 μg per mg of total lipid extract), and 6.8% w / w (68 μg per mg of total lipid extract). For 1 mg of total lipid extract or 695 nmol of structured lipids, this translates to 0% mol, 12.5% ​​mol, and 25% mol of cholesterol.

[0297] Exemplary generation of empty composite lipid particles As briefly described above, two main formulation processes for preparing composite lipid particles were used: 1) thin film rehydration and 2) microfluidic encapsulation.

[0298] In this example, the formulation process for thin film rehydration includes the following steps as shown in FIG. 1A. 1) Natural source lipids (natural lipids extracted from plants such as lemon) and exogenous lipids (i.e., cholesterol and PEGylated lipids discussed above at various concentrations) were solubilized in organic solvents (such as chloroform or ethanol). Natural lipids were solubilized at a given concentration (e.g., 5 mg / ml). 2) A thin film of mixed lipids was formed in a flask by evaporating the organic solvent in a rotary evaporator at 42° C. and further dried using a stream of nitrogen. 3) The thin film was hydrated in an aqueous buffer containing a cryoprotectant at 5 mg / ml lipid in the buffer for 30 min at 40° C. and then vortexed. The buffer was selected to facilitate downstream solubilization of the protein cargo (e.g., based on the isoelectric point of the protein cargo). For insulin, the buffer was citrate at pH 3 or sodium bicarbonate at pH 8.2. For GLP1 receptor agonists (e.g., semaglutide and exenatide), the buffer was 0.1 M sodium bicarbonate at pH 8.2. The cryoprotectant was 2-5% sucrose or 2-5% mannitol. Alternatively, the cryoprotectant can range from 0% to 5%. The cryoprotectant can be about 0.5-2%. 4) The lipid film was homogenized in aqueous buffer in a sonicator for the formation of small unilamellar vesicles (SUVs) containing native and exogenous lipids. The sonication process included two steps: sonication for 20 min at 42°C, brief mixing with a vortex, and sonication for a second time for 20 min at about 42°C. If aggregates were observed, the particle solution was filtered through a cotton filter. Particle morphological characterization was analyzed by dynamic light scattering. 5) The lipid solution was frozen in liquid nitrogen for 10 minutes. The lipid solution in aqueous buffer and cryoprotectant was lyophilized (freeze-dried) at room temperature overnight or for 12-48 hours, depending on the volume of the particle solution. The resulting complex lipid formulation containing natural source lipids and exogenous lipids was produced in dry powder form, which was stored at -20°C until use. Particles can be freeze-dried by an alternative method known as storage freeze-drying, where the particles are controlled throughout the freezing process using a vacuum rate. Alternatively, the particles were not freeze-dried.

[0299] The formulation process for the microfluidic encapsulation process included the following steps, which are shown in Figure 1B. 1) Natural and exogenous lipids (i.e., cholesterol and PEGylated lipids discussed above at various concentrations) were solubilized in organic solvent at a given concentration (e.g., at a concentration of 5 mg / ml). 2) The lipid mixture in organic solvent was co-injected into a microfluidic device (NanoAssemblr Ignite) with an aqueous buffer (e.g., 0.1 M sodium bicarbonate at a pH of 8.2 with the addition of 5% mannitol as a cryoprotectant). 3) The organic solvent was dialyzed overnight against an aqueous buffer (eg, 0.1 M sodium bicarbonate, pH 8.2, containing 5% mannitol as a cryoprotectant). 4) Particle morphological characterization was analyzed by dynamic light scattering. 5) The lipid solution was frozen in liquid nitrogen for 10 minutes. The lipid solution was lyophilized (freeze-dried) overnight at room temperature. The resulting composite lipid particles containing natural source lipids and exogenous lipids were produced in dry powder form, which was stored at -20°C until use. The particles can be freeze-dried by an alternative method known as storage freeze-drying, in which the particles are controlled through the freezing process using a vacuum rate. Alternatively, the particles were not freeze-dried.

[0300] Other known methods such as microfluidic T-junction generation may also be used.

[0301] Loading of polypeptides into empty composite lipid particles According to the formulation process described above, the composite lipid particles containing natural source lipids and exogenous lipids prepared in dry powder form, stored at -20°C, were harvested and formulated with an exemplary active biomolecule (e.g., a polypeptide such as insulin).

[0302] 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 for peptides of 5 kD or less with a lipid to active biomolecule ratio of 1:1 w / w, or 1 mg / mL for proteins of 5 kD or more with a lipid:peptide mass ratio of 1:5 w / w. After rehydration, the particles were briefly homogenized by sonication at 40° C. for 20 minutes. This rehydration buffer was similar to that used to hydrate the thin film (step 3 above) and was selected based on its properties for solubilizing the active biomolecule. Again, for insulin, the buffer was citrate at pH 3 or sodium bicarbonate at pH 8.2, and for GLP1 receptor agonists (e.g., semaglutide or exenatide), the buffer was 0.1 M sodium bicarbonate at pH 8.2. Particle morphological characterization was analyzed by dynamic light scattering. Alternatively, if the particles were not lyophilized, the empty particles were mixed with an aqueous buffer containing an active biomolecule (eg, a polypeptide such as insulin) in a manner similar to that described above.

[0303] The mixture of rehydrated particles containing active biomolecules was then purified and separated from free active biomolecules not loaded on the particles by a dialysis step overnight. Dialysis was performed in a 100 kD dialysis membrane in the buffer used in the formulation process discussed above with at least 2000x the volume of the solution to be purified (e.g., 0.1 M sodium bicarbonate buffer, pH 8.2). The dialysate was concentrated to the desired concentration in a centrifuge unit. Alternatively, the purification and separation steps can be performed using tangential flow filtration (TFF). The purification steps described are optional and were not performed for all formulations. Particle morphology characterization was analyzed by dynamic light scattering. Particle loading efficiency was quantified by bicinchoninic acid assay (BCA).

[0304] The above process for loading bioactive molecules into empty composite lipid particles containing natural source lipids and exogenous lipids is illustrated in FIG.

[0305] An alternative method for loading bioactive molecules into composite lipid particles containing natural lipids and exogenous lipids includes solubilizing the lipid mixture in an organic solvent together with an aqueous buffer containing the active biomolecule before homogenizing the mixed solution via microfluidics to directly generate the loaded particle formulation. Alternatively, the bioactive molecule may also be solubilized in the organic phase.

[0306] Characterization of polypeptide-loaded complex lipid formulations The efficacy of the complex lipid formulations prepared according to the above formulation and loading process was evaluated by a quantitative method that characterizes the size of the particles and the size distribution among the particle populations. The particle size was measured by dynamic light scattering (DLS) measurement. The results are shown in Table 5. Table 5 lists the particle diameter (nm) measured by DLS for the complex lipid formulations described above by varying the amount of added cholesterol (left column) and PEGylated lipid (top row) in the complex lipid formulation. [Table 5]

[0307] As shown in Table 5, the addition of cholesterol alone resulted in a much larger particle size, and the addition of PEGylated lipid also slightly increased the particle size when the amount of PEGylated lipid was 0.5% w / w. The insulin-loaded complex lipid formulation had a smaller particle size when the complex lipid formulation contained 2.5% w / w PEGylated lipid and 6.8% w / w cholesterol, based on the amount of total lemon lipid extract.

[0308] A complex lipid formulation containing 2.5% w / w PEGylated lipid and 6.8% w / w cholesterol was able to provide particle sizes in the range of 100-160 nm when the formulation and loading process described above was repeated multiple times (e.g., hundreds of times).

[0309] The insulin encapsulation efficiency (amount of insulin entrapped in the vesicles) of the complex lipid formulations was also characterized, and the results were consistently in the range of 25-35%. Similar characterization was measured for other formulations of complex lipid particles.

[0310] Stability of polypeptide-loaded complex lipid formulations. As shown in Table 5, as a control, the insulin-loaded complex lipid formulation without exogenous lipids (i.e., 0% w / w PEGylated lipid and 0% w / w cholesterol) initially had a small particle size of about 120 nm. However, this formulation was unstable over time, and the particle size increased to 400-500 nm after one day of storage at 4 °C. Furthermore, although lyophilization is typically used for longer time storage of the formulation (e.g., more than two weeks), the protein-loaded complex lipid formulation without exogenous lipids (i.e., 0% w / w PEGylated lipid and 0% w / w cholesterol) was unstable after a second lyophilization, and the particle size increased to 400-500 nm after a second lyophilization. Furthermore, the protein-loaded complex lipid formulation without exogenous lipids (i.e., 0% w / w PEGylated lipid and 0% w / w cholesterol) could not be reconstituted by rehydration of the powder with buffer.

[0311] On the other hand, the complex lipid formulation containing PEGylated lipid and cholesterol was stable for at least two weeks at 4°C. For longer storage time, lyophilization was used. The complex lipid formulation containing PEGylated lipid and cholesterol was stable after lyophilization. The complex lipid formulation containing PEGylated lipid and cholesterol could also be reconstituted upon rehydration of the powder and buffer. After reconstitution, the particle size of the complex lipid formulation could reach about 90% of the original particle size before lyophilization, and the insulin encapsulation efficiency of the complex lipid formulation could reach about 30% of the initial encapsulation efficiency before lyophilization. Similar stability measurements were used for other formulations of complex lipid particles.

[0312] Lipidomics of plant lipid extracts and empty complex lipid particles In this example, lipids were extracted from five plant sources (e.g., lemon, dragon fruit, spinach, kale, strawberry) as described in Example 1. These extracts were formulated into empty complex lipid particles or maintained as lipid extract compositions as described above in Example 2. Both the extracts and empty particles were subjected to lipidomic analysis.

[0313] Briefly, extracts or particles were solubilized in a compatible solvent and analyzed by MS / MS. The occurrence of determined lipid classes and subclasses identified in extracts vs. particles was analyzed. 46 different lipid classes / subclasses were annotated via lipidomics, including the following: acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesteryl ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acid, fatty acid ester of hydroxyl fatty acid, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylcholine, ... The lipids that were synthesized from the phosphoinositides were phosphoinositides, phospholipids ...

[0314] The number of lipids annotated in the lemon extract alone exceeded 800, and these lipids were classified into at least 20 of the classes or subclasses listed above. This highlights the complexity of lipid extract composition from natural sources, which was evident among all five plant sources tested. The occurrence of certain lipid classes or subclasses changed throughout the formulation process, as was evident when comparing the analyzed lipids present in the extracts or empty complex lipid particles. The overall diversity of lipid classes / subclasses was not lost, even with the change in the occurrence of lipid classes or subclasses during the formulation process.

[0315] Across the five tested empty particle formulations, an average of 24 of the enumerated lipid classes were represented during the positively charged particle analysis, and an average of 31 of the annotated lipid classes were represented during 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.

[0316] Of note, non-lipid components found within the lipid extracts were not examined within the lipidomics analysis. Thus, characterization of the lemon lipid extracts found that across multiple extracts, 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. Further characterization of the lemon lipid extracts found that the total dsDNA was less than 1% w / w of the extract composition as measured by Picogreen assay. In some cases, the total dsDNA was less than 0.05% w / w of the extract composition.

[0317] Example 3: Preparation of oral dosage forms of complex lipid formulations The following examples describe how to produce capsule or tablet dosage forms from complex lipid formulations.

[0318] First, for capsule dosage form, liquid complex lipid formulation is obtained according to the procedure described in Example 2. Additional excipients can 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 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 filled into capsules using standard manufacturing procedures. If the dried material is not free-flowing, such as in the case of freeze-dried cake, a procedure such as grinding is used to generate a powder from the cake. The powder is then filled into capsules using standard manufacturing procedures. The capsule shell can be uncoated or coated. The capsule can be bulk-packaged or individually packaged.

[0319] First, for tablet dosage form, liquid complex lipid formulation is obtained according to the procedure described in Example 2. Additional excipients can be added, including but not limited to cryoprotectants, lyoprotectants, stabilizers, fillers, 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 filled into tablets using standard manufacturing procedures. If the dried material is not free-flowing, such as in the case of freeze-dried cakes, a procedure such as milling is used to generate powder from the cake. The powder is then compressed into tablets using standard manufacturing procedures. The tablets can be uncoated or coated. The tablets can be bulk-packaged or individually packaged.

[0320] Example 4: In vivo intranasal and enteral delivery of protein-loaded complex lipid formulations An insulin-loaded complex lipid formulation was obtained according to Example 2, the complex lipid formulation containing 2.5% w / w PEGylated lipid and 6.8% w / w cholesterol based on the amount of total lemon lipid extract.

[0321] The animals used were 8-week-old female C57 mice (Jax #0664).

[0322] Intracolonic experiments Mice were administered the insulin-loaded conjugated lipid formulation via a 50 μL intrarectal dose (50 U / kg) according to the treatment schedule in Table 6. At various time points, blood was drawn, plasma samples were obtained, and insulin levels were quantified by ELISA assay, as shown in Table 6. [Table 6]

[0323] The results are shown in Figure 3. Figure 3 shows the concentration of insulin in the plasma of mice 1 hour after rectal administration of the insulin-loaded complex lipid formulation to mice, demonstrating the systemic absorption of the insulin-loaded complex lipid formulation. Compared to the benchmark of fused liposomes containing insulin administered directly to the colon (10 U / kg, 12 minutes after injection), rectal administration of the insulin-loaded complex lipid formulation resulted in significantly higher insulin plasma levels compared to the benchmark.

[0324] Intranasal experiment Mice were administered the insulin-loaded complex lipid formulation via a 40 μL intranasal dose (20 μL pernostril, 50 U / kg) according to the treatment schedule in Table 7. At various time points as shown in Table 7, brains were weighed and proteins were extracted using acidified ethanol. Insulin levels were quantified by ELISA assay. [Table 7]

[0325] The results are shown in Figure 4. Figure 4 shows the concentration of insulin in the brain of mice 2 hours after intranasal administration of insulin-loaded complex lipid particles to mice, demonstrating efficient brain delivery of insulin via intranasal administration of insulin-loaded complex lipid particles. Compared to the benchmarks of intranasal administration of free insulin as well as intranasal administration of insulin with a cell-penetrating peptide, intranasal administration of insulin-loaded complex lipid particles resulted in significantly higher insulin brain levels compared to both benchmarks.

[0326] Example 5: In vivo enteral delivery of protein-loaded complex lipid formulations Insulin-loaded complex lipid formulations were obtained according to Example 2, and the complex lipid particles were formulated as described in Table 8. [Table 8]

[0327] Intracolonic experiments The animals used were 8-week-old female C57BL6 mice (Jax #0664).

[0328] C57BL6 female 8-week-old mice (Jax #0664) were used (n=4-5 mice / formulation). Mice were weighed and pre-bleed samples were collected (through tail vein, retro-orbital, or submandibular means). Mice were fasted overnight and then administered 50 μL of a given insulin-loaded complex lipid formulation via intracolonic administration (Table 8). Non-terminal bleeds at 30 and 60 min post-administration were collected, alternating collection routes. A terminal bleed at 120 min post-administration via cardiac puncture was also collected. Whole blood samples from pre-bleed, 30 min, 60 min, and 120 min bleeds were processed for plasma and then stored at -80°C. Insulin levels were quantified by MSD assay. Particle characterization and insulin measurements are reported in Table 9. [Table 9]

[0329] Exposure to active biomolecules (e.g., insulin) was measured as the area under the curve (AUC) for the four measured time points, and dose was measured as BCA x dose volume (0.05 mL). Thus, the data in Table 9 for each mouse is presented as systemic levels of insulin (or exposure normalized by dose). As shown in Table 9, 22 / 22 different conjugated lipid formulations showed good systemic levels of insulin. Unexpectedly, 20 / 22 (>90%) different conjugated lipid formulations outperformed free IC insulin.

[0330] Example 6: In vivo jejunal delivery of protein-loaded complex lipid formulations Exenatide-loaded complex lipid formulations were obtained according to Example 2, and the complex lipid particles were formulated as described in Table 10. Mice were treated according to Table 11. [Table 10] [Table 11]

[0331] Intrajejunal experiment The animals used were 8-week-old female C57BL6 mice (Jax #0664).

[0332] C57BL6 female 8-week-old mice (Jax #0664) were used (n=5 mice / formulation). Mice were weighed and pre-bleed samples were collected (through tail vein, retro-orbital, or submandibular means). Mice were fasted overnight and then administered 50 μL (50 μg / mouse) of a given exenatide-loaded complex lipid formulation via intrajejunal administration (Table 10). Non-terminal bleeds were collected 30 and 60 minutes after administration, alternating collection routes. A terminal bleed was also collected 120 minutes after administration via cardiac puncture. Whole blood samples from pre-bleed, 30, 60, and 120 minute bleeds were processed for plasma and then stored at -80°C. Exenatide levels were quantified by ELISA assay. Formulations are reported in Table 10 and treatment schedules are shown in Table 11. Results of particle characterization and exenatide measurements are shown in Table 12. [Table 12]

[0333] Exposure to the active biomolecule (e.g., exenatide) was measured as the area under the curve (AUC) for the four measured time points, and dose was measured as BCA x dose volume (0.05 mL). Thus, the data in Table 12 for each mouse is presented as systemic levels of insulin (or exposure normalized by dose). As shown in Table 12, the results indicate that a complex lipid formulation incorporating plant lipid extracts from both broccoli and strawberry performed well when delivering exenatide.

[0334] Other embodiments The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding; however, the illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety. Other embodiments are within the scope of the following claims.

Claims

1. 1. A method for delivering a therapeutic peptide or protein to a human subject in need thereof, comprising administering to said human subject: (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 exogenous to said one or more plant sources; (iii) comprising polyethylene glycol (PEG) conjugated lipids; (iii) containing less than 10% w / w protein material endogenous to said one or more plant sources; and (iv) containing less than 10 mole % exogenous ionized lipids; (b) orally or enterally administering a pharmaceutical preparation comprising said therapeutic peptide or protein encapsulated in said composite lipid particle.

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

5. 2. The method of claim 1, wherein the composite lipid particles contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerol, diacylglyceryl glucuronide, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and sterols.

6. 6. The method of claim 5, wherein the composite lipid particle contains lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different subclasses.

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

8. 10. The method of claim 1, wherein the composite lipid particles contain less than 5 mole percent exogenous ionizable lipids.

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

10. The complex lipid particles are, based on the amount of total lipid in the complex lipid formulation, about 85-95% w / w of said plant lipids, about 5-8% w / w of said sterol, and 10. The method of claim 1, comprising about 1-3.5% w / w of the polyethylene glycol (PEG)-lipid conjugate.

11. A complex lipid formulation comprising: a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and and one or more exogenous peptides, polypeptides, or proteins encapsulated in said composite lipid particles, said composite lipid particles having the following characteristics: i) containing less than 50% w / w protein material endogenous to said one or more plant sources; and ii) containing 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. 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 tirzepatide.

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

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

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

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

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

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

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

26. 25. The complex lipid formulation of claim 24, wherein the complex lipid particle contains one or more sphingolipids selected from the group consisting of glycosyl inositol phosphoceramide (GIPC), glucosylceramide (GCer), ceramide (Cer), and free long chain base (LCB).

27. The complex lipid particles are 25. The complex lipid formulation of claim 24, containing one or more phytosterols selected from the group consisting of campesterol, stigmasterol, and sitosterol.

28. The composite lipid particles may be selected from the group consisting of acyldiacylglyceryl glucuronide, acylhexosylceramide, acylsteryl glycoside, bile acid, acylcarnitine, cholesteryl ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactosyldiacylglycerol, diacylglyceryl glucuronide, dilysocardiolipin, fatty acid, fatty acid ester of hydroxyl fatty acid, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylserine, monoga The complex lipid formulation according to claim 24, comprising one or more lipids belonging to one or more of the subclasses selected from the group consisting of lactosyldiacylglycerol, lysocardiolipin, N-acylethanolamine, N-acylglycine, N-acylglycylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, ceramide phosphoinositol, phosphatidylmethanol, phosphatidylserine, steryl esters, stigmasterol, sulfatides, sulfonolipids, sphingomyelin, sulfoquinovosyldiacylglycerol, sterols, and triacylglycerols.

29. The complex lipid particles contain 10 or more lipids belonging to one or more of the subclasses selected from the group consisting of acylsterylglycosides, ceramides, digalactosyldiacylglycerol, diacylglyceryl glucuronide, hemibismonoacylglycerophosphate, hexosylceramide, lysophosphatidylcholine, lysophosphatidylethanolamine, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, sulfoquinovosyldiacylglycerol, and sterols. The complex lipid formulation of claim 28.

30. 30. The complex lipid formulation of claim 28 or 29, wherein the complex lipid particle contains lipids from at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different subclasses.

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

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

33. 12. The complex lipid formulation of claim 11, wherein the complex lipid particle contains less than 20 mole percent exogenous ionizable lipid.

34. 34. The complex lipid formulation of claim 33, wherein the complex lipid particle contains less than 5 mole percent exogenous ionized lipid.

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

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

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

38. 38. The complex lipid formulation of claim 37, wherein the non-citrus plant is dragon fruit, 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. 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. 40. The complex lipid formulation of claim 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 additional synthetic lipid.

44. The complex lipid particles are, based on the amount of total lipid in the complex lipid formulation, about 10-95% w / w of said plant lipid, about 5-60% w / w of said sterol, and 40. The complex lipid formulation of claim 39, comprising about 0.5-15% w / w of the polyethylene glycol (PEG)-lipid conjugate.

45. The complex lipid particles are, based on the amount of total lipid in the complex lipid formulation, about 85-95% w / w of said plant lipids, about 5-8% w / w of said sterol, and 45. The complex lipid formulation of claim 44, comprising about 1-3.5% w / w of the polyethylene glycol (PEG)-lipid conjugate.

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

47. 47. The complex lipid formulation of claim 46, wherein the complex lipid particles have an average size of about 100-180 nm.

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

5.

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

4.

50. 12. The complex lipid formulation of claim 11, wherein the complex lipid particles further comprise 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 4°C for at least two weeks without lyophilization.

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

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

56. 1. A method for delivering a peptide, polypeptide, or protein to a mammalian cell or mammal, comprising: contacting said mammalian cells with a complex lipid formulation or administering said mammal to a complex lipid formulation under conditions sufficient to permit uptake of said complex lipid formulation by said mammalian cells or by said mammal; The complex lipid formulation comprises: a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and and one or more exogenous peptides, polypeptides, or proteins encapsulated in said composite lipid particles, said composite lipid particles having the following characteristics: i) containing less than 50% w / w protein material endogenous to said one or more plant sources; and ii) containing less than 50 mol % ionized lipids.

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

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

59. 57. 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. 57. The method of claim 56, wherein the mammalian cell is a brain cell.

61. 1. A method of treating or preventing a disease or disorder in a subject in need of a therapeutic agent, comprising: To the subject in need thereof, a plurality of composite lipid particles, each composite lipid particle of the plurality comprising at least five lipids extracted from one or more botanical sources and at least two exogenous lipids; and and administering an effective amount of a complex lipid formulation comprising one or more exogenous peptides, polypeptides, or proteins encapsulated in said complex lipid particles, said complex lipid particles having the following characteristics: i) containing less than 50% w / w protein material endogenous to said one or more plant sources; and ii) containing less than 50 mol % ionized lipids.

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

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

64. 1. A method of producing a complex lipid formulation comprising a plurality of complex lipid particles encapsulating an exogenous peptide, polypeptide, or protein, 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 loading the composite lipid particle with the exogenous peptide, polypeptide, or protein, whereby the exogenous peptide, polypeptide, or protein is encapsulated by the composite lipid particle, thereby forming the composite lipid formulation.

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

66. 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, dimethylsulfoxide:methanol, acetonitrile:ethanol, or ethyl acetate:ethanol.

67. 65. The method of claim 64, wherein the extraction process further comprises reducing to less than 50% w / w or removing protein material endogenous to the one or more plant sources.

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

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

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

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