Oral delivery of plant derived vesicles and uses thereof

Abstract

According to various aspects of this disclosure, the present disclosure relates to a composition or capsule comprising a plurality of plant derived vesicles (PDVs) which are coated with a polymer. In some aspects, the plurality of coated PDVs are lyophilized. In some aspects, the plurality of PDVs are derived from spinach, cabbage, or any combination thereof.

Description

ORAL DELIVERY OF PLANT DERIVED VESICLES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Application No. 63 / 501,590 filed May 11, 2023, the contents of which are incorporated herein by reference in their entirety.GOVERNMENT FUNDING

[0002] This invention was made with government support under grant number CA209904 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to plant derived vesicles.BACKGROUND

[0004] Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells, which can play important roles in intercellular communication. EVs can be used to transfer bioactive components, and the EVs themselves are being evaluated as potential therapeutic agents. However, EVs from cultured animal cells have many limitations (e.g., triggering an undesired immune response, difficulty in scale up, etc.).

[0005] Plant-derived vesicles (PDVs) are nano-sized vesicles that are produced by most plants. Different species of plants have PDVs containing phytochemicals and other compounds that have therapeutic or preventive value. Metabolomic analysis of PDVs has also been performed. For example, in PDVs from ginger, the phytochemical shogaol was identified (Zhuang, X., et al., J. Extracell Vesicles 4:28713 (2015)), while broccoli- derived PDVs contain sulforaphane, a compound of the isothiocyanate group (Deng, Z., et al., Mol. Ther. 25: 1641-1654 (2017)). The flavonoid naringenin has been found in grapefruit-EV (Wang, B., et al., Mol. Ther. 22:522-534 (2014)). Ascorbic acid has beenfound in strawberry-derived EVs (416 nanomoles / mg EVs) (Perut, F., et al., Biomolecules 11 :87 (2021)).

[0006] Studies have noted anti-cancer properties of certain PDVs from different plants, thus suggesting use as potential therapeutic compounds in combination with current treatments in cancer management (see Raimondo, S., et al., Oncotarget 6: 19514-19527 (2015); Cao, M., et al., J. Immunother. Cancer 7:326 (2019); Lee, R., et al. J. Extracell. Vesicles 9: 1703480 (2020); Kim, K., et al., J. Funct. Biomater. 11 :22 (2020); Yang, M., et al., J. Nanobiotechnol. 18: 100 (2020); Potesta, M., et al., Cell Death Discov., 6:43 (2020); Stanly, C., et al., Cells 9:2722 (2020)). For example, PDVs from citrus limon juice were able to inhibit cell proliferation of A549 (human lung carcinoma cell line), LAMA84 (human chronic myeloid leukemia cell line), and SW480 (human colorectal adenocarcinoma cell line). Lemon-derived PDVs have been shown to suppress cell growth and induce apoptosis in AGD and BGC-823 cells (Tamura, R.E., et al., Curr. Mol. Med. 12:634-651 (2012)). PDVs derived from four Citrus species: C. sinensis, C. limon, C. paradisi, and C. aur antium negatively influenced cell growth of A375 (human melanoma), A549, and MCF-7 (human breast carcinoma) but not of HaCat (human keratinocytes) cells (Stanly, C., et al., Cells 9:2722 (2020)).

[0007] Some plant-derived EVs have anti-inflammatory properties (see Ju, S., et al., Mol. Ther. 21 : 1345-1357 (2013); De Robertis, M., et al., Biomolecules 10:742 (2020); Chen, X., et al., Mol. Pharm. 16:2690-2699 (2019); Rahimi Ghiasi, M., et al., Adv. Biomed. Res. 7: 125 (2018); Deng, Z., et al., Mol. Ther. 25: 1641-1654 (2017); Mu, J., et al., Mol. Nutr. Food Res. 58: 1561-1573 (2014); Zhang, M., et al., Biomaterials 101 :321-340 (2016)). Different groups found that grape exosomes-like nanoparticles (GELNs) have a protective effect against dextran sulfate sodium (DSS)-induced colitis (Ju, S., et al., Mol. Ther. 21 : 1345-1357 (2013), Rahimi Ghiasi, M., et al., Adv. Biomed. Res. 7: 125 (2018)). In ginger-derived nanoparticles (GDNPs), a subpopulation called GDNPs 2 has been identified that possesses beneficial properties towards acute colitis and could prevent chronic colitis and colitis-associated cancer (Zhang, M., et al., Biomaterials 101 :321-340 (2016)). Broccoli-derived nanovesicles were able to contrast the increase of pro- inflammatory cytokines, such as TNF-a, IL-17A, and IFN-y, in colonic tissues of two colitis models (DSS-induced and T cell transfer model of colitis) (Deng, Z., et al., Mol. Ther. 25: 1641-1654 (2017)).

[0008] The effects of several PDVs (cilantro, aloe vera, grapefruit, garlic, turmeric, dandelion, lavender, cactus, and ginger) on NLRP3 inflammasome activation, a biological process involved in the initiation and progression of autoinflammatory, neurodegenerative, and metabolic diseases has also been investigated (Malik, A., J. Cell Sci. 130:3955-3963 (2017)). Ginger-derived exosome-like nanoparticles suppressed the oligomerization of the apoptotic speck protein containing a caspase recruitment domain; PDV activity seems to be attributed to their lipid content (Chen, X., et al., Mol. Pharm. 16:2690-2699 (2019)). Recent evidence shows that blueberries-derived exosome-like nanoparticles also have anti-inflammatory properties on human endothelial cells (EA.hy926) (De Robertis, M., et al., Biomolecules 10:742 (2020)). After oral administration, ginger PDVs conjugated with methotrexate (MTX) (GMTX) preferentially localized in macrophages of lamina propria and reduced body weight, colon length shortening, and colon tissue damages in treated mice with respect to the control groups (MTX alone and PBS groups) (Wang, B., et al., Mol. Ther. 22:522-534 (2014)). Additionally, ginger-derived exosomes such as nanovesicles (GDENs) coated with folic acid were able to target and deliver survivin siRNA to tumor sites in vivo (Garg, H., et al., Cancer Cell Int., 16:49 (2016)).

[0009] The harsh environment in gastrointestinal tract including stomach acidic pH and enzymes in the gastrointestinal tract can destroy the PDVs.BRIEF SUMMARY

[0010] Certain aspects of the disclosure are directed to a composition or capsule comprising a plurality of plant derived vesicles (PDVs) which are coated with a polymer. In some aspects, the plurality of coated PDVs are lyophilized.

[0011] Certain aspects of the disclosure are directed to a capsule comprising a plurality of plant derived vesicles (PDVs), wherein the capsule is coated with a polymer. In some aspects, the plurality of PDVs are coated with a polymer. In some aspects, the plurality of PDVs are lyophilized.

[0012] In some aspects, the plurality of PDVs are derived from spinach, cabbage, or any combination thereof.

[0013] In some aspects, the plurality of PDVs are derived from cabbage.

[0014] In some aspects, the plurality of PDVs are derived from spinach.

[0015] In some aspects, one or more of the plurality of PDVs comprises a therapeutic agent (e.g., a therapeutic agent is encapsulated in a PDV, e.g., a coated PDV).

[0016] In some aspects, the therapeutic agent is a siRNA, a miRNA, a mRNA, a protein, a plasmid, a DNA, or a small molecule

[0017] In some aspects, the therapeutic agent is an aminosalicylatea, a corticosteroid, an immunomodulator, an antibiotic or an antidiarrheal agent.

[0018] In some aspects, the polymer coating comprises a methacrylic acid-methyl methacrylate copolymer (e.g., a Eudragit L, S, FS or E polymer, such as Eudragit S100 or Eudragit L-100).

[0019] In some aspects, the polymer coating comprises 1 mg / ml to 4 mg / ml (e.g., 2 mg / ml) methacrylic acid-methyl methacrylate copolymer.

[0020] In some aspects, the polymer coating comprises 2% to 20% methacrylic acid- methyl methacrylate copolymer.

[0021] In some aspects, the polymer coating comprises 4% methacrylic acid-methyl methacrylate copolymer.

[0022] In some aspects, the polymer coating comprises 14% methacrylic acid-methyl methacrylate copolymer.

[0023] In some aspects, the plurality of PDVs coated with two or more layers of the polymer. In some aspects, the polymer coating comprises a layer of 4% methacrylic acid- methyl methacrylate copolymer and a layer of 14% methacrylic acid-methyl methacrylate copolymer.

[0024] In some aspects, one or more of the plurality of PDVs comprise an antiinflammatory compound (e.g., an anti-inflammatory compound is encapsulated in a PDV, e.g., a coated PDV). In some aspects, the anti-inflammatory compound is selected from one or more compounds in Table 2, Table 3, and / or Table 4.

[0025] In some aspects, the anti-inflammatory compounds comprise choline; betaine; trigonelline; DL-tryptophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L-cysteine-S- oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L-glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL- serine; DL-homoserine; DL-arginine; and / or combinations thereof.

[0026] In some aspects, the anti-inflammatory compounds comprise choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4-indolecarbaldehyde; trans- 3 -indoleacrylic acid; Y-aminobutyric acid (GABA); and / or combinations thereof.

[0027] In some aspects, the capsule is suitable for oral administration.

[0028] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least70% of the coated plurality of PDVs remain intact at a pH of 1 to 2 for at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes.

[0029] In some aspects, at least 70% of the plurality of coated PDVs remain intact at a pH of 1 to 2 for at least 30 minutes.

[0030] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact for at least 30 minutes to 4 hours, at least 1 to 4 hours, at least 1 to 3 hours or at least 1 to 2 hours at a pH of 1 to 2. In some aspects, the at least 70% of the plurality of coated PDVs remain intact for at least 30 minutes at a pH of 1 to 2.

[0031] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.

[0032] Certain aspects of the disclosure are directed to the use of a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) disclosed herein for treating a subject suffering from an inflammatory disease.

[0033] In some aspects, the inflammatory disease is inflammatory bowel disease, colitis, ulcerative colitis, Crohn’s Disease, Rheumatoid arthritis, Psoriasis, Chronic obstructive pulmonary disease (COPD), or vasculitis. In some aspects, the use is for delivery to the colon of the subject, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon of the subject.

[0034] Certain aspects of the disclosure are directed to a method of preparing a capsule for oral delivery comprising (a) isolating a plurality of plant derived vesicles (PDVs) from cabbage or spinach; (b) coating one of more of the plurality of PDVs with a methacrylic acid-methyl methacrylate copolymer (e.g., 2 mg / ml methacrylic acid-methyl methacrylate copolymer); (c) lyophilizing the plurality of coated PDV; (d) filling a capsule with the lyophilized plurality of coated PDV; and optionally, (e) coating the capsule. In some aspects, the coated plurality of PDVs remain intact at a pH of 1 to 2 longer than a plurality of uncoated PDVs. In some aspects, the coated capsule to remain intact at a pH of 1 to 2 longer than an uncoated capsule.

[0035] In some aspects, the plurality of PDVs is derived from cabbage.

[0036] In some aspects, the plurality of PDVs is derived from spinach.

[0037] In some aspects, the isolating comprises (i) disinfecting the cabbage or spinach;(ii) blending the cabbage or spinach; (iii) filtering the blended cabbage or spinach to form a cabbage or spinach juice; (iv) sequentially centrifuging the cabbage or spinach juice to form a supernatant; (v) centrifuging the supernatant into a pellet comprising the plurality of PDVs; (vi) suspending the plurality of PDVs in solution, optionally phosphate buffered saline (PBS); or any combination thereof.

[0038] In some aspects, the isolating comprises the steps of FIG. 1.

[0039] In some aspects, the isolating comprises (i) blending the cabbage or spinach (e.g., the leaves) to form a juice; (ii) sequentially centrifuging the cabbage or spinach juice to form a supernatant comprising a plurality of PDVs; (iii) centrifuging the supernatant of step (ii) to form a pellet comprising the plurality of PDVs; (iv) suspending the pellet comprising the plurality of PDVs in solution; (v) washing the plurality the suspension of PDVs of step (iv); and (vi) filtering the washed PDVs of step (v), wherein the flow through from the filter comprises the isolated plurality of PDVs.

[0040] In some aspects, the lyophilizing comprises (i) mixing the plurality of PDVs to create a mixture; (ii) freezing the mixture in liquid nitrogen; and (iii) lyophilizing the frozen mixture to obtain the lyophilized plurality of PDVs.

[0041] In some aspects, the coating comprises a methacrylic acid-methyl methacrylate copolymer (e.g., a Eudragit L, S, FS or E polymer, such as Eudragit SI 00 or Eudragit L- 100).

[0042] In some aspects, the coating comprises 1 mg / mL - 4 mg / ml methacrylic acid- methyl methacrylate copolymer.

[0043] In some aspects, the coating comprises 2 mg / ml methacrylic acid-methyl methacrylate copolymer.

[0044] In some aspects, the coating comprises 2% to 20% methacrylic acid-methyl methacrylate copolymer. In some aspects, the coating comprises 14% methacrylic acid- methyl methacrylate copolymer.

[0045] In some aspects, the coating comprises a first coat with 4% methacrylic acid- methyl methacrylate copolymer and a second coat with 14% methacrylic acid-methyl methacrylate copolymer.

[0046] In some aspects, the coating comprises a first coat with 4% methacrylic acid- methyl methacrylate copolymer, a second coat with 4% methacrylic acid-methylmethacrylate copolymer, a third coat with 14% methacrylic acid-methyl methacrylate copolymer, and a fourth coat with 14% methacrylic acid-methyl methacrylate copolymer.

[0047] In some aspects, one or more of the plurality of coated PDVs comprises an antiinflammatory compound (e.g., the anti-inflammatory compound is encapsulated in the PDV). In some aspects, the anti-inflammatory compound is selected from one or more compounds in Table 2, Table 3, and / or Table 4.

[0048] In some aspects, the anti-inflammatory compound comprises choline; betaine; trigonelline; DL-tryptophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L-cysteine-S- oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L-glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL- serine; DL-homoserine; DL-arginine; or any combination thereof.

[0049] In some aspects, the anti-inflammatory compound comprises choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4-indolecarbaldehyde; trans- 3 -indoleacrylic acid; Y-aminobutyric acid (GABA); or any combination thereof.

[0050] In some aspects, the one or more of the plurality of PDVs comprise a therapeutic agent (e.g., a therapeutic agent is encapsulated in a PDV).

[0051] In some aspects, provided herein is a composition comprising a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) produced by any of the methods disclosed herein.

[0052] In some aspects, provided herein is a method of treating inflammation in the intestine of a subject in need thereof comprising administering to the subject a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) disclosed herein. In some aspects, the administration is oral. In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.

[0053] In some aspects, the inflammation is caused by an inflammatory disease.

[0054] In some aspects, the inflammatory disease is an inflammatory bowel disease, colitis, ulcerative colitis, Crohn’s Disease, Rheumatoid arthritis, Psoriasis, Chronic obstructive pulmonary disease (COPD), or vasculitis.DESCRIPTION OF FIGURES

[0055] Figure 1 (FIG. 1) shows an exemplary schematic for plant derived vesicle (PDV) isolation.

[0056] Figures 2A-2D (FIGs. 2A-2D) show PDVs exposed to conditions mimicking the GI System. FIG. 2A shows Transmission electron microscopy (TEM) imaging and quantification of PDVs exposed to different GI pH mimicking conditions as indicated, with arrows representing broken vesicles (n=3). FIG. 2B shows Nanoparticle Tracking Analysis (NTA) particle number in samples exposed to pH changes mimicking the GI system (n=3). FIG. 2C shows cryo-EM images of PDVs after 30 minutes at 37 °C in phosphate buffered saline (PBS) control (untreated) or PBS at a pH of 1.5 (left panels), as well as the percentages of PDVs determined by cryo-EM imaging to be misshapen / broke, or intact after treatment with PBS control or PBS at a pH of 1.5 (right panel). FIG. 2D shows structural appearance and quantitative analysis of cabbage plant-derived vesicles (PDVs) after acidic pH and enzyme treatment via TEM, with red arrow heads indicated broken and destroyed PDVs (n=3) (bars = 50 nm) (error bars in SD).

[0057] Figures 3A-3D (FIGs. 3A-3D) show structural appearance and quantitative analysis of cabbage plant-derived vesicles (PDVs) that are either uncoated or coated and subjected to lyophilization. Analysis was done via TEM (FIGs. 3A-3B) and NTA (FIGs. 3C-3D). n=3, bars = 50 nm, and error bars are in SD.

[0058] Figures 4A-4D (FIGs. 4A-4D) show average mouse body weight (FIG. 4A, error bars in SEM), colon length (FIG. 4B), H&E staining and histoscore after treatment (FIG. 4C, bars = 100 pm), IL 1-P levels (FIG. 4D, left panel), IL-6 levels (FIG. 4D, middle panel) and TNF-a levels (FIG. 4D, right panel) relative to 36B4 from colon after 1) a no treatment control group, 2) mice treated with DSS and PBS, 3) mice treated with DSS and PDV in PBS, and 4) mice treated with DSS and capsulized lyophilized PDVs (CapPDVs) (n=5, dose is 1.6 mg). Error bars for FIGs. 4B-4D are in SD.

[0059] Figures 5A-5F (FIGs. 5A-5F) show coated PDV characterization. FIG. 5A shows TEM imaging and quantification of PDVs after Eudragit SI 00 coat (Error bars in SD, n=3, bars = 100 nm). FIG. 5B shows NTA for size and particle number change after Eudragit SI 00 coating (Error bars in SD, n=3). FIG. 5C shows Cytek Aurora flow cytometry test Eudragit S100 coating efficiency to PDVs. FIG. 5D shows TEM imaging and quantification of cPDVs exposed to upper GI pH and pepsin. FIG. 5E and FIG. 5Fshow IL 1-P levels (FIG. 5E) and TNF-a levels (FIG. 5F) after treatment with PDV in capsule.

[0060] Figures 6A-6B (FIGs. 6A-6B) show comparative tissue and organ distribution between PDVs in PBS and capsules containing coated PDVs (CAP-cPDVs). FIG. 6A shows fluorescent imaging of DAPI stained nuclei and red fluorescence showing CELLMask Deep Red PDVs (bars = 5 pm). FIG. 6B shows IVIS imaging (n=3, dose is 1 6 mg).

[0061] Figures 7A-7E (FIGs. 7A-7E) show Cap-cPDVs had an enhanced therapeutic effect on DSS-induced colitis than PDVs in PBS. FIG. 7A shows average mice body weight and SEM after PDVs in PBS and CAP-cPDVs treatment (error bars in SEM). FIG. 7B shows colon length comparison with control, DSS, DSS+PDVs, DSS+cap-cPDVs. FIGs. 7C and 7D show histoscore and H&E comparison between the four groups (bars = 100 pm). FIG. 7E shows relative cytokine expression level to 36B4 from colon after treatments (n=5, dose is 1.6 mg). Error bars for FIGs. 7B-7E are in SD.

[0062] Figure 8 (FIG. 8) shows TEM imaging and quantification of PDVs exposed in pancreatin (error bars in SD, n=3, bars = 50 nm).

[0063] Figures 9A-9B (FIGs. 9A-9B) show compounds present in PDVs. FIG. 9 A shows different compounds as measured by LC-MS (n=3). FIG. 9B shows further LC-MS analysis for choline and I3C by standards of comparison. Error bars in SD.

[0064] Figure 10 (FIG. 10) shows PDV yield comparison between cabbage, spinach, mushroom, and ginger (Error bars in SD, n=3).

[0065] Figure 11 (FIG. 11) shows extracellular vesicle protein marker test for cabbage PDVs with proteomic tests. Y axis shows peptide-spectrum match (PSM). Three extracellular vesicle positive marker Tetraspanin-3, Annexin DI, Heat Shock 70 kDa and one negative marker Apoliprotein Al are shown in the figure (error bars in SD, n=2).

[0066] Figure 12 (FIG. 12) shows DLS size of PDVs suspended in PBS to PDVs subjected to lyophilization followed by resuspension in PBS.

[0067] Figures 13A-13F (FIGs. 13A-13F) show the process of gavaging the mice with capsulized non-coated PDVs.

[0068] Figures 14A-14B (FIGs. 14A-14B) show biodistribution of PDVs using cooper- 64 labeled cabbage PDVs and PET scanning.

[0069] Figures 15A-15C (FIGs. 15A-15C) show mouse body weight (FIG. 15 A), colon length (FIGs. 15B), and H&E staining and pathology evaluation of colon (FIG. 15C) formice treated with DSS for inducing colitis at 2%, 3%, or 4% DSS. Error bars in SD, n=3, bars = 100 gm.

[0070] Figures 16A-16B (FIGs. 16A-16B) show CellMask Deep Red stained cabbage PDVs biodistribution in mice. FIG. 16A shows fluorescence detection in mouse organs after oral gavage of CellMask Deep Red-dyed cabbage PDVs that were encapsulated and left uncoated or were coated with Eudragit S-100 or Eudragit L-100. FIG. 16B shows In Vivo Imaging System (IVIS) images of fluorescence in stomach, small intestine, and colon of mice gavaged with dyed cabbage PDVs suspended in PBS or mice gavaged with Eudragit S100 coated capsule loaded with cabbage PDVs. n=3, bars = 50 gm.

[0071] Figures 17A-17G (FIGs. 17A-17G) show cabbage PDV lyophilization, encapsulation, coat application, and gavage delivery. FIG. 17A shows cabbage PDVs in PBS. FIG. 17B shows cabbage PDVs after lyophilization. FIG. 17C shows negativestaining transmission electron microscopy (TEM) of cabbage PDVs suspended in PBS. FIG. 17D shows TEM of vesicles intact after lyophilization and re-suspension. FIG. 17E shows an M-size capsule funnel kit that was used to fill capsules with premeasured powder. FIG. 17F capsules coated with Eudragit and held by an X-HOLD M holder. FIG. 17G shows a lab-modified capsule gavage needle with a Eudragit-coated capsule at the tip.

[0072] Figures 18A-18D (FIGS. 18A-18D) show cabbage PDVs for mice colitis treatment. FIG. 18A shows body weight comparisons mice with dextran sulfate sodium (DSS) induced colitis treated with PBS, mice with DSS induced colitis treated with Cabbage PDV in PBS, and no-treatment control. FIG. 18B shows colon length comparisons of mice with DSS induced colitis treated with PBS, mice with DSS induced colitis treated with Cabbage PDV in PBS, and no-treatment control. FIG. 18C shows body weight comparison between mice with DSS induced colitis treated with coated capsulized cabbage PDV and mice with DSS induced colitis treated with cabbage PDV in PBS with an empty capsule daily. FIG. 18D show colon length comparison of mice with DSS induced colitis treated with coated capsulized cabbage PDV and mice with DSS induced colitis treated with cabbage PDV in PBS with an empty capsule daily.

[0073] Figures 19A-19B (FIGs. 19A-19B) show cabbage PDV in PBS and Eudragit SI 00 coated capsule loaded lyophilized cabbage PDV biodistribution in mice. FIG. 19A fluorescence biodistribution in stomach, small intestine, and colon. FIG. 19B shows colonlength in mice with 1) control, 2) DSS treatment, 3) DSS and PDV treatment, and 4) DSS and lyophilized coated PDV (Cap-cPDV) treatment.DETAILED DESCRIPTION

[0074] In some aspects, provided herein is a composition or capsule comprising a plurality of plant derived vesicles (PDVs) which are coated with a polymer. In some aspects, the plurality of coated PDVs are lyophilized. In some aspects, the plurality of PDVs are lyophilized and the plurality of PDVs are derived from spinach, cabbage, or a combination thereof. In some aspects, the PDVs are derived from spinach. In some aspects, the PDVs are derived from cabbage.

[0075] In some aspects, provided herein is a capsule comprising a plurality of plant derived vesicles (PDVs), wherein the capsule is coated with a polymer.

[0076] In some aspects, provided herein is a method of preparing a capsule for oral delivery comprising (a) isolating a plurality of plant derived vesicles (PDVs) from cabbage or spinach; (b) coating one of more of the plurality of PDVs with a methacrylic acid-methyl methacrylate copolymer (e.g., 2 mg / ml methacrylic acid-m ethyl methacrylate copolymer); (c) lyophilizing the plurality of coated PDV; (d) filling a capsule with the lyophilized plurality of coated PDV; and optionally, (e) coating the capsule. In some aspects, the coated plurality of PDVs remain intact at a pH of 1 to 2 longer than a plurality of uncoated PDVs. In some aspects, the coated capsule to remain intact at a pH of 1 to 2 longer than an uncoated capsule.

[0077] In some aspects, provided herein is a method of treating inflammation in the intestine of a subject in need thereof comprising administering to the subject a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) disclosed herein. In some aspects, the administration is oral. In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.I. Definitions

[0078] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application including the definitionswill control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0079] Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description and from the claims.

[0080] In order to further define this disclosure, the following terms and definitions are provided.

[0081] The singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein. In certain aspects, the term "a" or "an" means "single." In other aspects, the term "a" or "an" includes "two or more" or "multiple."

[0082] The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower).

[0083] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Numeric ranges recited are inclusive of the numbers defining the range and include each integer within the defined range.

[0084] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within the disclosure. Thus, ranges recited herein are understood to be shorthand for all of the values within the range, inclusive of the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0085] Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of the disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of a disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0086] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0087] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, formulations, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0088] The term "excipient" refers to any substance, not itself a therapeutic agent, which may be used in a composition for delivery of an active therapeutic agent to a subject or combined with an active therapeutic agent (e.g., to create a pharmaceutical composition) to improve its handling or storage properties or to permit or facilitate formation of a dose unit of the composition (e.g., a capsule comprising a PDV). Excipients include, but are not limited to, solvents, penetration enhancers, wetting agents, antioxidants, lubricants, emollients, substances added to improve appearance or texture of the composition and substances used to form hydrogels. Any such excipients can be used in any dosage forms according to the present disclosure. The foregoing classes of excipients are not meant to be exhaustive but merely illustrative as a person of ordinary skill in the art would recognize that additional types and combinations of excipients could be used to achieve the desired goals for delivery of a drug. The excipient can be an inert substance, an inactive substance, and / or a not medicinally active substance. The excipient can serve various purposes. A person skilled in the art can select one or more excipients with respect to the particular desired properties by routine experimentation and without any undue burden. The amount of each excipient used can vary within ranges conventional in the art. Techniques and excipients which can be used to formulate dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005). In some aspects, the excipient comprises trehalose, polyethylene glycol (PEG), a cholesterol, a phospholipid, a surfactant, or any combination thereof.

[0089] The term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" as used herein refers to the amount or quantity of a drug or pharmaceutically active substance which is sufficient to elicit the required or desired therapeutic response, or in other words, the amount which is sufficient to elicit an appreciable biological response when administered to a patient.

[0090] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation,phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain aspects, the polypeptides can occur as single chains or associated chains.

[0091] The terms "administer," "administering," "administration," and the like, as used herein, refer to methods that may be used to enable delivery of a drug, e.g., a polymer coated capsule comprising a plurality of plant derived vesicles to the desired site of biological action (e.g., intravenous administration). Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0092] The terms "treat," "treatment," or "treating," as used herein refers to, e.g., the reduction in severity of a disease or condition; the reduction in the duration of a disease course; the amelioration or elimination of one or more symptoms associated with a disease or condition; the provision of beneficial effects to a subject with a disease or condition, without necessarily curing the disease or condition. The term also include prophylaxis or prevention of a disease or condition or its symptoms thereof. In some aspects, the term "treating" or "treatment" means reducing inflammation (e.g. inflammation caused by inflammatory diseases such as inflammatory bowel disease).

[0093] The terms "subject," "patient," "individual," and "host," and variants thereof are used interchangeably herein and refer to any mammalian subject, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like), and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like) for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. As used herein, the phrase "subject in need thereof' includes subjects, such as mammalian subjects, that would benefit from administration of a therapeutic agent, e.g., a polymer coated capsule comprising a plurality of plant derived vesicles.

[0094] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0095] As used herein, the terms "derived from" or "derivative" refer to a component that is isolated from or made using a specific plant (e.g., a cabbage plant).

[0096] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle comprising a membrane that encloses an internal space. Generally extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular payload either within the internal space (i.e., lumen), displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. Said payload can comprise nucleic acids, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. In some aspects, an extracellular vesicle comprises an antiinflammatory compound. Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, prokaryotic or eukaryotic cells, and / or cultured cells.

[0097] As used herein, the term "plant derived vesicles" or "plant-derived vesicles" refer to a vesicle (e.g., an extracellular vesicle or nanovesicle) that is derived from plant cells (e.g., cabbage or spinach species) or fungi cells (e.g., mushroom species). In some aspects, the plant-derived vesicles range in diameter from about 100 nm to about 300 nm. In some aspects, the plant-derived vesicles range in diameter from about 100 nm to about 250 nm.

[0098] As used herein, the term "remains intact" refer to plant-derived vesicles who maintain the structural integrity of their phospholipid membrane after exposure to certain conditions (e.g., low pH levels).II. Plant Derived Vesicles

[0099] Plant-derived vesicles (PDVs) can contain functional biomolecules such as proteins, lipids, RNAs, and metabolites, which can mediate cell-cell communication. The physiological role of PDVs appears to be related mainly to plant immune response (Urzi, O., et al., In. J. Mol. Sci. 22(10):5366 (2021); Raimondo, S., et al., Metabolites 11(5):276 (2021); Rutter, B.D., et al., Plant Physiol. 173:728-741 (2017); Woith E., et al., Int J Mol Sci. 22(7):3719 (2021)) and plant-microbe symbiosis (Ivanov, S. et al., Nat. Plants 5: 194-203 (2019); Roth R., et al., Nat. Plants 5:204-211 (2019)). However, PDVs have been shown to interact with mammalian cells.

[0100] The major lipid species found in PDVs are phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Phospatidic acid was described in the vesicular fraction of sunflower apoplastic fluid (Regente, M., et al., FEBS Lett. 583:3363-3366 (2009)), and it is enriched in grape-PDVs compared to the whole juice (Ju, S., et al., Mol. Ther. 21 : 1345-1357 (2013)), as well as in ginger-derived PDVs (Teng Y., Cell Host Microbe 24:637-652 (2018)).

[0101] Phospatidic acid was recently reported in nanovesicles from Uvae-ursi folium, Craterostigma plantagineum, and Zingiberis rhizoma (Woith E., et al., Int J Mol Sci. 22(7):3719 (2021)). Phosphatidylcholine was described in grapefruit (Wang, B., et al., Mol. Ther. 22:522-534 (2014)), together with PE that was also found in grape-PDVs (Ju, S., et al., Mol. Ther. 21 : 1345-1357 (2013)), and in nanovesicles from C. plantagineum (Woith E., et al., Int J Mol Sci. 22(7):3719 (2021)).

[0102] Lipidomic analysis of Arabidopsis rosette leaf PDVs and the whole leaf tissues allowed the identification of 23 classes and 279 species of lipids in PDVs. Interestingly, the PDV-lipid profile showed enrichment in sphingolipids (around 46%) in particular of glycosylinositolphosphoceramides compared to the leaf tissue (Liu, N.J., et al., Mol. Plant 13: 1523-1532 (2020)).

[0103] PDVs can interact with the microbiota and the result of this cross-talk may offer opportunities for their use as therapeutic agents (Sundaram, K., et al., iScience 21 :308- 327 (2019); Teng Y., Cell Host Microbe 24:637-652 (2018), Lei, C., iScience 23: 101571 (2020)).

[0104] PDVs as disclosed herein can be used for drug delivery because they possess several advantageous properties including safety, non-toxicity, and low immunogenicity. PDVs can also be produced on large scale and several studies have demonstrated that they are stable and resistant in the stomach- and intestinal-like solutions (Rahimi Ghiasi, M., et al., Adv. Biomed. Res. 7: 125 (2018); Zhuang, X., et al., J. Extracell Vesicles 4:28713 (2015); Wang, B., et al., Mol. Ther. 22:522-534 (2014); Zhang, M., et al., Biomaterials 101 :321-340 (2016).

[0105] Several plant species were considered including those having anti-inflammatory properties. Among the most consumed vegetables, cabbage, mushrooms, spinach and ginger have been shown to contain anti-inflammatory properties (Rokayya, S., et al.,Asian Pacific Journal of Cancer Prevention 14:6657-6662 (2013); Lomnitski, L., et al., Nutrition and Cancer 46:222-231 (2003); Muszynska, B., et al., Food Chemistry 243:373- 381 (2018); and Shayesteh, F., et al., Trials 21 :1-6 (2020)). In some aspects, provided herein is a composition or capsule comprising a plurality of plant derived vesicles (PDVs) which are coated with a polymer. In some aspects, the plurality of coated PDVs are lyophilized.

[0106] In some aspects, provided herein is a capsule comprising a plurality of plant derived vesicles (PDVs), wherein the capsule is coated with a polymer. In some aspects, the plurality of PDVs are coated with a polymer. In some aspects, the plurality of PDVs are lyophilized.

[0107] In some aspects, the plurality of PDVs are derived from cabbage. In some aspects, the cabbage species is Brassica oleracea. Brassica oleracea is a capitata L. cultivar. In some aspects, the cabbage is Brassica oleracea var. capitata L. The Capitata Group of Brassica oleracea has hearty leaf plants and grows best at cooler temperatures, typically around 60 degrees. In some aspects, the best growing conditions include full sun, as shade can slow the growing process. Common cabbage has dark green leaves. Dutch cabbage has a larger heart and leaves and a milder flavor and is pale green or even white. Red cabbage has purplish colored leaves. They are in flower from May to August and ready for harvest from July to September. A head of cabbage can weigh between 1 to 9 lbs. A versatile plant, it can be pickled, fermented, steamed, stewed, braised, sauteed, or eaten raw. The shredded leaves can be used in salads. Dutch cabbage is the tenderer and sweeter variety, most suitable for eating raw. Red cabbage is used for pickling. Cabbages have a large terminal bud that is edible. Savoy cabbage has puckered leaves that can be shredded to make coleslaw. In some aspects, the cabbage is a Savoy Cabbage (e.g., Brassica oleracea var. sabauda L), a Green Cabbage, a Red Cabbage, a White cabbage, an Ornamental cabbage (e.g., Brassica oleracea var. acephala), an Alcosa cabbage, a Blue Dynasty cabbage, a Blue Vantage cabbage, a Caraflex cabbage, a Charleston Wakefield cabbage, a Cheers cabbage, a Dynamo cabbage, an Early Jersey Wakefield cabbage, a Famosa cabbage, a Farao cabbage, a Golden Acre cabbage, a Red Acre cabbage, a Red Express cabbage, a Rubyball cabbage, a Ruby Perfection cabbage, a Samantha cabbage, or a Savory Ace cabbage. In some aspects, the cabbage is a Red Cabbage.

[0108] In some aspects, the plurality of PDVs derived from cabbage comprise antiinflammatory compounds. In some aspects, the anti-inflammatory compounds comprisecholine; betaine; trigonelline; DL-tryptophan; L-isoleucine; L-phenyl alanine; I3C; S- methyl-L-cysteine-S-oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L-glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L- aspartic acid; DL-serine; DL-homoserine; DL-arginine; and / or combinations thereof. In some aspects, the anti-inflammatory compounds are disclosed in Table 1 below.

[0109] In some aspects, S-methyl-L-cysteine-S-oxide is also a cruciferous biomarker. In some aspects, 9S,13R-12-oxophytodienoic acid (12-OPDA) and trigonelline are also plant hormone related. In some aspects, Indole-3 -Carbinol, DL-tryptophan, iberin, sulforaphane, L-iditol, and pentaerythritol are also secondary metabolites. In some aspects, choline, betaine, L-glutamic acid, valine, L-(+)-citrulline, D-(+)-proline, D-(+)- pyroglutamic acid, L-phenylalanine, L-aspartic acid, L-isoleucine, DL-serine, DL- homoserine, and DL-arginine are involved in metabolism.

[0110] In some aspects, the plurality of PDVs are derived from spinach. In some aspects, the spinach is Spinacia oleracea. In some aspects, spinach comprises cultivated spinach, such as breeding lines (e.g. backcross lines, inbred lines), cultivars and varieties (open pollinated or hybrids). This includes any type of spinach, such as savoy, flat- or smoothleaf spinach or semi-savoy types. In some aspects, spinach comprises wild spinach (i.e., not cultivated spinach).

[0111] Spinach is an annual vegetable in the Amaranth family that is widely grown for use as a leafy green in salads and cooked dishes. Spinach can be grown at home in containers or beds and tolerates areas too shady for most other vegetables to thrive. Spinach grows best in moist, rich, well-drained soil in full sun to light shade. Spinach can tolerate 3 to 4 hours of sun a day, and some shade can be beneficial in hot weather.

[0112] In some aspects, the plurality of PDVs derived from spinach comprise antiinflammatory compounds. In some aspects, the anti-inflammatory compounds comprise choline, betaine, trigonelline, hypoxanthine, D-(+)-tryptophan, L-isoleucine, 4- indolecarbaldehyde, trans-3 -indoleacrylic acid, Y-aminobutyric acid (GABA), and / or combinations thereof.

[0113] In some aspects, the anti-inflammatory compounds comprise Choline, Betaine, L- Isoleucine, L-phenylalanine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, D-(+)- Tryptophan, Ecdysterone, a-Eleostearic acid, Adenosine, Guanine, Adenine, Spermine, Oleamide, and / or combinations thereof.

[0114] In some aspects, the anti-inflammatory compound comprises one or more compounds disclosed in Table 2 herein.

[0115] In some aspects, the plurality of PDVs are derived from mushroom. In some aspects, the mushroom is a Bella mushroom, a Shiitake mushroom, a Button White mushroom, a remini (Italian Brown) Crimini mushroom, a Cordyceps mushroom, a Lion's mane mushroom, a Mai take mushroom, or a Reishi mushroom.

[0116] In some aspects, the PDVs derived from mushroom comprise anti-inflammatory compounds. In some aspects, the anti-inflammatory compounds comprise Adenosine, Guanine, D-(+)-Tryptophan, L-Isoleucine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, L-Phenylalanine, Choline, Adenine, Betaine, Spermidine, Spermine, DL-arginine, Oleamide, Trigonelline, Hypoxanthine, Tangeritin, Isoquinoline, and / or combinations thereof.

[0117] In some aspects, the anti-inflammatory compound comprises one or more compounds disclosed in Table 3 herein.

[0118] In some aspects, the plurality of PDVs are derived from ginger. In some aspects, the PDVs derived from ginger comprise anti-inflammatory compounds. In some aspects, the anti-inflammatory compounds comprise 4-Methoxybenzaldehyde, Spermine, Isoquinoline, p-cymene, Crotonic acid, Isoleucine, (-)-trans-Methly dihydroj asm onate, Trans- Anethole, Ibuprofen, Pelubiprofen, L-(-)-Serine, and / or combinations thereof.

[0119] In some aspects, the anti-inflammatory compound comprises one or more compounds disclosed in Table 4 herein.III. Coated PDVs and / or Coated Capsules

[0120] Given the extensive destructive effects of the gastrointestinal tract on PDVs, a strategy was developed to protect the PDVs for successful oral delivery.

[0121] In some aspects, provided herein is a composition or capsule comprising a plurality of plant derived vesicles (PDVs) which are coated with a polymer. In some aspects, the plurality of coated PDVs are lyophilized.

[0122] In some aspects, provided herein is a capsule comprising a plurality of plant derived vesicles (PDVs), wherein the capsule is coated with a polymer. In some aspects, the plurality of PDVs are coated with a polymer. In some aspects, the plurality of PDVs are lyophilized.

[0123] In some aspects, the plurality of PDVs are derived from cabbage.

[0124] In some aspects, the plurality of PDVs are derived from spinach.

[0125] In some aspects, one or more of the plurality of PDVs comprises a therapeutic agent (e.g., a therapeutic agent is encapsulated in a PDV, e.g., a coated PDV).

[0126] In some aspects, the therapeutic agent is a siRNA, a miRNA, a mRNA, a protein, a plasmid, a DNA, or a small molecule.

[0127] In some aspects, the therapeutic agent is an aminosalicylatea, a corticosteroid, an immunomodulator, an antibiotic or an antidiarrheal agent.

[0128] In some aspects, the polymer coating comprises Poly(methacrylic acid-co-methyl methacrylate) (Eudragit L), Hydroxypropyl methylcellulose phthalate (HPMCP), Poly(N- isopropyl acrylamide) (PNIPAM), Poly(ethylene glycol)-block-poly(acrylic acid) (PEG-b- PAA), Polylactic acid (PLA), Polyethylene glycol (PEG), Poly(lactide-co-glycolide) (PLGA), or Chitosan.

[0129] In some aspects, the polymer coating comprises a methacrylic acid-methyl methacrylate copolymer (e.g., a Eudragit L, S, FS or E polymer, such as Eudragit S100 or Eudragit L-100).

[0130] In some aspects, the polymer coating comprises 1 mg / ml to 4 mg / ml (e.g., 2 mg / ml) methacrylic acid-methyl methacrylate copolymer.

[0131] In some aspects, the polymer coating comprises 2% to 20% methacrylic acid- methyl methacrylate copolymer.

[0132] In some aspects, the polymer coating comprises 4% methacrylic acid-methyl methacrylate copolymer.

[0133] In some aspects, the polymer coating comprises 14% methacrylic acid-methyl methacrylate copolymer.

[0134] In some aspects, the plurality of PDVs coated with two or more layers of the polymer. In some aspects, the polymer coating comprises a layer of 4% methacrylic acid- methyl methacrylate copolymer and a layer of 14% methacrylic acid-methyl methacrylate copolymer.

[0135] In some aspects, the polymer coating comprises between about 3% and about 15%, between about 4% and about 15%, between about 5% and about 15%, between about 8% and about 15%, between about 10% and about 15%, between about 12% and about 15%, between about 14% and about 15%, between about 4% and about 14%, between about 4% and about 8%, between about 4% and about 10%, between about 8%and about 14%, or between about 10% and about 14% of a methacrylic acid-m ethyl methacrylate copolymer.

[0136] In some aspects, the polymer coating comprises about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% of methacrylic acid-methyl methacrylate copolymer.

[0137] In some aspects, the polymer coating comprises at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14% or at least 15% of methacrylic acid-methyl methacrylate copolymer.

[0138] In some aspects, the polymer coating comprises at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14% or at least about 15% of methacrylic acid-methyl methacrylate copolymer.

[0139] In some aspects, the polymer coating comprises about 2 mg / mL methacrylic acid- methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 1 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 3 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 4 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 5 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises 1, 2, 3, 4, or 5 mg / mL methacrylic acid-methyl methacrylate copolymer.

[0140] In some aspects, the polymer coating comprises between about 1 mg / L to about 5 mg / mL of methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises between 1 mg / L to 5 mg / mL of methacrylic acid-methyl methacrylate copolymer.

[0141] EudragH® Polymers

[0142] The coating of capsules offers many advantages over uncoated counterparts, such as higher physicochemical stability, better compliance and increased therapeutic efficiency of the active ingredients. Indeed, the effectiveness of a medication depends not only on the actives it contains, but also on formulation and processing.

[0143] Poly(meth)acrylates (e.g., a methacrylic acid-methyl methacrylate copolymer) have proven particularly suitable as coating materials. These polymers, typically used inamounts of only a few milligrams, are pharmacologically inactive, i.e. are excreted unchanged.

[0144] EUDRAGIT® is the trade name for copolymers derived from esters of acrylic and methacrylic acid, whose properties are determined by functional groups. The individual EUDRAGIT® grades differ in their proportion of neutral, alkaline or acid groups and thus in terms of physicochemical properties. The skillful use and combination of different EUDRAGIT® polymers offers ideal solutions for controlled drug release in various pharmaceutical and technical applications. EUDRAGIT® provides functional films for sustained-release tablet and pellet coatings. The polymers are described in international pharmacopeias such as Ph.Eur., USP / NF, DMF and JPE.

[0145] EUDRAGIT® polymers can provide the following possibilities for controlled drug release:Gastrointestinal tract targeting (gastroresi stance, release in the colon), Protective coatings (taste and odor masking, protection against moisture), and Delayed drug release (sustained-release formulations).

[0146] EUDRAGIT® polymers are available in a wide range of different concentrations and physical forms, including aqueous solutions, aqueous dispersion, organic solutions, and solid substances.

[0147] The pharmaceutical properties of EUDRAGIT® polymers are determined by the chemical properties of their functional groups. A distinction is made between: poly(meth)acrylates, soluble in digestive fluids (by salt formation) and poly(meth)acrylates, insoluble in digestive fluids.

[0148] EUDRAGIT® L, S, FS and E polymers with acidic or alkaline groups enable pH- dependent release of the active ingredient and are soluble in digestive fluids.

[0149] Applications: from simple taste masking via resistance solely to gastric fluid, to controlled drug release in) all sections of the intestine.

[0150] EUDRAGIT® RL and RS polymers with alkaline and EUDRAGIT® NE polymers with neutral groups enable controlled time release of the active by pH-independent swelling and are insoluble in digestive fluids

[0151] Enteric Coatings

[0152] Enteric EUDRAGIT® coatings provide protection against release of the PDVs in the stomach and enable controlled release in the intestine. In some aspects, this dosage form allows the PDVs to survive the harsh environment of the stomach, so that the anti-inflammatory compounds may be delivered to the colon. The dominant criterion for release is the pH-dependent dissolution of the coating, which takes place in a certain section of the intestine (pH 5 to over 7) rather than in the stomach (pH 1-5). For these applications, anionic EUDRAGIT® grades containing carboxyl groups can be mixed with each other. This makes it possible to finely adjust the dissolution pH, and thus to define the drug release site in the intestine. EUDRAGIT® L and S grades are suitable for enteric coatings. Eudragit L-100 and S-100 dissolve above pH 6, which can allow for pH- dependent release of active ingredients and therefore potentially better colon-targeted delivery (Coban, O. et al., Colloids and Surfaces B: Biointerfaces 197: 111391 (2021); Khan, M.Z.I., et al., Journal of Controlled Release 58:215-222 (1999); Khan, M.Z.I., et al., Drug Development and industrial pharmacy 26:549-554 (2000); Subudhi, M.B., et al., Materials 8:832-849 (2015)).

[0153] In some aspects, EUDRAGIT® FS 30 D is specifically used for controlled release in the colon. In some aspects, the capsules are coated by EUDRAGIT® S-100.

[0154] Application benefits of enteric EUDRAGIT® coatings include: pH-dependent drug release, protection of actives sensitive to gastric fluid, protection of the gastric mucosa from aggressive actives, increase in drug effectiveness, good storage stability, and controlled release in the colon / GI targeting.

[0155] In some aspects, the PDV remains intact at a pH of 1 to 2. In some aspects, the PDV remains intact at a pH of 1 to 4. In some aspects, the PDV remains intact at a pH of1 to 3. In some aspects, the PDV remains intact at a pH of 1.5 to 3.5. In some aspects, the PDV remains intact at a pH of 2 to 4. In some aspects, the PDV remains intact at a pH of2 to 3. In some aspects, the PDV remains intact at a pH of 1.5 to 2.5. In some aspects, the PDV remains intact at a pH of 2.5 to 3.5.

[0156] In some aspects, the PDV remains intact at a pH of about 1 to about 2. In some aspects, the PDV remains intact at a pH of about 1 to about 4. In some aspects, the PDV remains intact at a pH of about 1 to about 3. In some aspects, the PDV remains intact at a pH of about 1.5 to about 3.5. In some aspects, the PDV remains intact at a pH of about 2 to about 4. In some aspects, the PDV remains intact at a pH of about 2 to about 3. In someaspects, the PDV remains intact at a pH of about 1.5 to about 2.5. In some aspects, the PDV remains intact at a pH of about 2.5 to about 3.5.

[0157] In some aspects, the PDV remains intact at a pH of about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, or about 4. In some aspects, the PDV remains intact at a pH of at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, or at least 4. In some aspects, the PDV remains intact at a pH of at least about 1, at least about 1.5, at least about 2, at least about 2.5, at least about 3, at least about 3.5, or at least about 4.

[0158] In some aspects, the PDV remains intact for about 2 hours. In some aspects, the PDV remains intact for about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours.

[0159] In some aspects, the PDV remains intact for at least 2 hours. In some aspects, the PDV remains intact for at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least out 4 hours, or at least 5 hours.

[0160] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the coated plurality of PDVs remain intact at a pH of 1 to 2 for at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes.

[0161] In some aspects, at least 70% of the plurality of coated PDVs remain intact at a pH of 1 to 2 for at least 30 minutes.

[0162] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact for at least 30 minutes to 4 hours, at least 1 to 4 hours, at least 1 to 3 hours or at least 1 to 2 hours at a pH of 1 to 2. In some aspects, the at least 70% of the plurality of coated PDVs remain intact for at least 30 minutes at a pH of 1 to 2.

[0163] In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.

[0164] Anti-inflammatory compounds

[0165] In some aspects, one or more of the plurality of PDVs comprise an antiinflammatory compound (e.g., an anti-inflammatory compound is encapsulated in a PDV, e.g., a coated PDV). In some aspects, the anti-inflammatory compound is selected from one or more compounds in Table 2, Table 3, and / or Table 4.

[0166] In some aspects, the anti-inflammatory compounds comprise choline; betaine; trigonelline; DL-tryptophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L-cysteine-S-oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L-glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL- serine; DL-homoserine; DL-arginine; and / or combinations thereof.

[0167] In some aspects, the anti-inflammatory compounds comprise choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4-indolecarbaldehyde; trans- 3 -indoleacrylic acid; Y-aminobutyric acid (GABA); and / or combinations thereof. In some aspects, the anti-inflammatory compounds comprise Choline, Betaine, L-Isoleucine, L- phenylalanine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, D-(+)-Tryptophan, Ecdysterone, a-Eleostearic acid, Adenosine, Guanine, Adenine, Spermine, Oleamide, and / or combinations thereof.

[0168] In some aspects, the anti-inflammatory compounds comprise Adenosine, Guanine, D-(+)-Tryptophan, L-Isoleucine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, L- Phenylalanine, Choline, Adenine, Betaine, Spermidine, Spermine, DL-arginine, Oleamide, Trigonelline, Hypoxanthine, Tangeritin, Isoquinoline, and / or combinations thereof.

[0169] In some aspects, the PDVs derived from ginger comprise anti-inflammatory compounds. In some aspects, the anti-inflammatory compounds comprise 4- Methoxybenzaldehyde, Spermine, Isoquinoline, p-cymene, Crotonic acid, Isoleucine, (-)- trans-Methly dihydrojasmonate, Trans-Anethole, Ibuprofen, Pelubiprofen, L-(-)-Serine, and / or combinations thereof.

[0170] In some aspects, the capsule is suitable for oral administration.

[0171] Certain aspects of the disclosure are directed to the use of a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) disclosed herein for treating a subject suffering from an inflammatory disease.

[0172] In some aspects, the inflammatory disease is inflammatory bowel disease, colitis, ulcerative colitis, Crohn’s Disease, Rheumatoid arthritis, Psoriasis, Chronic obstructive pulmonary disease (COPD), or vasculitis. In some aspects, the use is for delivery to the colon of the subject, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon of the subject.

[0173] Methods of Preparation

[0174] In some aspects, provided herein is a method of preparing a capsule for oral delivery comprising (a) isolating a plurality of plant derived vesicles (PDVs) fromcabbage or spinach; (b) coating one of more of the plurality of PDVs with a methacrylic acid-methyl methacrylate copolymer (e.g., 2 mg / ml methacrylic acid-m ethyl methacrylate copolymer); (c) lyophilizing the plurality of coated PDV; (d) filling a capsule with the lyophilized plurality of coated PDV; and optionally, (e) coating the capsule. In some aspects, the coated plurality of PDVs remain intact at a pH of 1 to 2 longer than a plurality of uncoated PDVs. In some aspects, the coated capsule to remain intact at a pH of 1 to 2 longer than an uncoated capsule.

[0175] In some aspects, the capsule is filled with about 1.6 mg of the lyophilized PDV. In some aspects, the capsule is filled with about 1 mg, about 2 mg, about 3 mg, or about 4 mg of the lyophilized PDV. In some aspects, the capsule is filled with between about 1 mg and about 3 mg of the lyophilized PDV.

[0176] In some aspects, the plurality of PDVs is derived from cabbage.

[0177] In some aspects, the plurality of PDVs is derived from spinach.

[0178] In some aspects, the plurality of PDVs is derived from mushroom.

[0179] In some aspects, the isolating comprises (i) disinfecting the cabbage or spinach;(ii) blending the cabbage or spinach; (iii) filtering the blended cabbage or spinach to form a cabbage or spinach juice; (iv) sequentially centrifuging the cabbage or spinach juice to form a supernatant; (v) centrifuging the supernatant into a pellet comprising the plurality of PDVs; (vi) suspending the plurality of PDVs in solution, optionally phosphate buffered saline (PBS); or any combination thereof.

[0180] In some aspects, the isolating comprises the steps of FIG. 1.

[0181] In some aspects, the isolating comprises (i) blending the cabbage or spinach (e.g., the leaves) to form a juice; (ii) sequentially centrifuging the cabbage or spinach juice to form a supernatant comprising a plurality of PDVs; (iii) centrifuging the supernatant of step (ii) to form a pellet comprising the plurality of PDVs; (iv) suspending the pellet comprising the plurality of PDVs in solution; (v) washing the plurality the suspension of PDVs of step (iv); and (vi) filtering the washed PDVs of step (v), wherein the flow through from the filter comprises the isolated plurality of PDVs.

[0182] In some aspects, the disinfecting of step (a) comprises washing cabbage leaves or spinach leaves with 10% bleach. In some aspects, the disinfecting of step (a) comprises washing cabbage leaves or spinach leaves with antibacterial soap. In some aspects, the disinfecting of step (a) comprises washing cabbage leaves or spinach leaves with 10%bleach, followed by antibacterial soap. In some aspects, the disinfected leaves are rinsed with water after being washed with 10% bleach and / or antibacterial soap.

[0183] In some aspects, the blending of step (b) comprises blending the disinfected leaves for about 2 minutes to generate tiny leaf slices. In some aspects, the tiny leaf slices are further blended for about 3 minutes.

[0184] In some aspects, the filtering of step (c) comprises filtering blended tiny leaf slices with a mesh nylon filter bag. In some aspects, the mesh nylon filter bag has a pore size of 8 pm x 12 pm.

[0185] In some aspects, the sequentially centrifuging of step (d) comprises a first centrifuging step at about 4 °C and about 700 x g for about 10 minutes. In some aspects, the sequentially centrifuging of step (d) further comprises a second centrifuging step at about 2,000 x g for about 20 minutes. In some aspects, the sequentially centrifuging of step (d) further comprises a third centrifuging step at about 10,000 x g for about 30 minutes.

[0186] In some aspects, the centrifuging the supernatant of step (e) comprises centrifuging the supernatant at about 100,000 x g for about 2 hours to pellet the remaining PDVs. In some aspects, the centrifuging the supernatant of step (e) further comprises suspending the pelleted PDVs in PBS and centrifuging a second time at about 4 °C and about 10,000 x g for about 30 minutes to pellet large vesicles and remaining cellular debris. In some aspects, the centrifuging the supernatant of step (e) further comprises centrifuging the supernatant at about 4 °C and about 100,000 x g for about 2 hours to pellet the desired PDVs.

[0187] In some aspects, the suspending of step (f) comprises suspending in PBS, filtering through a filter, and storing the suspension at about -80 °C. In some aspects, the filter is a 0.22 pm filter.

[0188] In some aspects, the lyophilizing comprises (i) mixing the plurality of PDVs to create a mixture; (ii) freezing the mixture in liquid nitrogen; and (iii) lyophilizing the frozen mixture to obtain the lyophilized plurality of PDVs.

[0189] In some aspects, the mixing of step (a) comprises mixing the PDVs in excess tertiary butanol. In some aspects, the PDVs were suspending in Trehalose in PBS prior to the mixing of step (a). In some aspects, the concentration of Trehalose is 25 mM.

[0190] In some aspects, the mixture was vortexed prior to the freezing of step (b).

[0191] In some aspects, the lyophilized PDVs were obtained by scrapping the lyophilized frozen mixture off the walls of a tube and centrifuging for about 10,000 x g for about 10 minutes.

[0192] In some aspects, the coating comprises a methacrylic acid-methyl methacrylate copolymer (e.g., a Eudragit L, S, FS or E polymer, such as Eudragit SI 00 or Eudragit L- 100).

[0193] In some aspects, the coating comprises 1 mg / mL - 4 mg / ml methacrylic acid- methyl methacrylate copolymer.

[0194] In some aspects, the coating comprises 2 mg / ml methacrylic acid-methyl methacrylate copolymer.

[0195] In some aspects, the coating comprises 2% to 20% methacrylic acid-methyl methacrylate copolymer. In some aspects, the coating comprises 14% methacrylic acid- methyl methacrylate copolymer. In some aspects, the coating comprises 4% methacrylic acid-methyl methacrylate copolymer.

[0196] In some aspects, the coating comprises a first coat with 4% methacrylic acid- methyl methacrylate copolymer and a second coat with 14% methacrylic acid-methyl methacrylate copolymer.

[0197] In some aspects, the coating comprises a first coat with 4% methacrylic acid- methyl methacrylate copolymer, a second coat with 4% methacrylic acid-methyl methacrylate copolymer, a third coat with 14% methacrylic acid-methyl methacrylate copolymer, and a fourth coat with 14% methacrylic acid-methyl methacrylate copolymer.

[0198] In some aspects, the methacrylic acid-methyl methacrylate copolymer is EUDRAGIT® S-100.

[0199] In some aspects, the polymer coating comprises between about 3% and about 15%, between about 4% and about 15%, between about 5% and about 15%, between about 8% and about 15%, between about 10% and about 15%, between about 12% and about 15%, between about 14% and about 15%, between about 4% and about 14%, between about 4% and about 8%, between about 4% and about 10%, between about 8% and about 14%, or between about 10% and about 14% of a methacrylic acid-methyl methacrylate copolymer.

[0200] In some aspects, the polymer coating comprises about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% of methacrylic acid-methyl methacrylate copolymer.

[0201] In some aspects, the polymer coating comprises at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14% or at least 15% of methacrylic acid-methyl methacrylate copolymer.

[0202] In some aspects, the polymer coating comprises at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14% or at least about 15% of methacrylic acid-methyl methacrylate copolymer.

[0203] In some aspects, the polymer coating comprises about 2 mg / mL methacrylic acid- methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 1 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 3 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 4 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 5 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises 1, 2, 3, 4, or 5 mg / mL methacrylic acid-methyl methacrylate copolymer.

[0204] In some aspects, the polymer coating comprises between about 1 mg / L to about 5 mg / mL of methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises between 1 mg / L to 5 mg / mL of methacrylic acid-methyl methacrylate copolymer.

[0205] In some aspects, one or more of the plurality of coated PDVs comprises an antiinflammatory compound (e.g., the anti-inflammatory compound is encapsulated in the PDV). In some aspects, the anti-inflammatory compound is selected from one or more compounds in Table 2, Table 3, and / or Table 4.

[0206] In some aspects, the anti-inflammatory compound comprises choline; betaine; trigonelline; DL-tryptophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L-cysteine-S- oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L-glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL- serine; DL-homoserine; DL-arginine; or any combination thereof.

[0207] In some aspects, the anti-inflammatory compound comprises choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4-indolecarbaldehyde; trans- 3 -indoleacrylic acid; Y-aminobutyric acid (GABA); or any combination thereof.

[0208] In some aspects, the anti-inflammatory compounds comprise Choline, Betaine, L- Isoleucine, L-phenylalanine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, D-(+)- Tryptophan, Ecdysterone, a-Eleostearic acid, Adenosine, Guanine, Adenine, Spermine, Oleamide, and / or combinations thereof.

[0209] In some aspects, the anti-inflammatory compounds comprise Adenosine, Guanine, D-(+)-Tryptophan, L-Isoleucine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, L- Phenylalanine, Choline, Adenine, Betaine, Spermidine, Spermine, DL-arginine, Oleamide, Trigonelline, Hypoxanthine, Tangeritin, Isoquinoline, and / or combinations thereof.

[0210] In some aspects, the plurality of PDVs derived from ginger comprise antiinflammatory compounds. In some aspects, the anti-inflammatory compounds comprise 4-Methoxybenzaldehyde, Spermine, Isoquinoline, p-cymene, Crotonic acid, Isoleucine, (- )-trans-Methly dihydrojasmonate, Trans- Anethole, Ibuprofen, Pelubiprofen, L-(-)-Serine, and / or combinations thereof.

[0211] In some aspects, the one or more of the plurality of PDVs comprise a therapeutic agent (e.g., a therapeutic agent is encapsulated in a PDV).

[0212] In some aspects, provided herein is a composition comprising a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) produced by any of the methods disclosed herein.

[0213] In some aspects, provided herein is a composition comprising a capsule produced by any of the methods of preparing disclosed herein.IV. Methods of Treatment

[0214] In some aspects, provided herein is a method of treating inflammation in the intestine of a subject in need thereof comprising administering to the subject a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) disclosed herein. In some aspects, the administration is oral. In some aspects, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.

[0215] In some aspects, the plurality of PDVs are lyophilized and the plurality of PDVs are derived from spinach, cabbage, or a combination thereof.

[0216] In some aspects, the plurality of PDVs is derived from a cabbage.

[0217] In some aspects, the plurality of PDVs is derived from a spinach.

[0218] In some aspects, the plurality of PDVs is derived from mushroom.

[0219] In some aspects, the coating comprises a methacrylic acid-methyl methacrylate copolymer.

[0220] In some aspects, the coating comprises 4% methacrylic acid-methyl methacrylate copolymer.

[0221] In some aspects, the coating comprises 14% methacrylic acid-methyl methacrylate copolymer.

[0222] In some aspects, the polymer coating comprises between about 3% and about 15%, between about 4% and about 15%, between about 5% and about 15%, between about 8% and about 15%, between about 10% and about 15%, between about 12% and about 15%, between about 14% and about 15%, between about 4% and about 14%, between about 4% and about 8%, between about 4% and about 10%, between about 8% and about 14%, or between about 10% and about 14% of a methacrylic acid-methyl methacrylate copolymer.

[0223] In some aspects, the polymer coating comprises about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% of methacrylic acid-methyl methacrylate copolymer.

[0224] In some aspects, the polymer coating comprises at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14% or at least 15% of methacrylic acid-methyl methacrylate copolymer.

[0225] In some aspects, the polymer coating comprises at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14% or at least about 15% of methacrylic acid-methyl methacrylate copolymer.

[0226] In some aspects, the polymer coating comprises about 2 mg / mL methacrylic acid- methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 1 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 3 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 4 mg / mL methacrylic acid-methylmethacrylate copolymer. In some aspects, the polymer coating comprises about 5 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises 1, 2, 3, 4, or 5 mg / mL methacrylic acid-methyl methacrylate copolymer.

[0227] In some aspects, the polymer coating comprises between about 1 mg / L to about 5 mg / mL of methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises between 1 mg / L to 5 mg / mL of methacrylic acid-methyl methacrylate copolymer.

[0228] In some aspects, the plurality of PDVs comprises an anti-inflammatory compound.

[0229] In some aspects, the anti-inflammatory compounds comprises choline; betaine; trigonelline; DL-tryptophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L-cysteine-S- oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L-glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL- serine; DL-homoserine; DL-arginine; or any combination thereof.

[0230] In some aspects, the anti-inflammatory compounds comprises choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4-indolecarbaldehyde; trans- 3 -indoleacrylic acid; Y-aminobutyric acid (GABA); or any combination thereof.

[0231] In some aspects, the anti-inflammatory compounds comprise Choline, Betaine, L- Isoleucine, L-phenylalanine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, D-(+)- Tryptophan, Ecdysterone, a-Eleostearic acid, Adenosine, Guanine, Adenine, Spermine, Oleamide, and / or combinations thereof.

[0232] In some aspects, the anti-inflammatory compounds comprise Adenosine, Guanine, D-(+)-Tryptophan, L-Isoleucine, 4-Indolecarbaldehyde, trans-3 -Indoleacrylic acid, L- Phenylalanine, Choline, Adenine, Betaine, Spermidine, Spermine, DL-arginine, Oleamide, Trigonelline, Hypoxanthine, Tangeritin, Isoquinoline, and / or combinations thereof.

[0233] In some aspects, the PDVs derived from ginger comprise anti-inflammatory compounds. In some aspects, the anti-inflammatory compounds comprise 4- Methoxybenzaldehyde, Spermine, Isoquinoline, p-cymene, Crotonic acid, Isoleucine, (-)- trans-Methly dihydrojasmonate, Trans-Anethole, Ibuprofen, Pelubiprofen, L-(-)-Serine, and / or combinations thereof.

[0234] In some aspects, the PDV comprises a therapeutic agent. In some aspects, the therapeutic agent is a siRNA, a miRNA, a mRNA, a protein, a plasmid, a DNA, or asmall molecule. In some aspects, the therapeutic agent is an aminosalicylatea, a corticosteroid, an immunomodulator, an antibiotic or an antidiarrheal agent.

[0235] In some aspects, the inflammatory disease is an inflammatory bowel disease, colitis, ulcerative colitis, Crohn’s Disease, Rheumatoid arthritis, Psoriasis, Chronic obstructive pulmonary disease (COPD), or vasculitis.

[0236] In some aspects, the inflammation is caused by an inflammatory disease.

[0237] In some aspects, the inflammatory disease is an inflammatory bowel disease. In some aspects, the inflammatory bowel disease is ulcerative colitis. In some aspects, the inflammatory bowel disease is Crohn's disease. In some aspects, the inflammatory bowel disease is Rheumatoid arthritis. In some aspects, the inflammatory bowel disease is Psoriasis. In some aspects, the inflammatory bowel disease is Chronic obstructive pulmonary disease (COPD). In some aspects, the inflammatory bowel disease is Vasculitis.V. Kits

[0238] The present disclosure also provides kits, or products of manufacture, comprising (i) a composition or a capsule of the present disclosure, and (ii) optionally instructions for use (e.g., a package insert with instructions to perform any of the methods described herein), wherein the composition or capsule comprises a plurality of plant derived vesicles (PDVs) which are coated with a polymer.. In some aspects, the plurality of PDVs are lyophilized and the plurality of PDVs are derived from spinach, cabbage, or a combination thereof.

[0239] The present disclosure also provides kits, or products of manufacture, comprising(i) a capsule of the present disclosure, and (ii) optionally instructions for use (e.g., a package insert with instructions to perform any of the methods described herein), wherein the capsule comprises a plurality of plant derived vesicles (PDVs), wherein the capsule is coated with a polymer. In some aspects, the plurality of PDVs are lyophilized and the plurality of PDVs are derived from spinach, cabbage, or a combination thereof.

[0240] In some aspects, the plurality of PDVs is derived from a cabbage.

[0241] In some aspects, the plurality of PDVs is derived from a spinach.

[0242] In some aspects, the plurality of PDVs is derived from mushroom.

[0243] In some aspects, the kit or product of manufacture comprises (i) an empty capsule,(ii) a plurality of plant derived vesicles (PDVs), (iii) a polymer coating the capsule, and(iv) optionally, instructions for use (e.g., a package insert with instructions to perform any of the methods described herein are also contemplated).

[0244] In some aspects, the kit or product of manufacture comprises (i) an empty capsule, (ii) a plurality of plant derived vesicles (PDVs) which are coated with a polymer, and (iii) optionally, instructions for use (e.g., a package insert with instructions to perform any of the methods described herein are also contemplated).

[0245] In some aspects, the polymer comprises a methacrylic acid-methyl methacrylate copolymer.

[0246] In some aspects, the polymer comprises 4% methacrylic acid-methyl methacrylate copolymer.

[0247] In some aspects, the polymer comprises 14% methacrylic acid-methyl methacrylate copolymer.

[0248] In some aspects, the polymer coating comprises between about 3% and about 15%, between about 4% and about 15%, between about 5% and about 15%, between about 8% and about 15%, between about 10% and about 15%, between about 12% and about 15%, between about 14% and about 15%, between about 4% and about 14%, between about 4% and about 8%, between about 4% and about 10%, between about 8% and about 14%, or between about 10% and about 14% of a methacrylic acid-methyl methacrylate copolymer.

[0249] In some aspects, the polymer coating comprises about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% of methacrylic acid-methyl methacrylate copolymer.

[0250] In some aspects, the polymer coating comprises at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14% or at least 15% of methacrylic acid-methyl methacrylate copolymer.

[0251] In some aspects, the polymer coating comprises at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14% or at least about 15% of methacrylic acid-methyl methacrylate copolymer.

[0252] In some aspects, the polymer coating comprises about 2 mg / mL methacrylic acid- methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 1mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 3 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 4 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises about 5 mg / mL methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises 1, 2, 3, 4, or 5 mg / mL methacrylic acid-methyl methacrylate copolymer.

[0253] In some aspects, the polymer coating comprises between about 1 mg / L to about 5 mg / mL of methacrylic acid-methyl methacrylate copolymer. In some aspects, the polymer coating comprises between 1 mg / L to 5 mg / mL of methacrylic acid-methyl methacrylate copolymer.

[0254] In some aspects, the methacrylic acid-methyl methacrylate copolymer is EUDRAGIT® S-100.

[0255] One skilled in the art will readily recognize that PDVs and capsules of the present disclosure, can be readily incorporated into one of the established kit formats which are well known in the art.

[0256] The following examples are illustrative and do not limit the scope of the claimed aspects.EXAMPLESExample 1. Plant Derived Vesicles Isolation from Cabbage

[0257] Plant Derived Vesicle Isolation

[0258] Whole cabbage leaves were washed sequentially with 10% Clorox bleach and diluted antibacterial soap, and were then thoroughly rinsed with tap water. Additionally, the same disinfecting and cleaning process was applied to all blending vessels and centrifuge tubes used during the isolation of vesicles. The cabbage leaves were blended with a Ninja blender (Model BL610) for about 2 minutes to generate tiny leaf slices. The tiny leaf slices were then subjected to a cold press juicer (Kuhaus) and filtered with a mesh nylon filter bag (Groupcow) with a pore size of 8 x 12 pm. The resultant cabbage juice was sequentially centrifuged at 700 x g for 10 minutes and 2,000 x g for 20 minutes at 4 °C with an Eppendorf benchtop centrifuge (Model 5810), rotor A-4-81. The remaining tissue fragments and large cellular debris were removed using a Beckman Optima XE Ultracentrifuge (rotor Ti45) at 4 °C, 10,000 x g for 30 minutes. Theremaining supernatant was centrifuged at 100,000 x g for 2 hours to pellet remaining vesicles. The pellet was suspended in Phosphate Buffered Saline (PBS), and then centrifuged at 4 °C, 10,000 x g for 30 minutes to remove the aggregated PDVs and contaminants. The supernatant was subjected to a final centrifugation at 4 °C and 100,000 x g for 2 hours. The pellets were resuspended in PBS as the final product, filtered through 0.22 pm syringe filter unit (Millipore), and stored at -80 °C. The same isolation method was used for spinach and mushroom PDV isolation. See FIG. 1.

[0259] Lyophilization

[0260] Cabbage PDVs were suspended in 25 mM D-trehalose (Sigma) before lyophilization to avoid disruption from the higher osmotic pressure caused by the increase in salt concentration during the lyophilizing process (C. Charoenviriyakul et al., Int J Pharm. 553(1 -2): 1 -7 (2018)). After isolation, PDVs were preserved by directly freezing them overnight (about 16 hours) in cry opreservation tubes wrapped in heat insulating material at -80 °C. The mixture was vortexed and every 2 mL mixture was placed in a borosilicate glass vial to be frozen in an acetone-dry ice bath (-78 °C). The vials were tilted during freezing to uniformly freeze the sample on the vial walls and facilitate the freeze-dry process. The lyophilization was performed with a Labconco dry system FreeZone 4.5 plus, according to the manufacturer’s guidelines. Lyophilized powder sample was scraped from the tube walls and then centrifuged at 10,000 * g for 10 minutes to gather the powder at the bottom of the tube.

[0261] Capsule filling, coating and storage

[0262] Capsules (Braintree Scientific Inc., size A-CT M) were filled with supplied funnel kit, and static electricity during whole filling operation was avoided. Capsule weight was measured before and after filling to ensure similar amounts of lyophilized powder were contained in each capsule.

[0263] Coating of cabbage PD Vs with Eudragit S-100.

[0264] Under magnetic stirring at 900 rpm, 3 mL of Eudragit S-100 (Evonik) solution (2 mg / mL) was slowly added to the 3 mL of ~1.0 x 1013PDV suspension (Xu, B., et al., International journal of biological macromolecules 113:534-542 (2018)). Stirring was continued for 12 hours in 4 °C to allow the ethanol of the Eudragit S-100 solution to evaporate. Lyophilization and encapsulation was performed afterwards.

[0265] Eudragit-SlOO coating efficiency to cabbage PDVs.

[0266] Eudragit-SlOO was labeled with Sulfo-Cy3-Amide-C6-Amine followed previous research (Vollrath, A., et al., Macromolecular rapid communications 31 :2053-2058 (2010)). 0.1 g Eudragit-SlOO and 3.86 mg of N,N,N’,N’-Tetramethyl-O-(N-succinimidyl) uronium tetrafluoroborate (ThermoFisher) was dissolved in 2.5 mL Dimethylformamide and 4 pL N,N-Diisopropylethylamine (ThermoFisher) were added, stirred for 2 hours at 27 °C for activation. 1 mg of Sulfo-Cy3-Amine dye (Broadpharm, 0.0012 mmol, Mw: 867) was added, stirred overnight at 27 °C. Particles were washed by deionized water and dried with vacuum machine. Dissolved product in 0.5 mL Dimethylformamide, purified by Sephadex LH-20 column, collected Cy3 labeled Eudragit SI 00 conjugate fractions, dried with vacuum machine. Cy3 labeled Eudragit SI 00 was used to coat CellMask Deep Red dyed PDVs. Incorporation efficiency was assessed via Cytek Aurora system.

[0267] The filled capsules were loaded into the capsule holder X-HOLD M with the body side up and 2-3 mm protruding from the top of holder (Silicone rubber sheet). A 4% concentration coating solution was placed in a petri dish. The coating holder was dipped into coating solution so as to coat the body and joint halfway up the capsule bottom. During coating, care was taken to ensure that enteric coating solution did not touch silicon rubber sheet of coating holder. After about 5 seconds, any extra solution was allowed to drip from capsule body ends, and then the petri dish was covered. Capsules on the holder were then left to dry for 30-40 minutes. The dried capsules were removed from the holder and reloaded with the cap side up, then coat the cap + joint part as described. This is a full procedure for one coating. Coating was performed twice with 4% Eudragit S-100 solution, and then twice with 14% Eudragit S-100 as a full procedure (See FIGs. 17A-17G).Example 2. Characterization of Coated Plant Derived Vesicles

[0268] A series of experiments to determine the effects of conditions that mimic the upper digestive system on PDVs were conducted. For proof-of-concept, cabbage PDVs were selected since cabbage is a commonly consumed vegetable that is known to have anti-inflammatory properties (Rokayya, S., et al., Asian Pacific Journal of Cancer Prevention 14:6657-6662 (2013)).

[0269] Negative staining and transmission electron microscopy (TEM). Negative staining and TEM were performed according to the method in Liu et al., Nanomedicine (2020). Briefly, Formvar / carbon-coated 100-mesh copper grids (Sigma) were treated withpoly-L-lysine for approximately 1 hour and then PDV samples were placed on the grids. Samples were negatively stained with Millipore-filtered aqueous 1% uranyl acetate for 1 minute. The stain was blotted dry from the grids with filter paper, and the samples were allowed to dry. The samples were then imaged using a JEM 1010 TEM (JEOL, USA, Inc.) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp.).

[0270] The detailed changes in vesicle shape and particle number when exposed to stomach and intestinal pH were investigated.

[0271] pH treatment. Solutions of phosphate-buffered saline (PBS) were adjusted to pH 1.5 with hydrochloric acid and to pH 7.5 with sodium hydroxide. Three samples containing 4 mL of cabbage PDVs were centrifuged at 4 °C, 100,000 x g for 2 hours; the resultant pellets were suspended in PBS at pH 7 as a control and at pH 1.5 or pH 7.5 as treatment groups. Samples were incubated at 37 °C for 30 min. Finally, the samples were visualized by cryogenic electron microscopy (cryo-EM) and diluted 10,000-fold; particle numbers were determined by a NanoSight NS500 instrument (Malvern).

[0272] For the statistics of cryo-EM, 49 images were taken from random spots. The number of PDVs were counted, the PDV number was reduced in the pH=l .5 treatment; intact and broken PDVs were identified. For the statistics of NanoSight particle number changes, the untreated PDV group was set as 100% and compared the particle number of various pH treatments with untreated to determine the percentages.

[0273] Cryo-EM. Cryo-EM studies were conducted according to procedures described in detail in a previously published article (Liu, Y., et al., Nanomedicine: Nanotechnology, Biology, and Medicine 29: 102271 (2020)). Briefly, 3-pL PDV samples were placed on a glow-discharged Lacey carbon 400-mesh Cu grid, after which the grid was plunge-frozen using a Vitrobot specimen-preparation unit (ThermoFisher Scientific). The freezing conditions were as follows: 100% humidity, 4 °C, 5 second wait time, 2 second blot time, and 0 blot force. Micrographs were collected on an FEI Titan Krios operating at 300 kV coupled with a Gatan K2 direct electron detector via Serial-EM software (Mastronarde N. J Struct Biol 2005). Each exposure image was collected at 18,000* nominal magnification resulting in a pixel size of 1.36 A / pixel in the counted mode, using a dose rate of 13 e- / pixel per second and 200 ms exposure per frame for a total of 40 frames. The total dose in the EM data collection was 55 e- / A2. The nominal defocus used was -2.0 pm. For statistical analysis of cryo-EM data, 49 images from random spots weretaken. The number of PDVs were counted, and intact and broken PDVs were identified. The control PDV group was set as 100% and compared the vesicle number of pH=1.5 treatment with the untreated group to determine the percentages.

[0274] Nanoparticle tracking analysis (NTA). Nanoparticle size and number were determined by using NTA, performed with a NanoSight instrument, NS500 (Malvern) with a green laser 532, SCMOS model. Details of the analysis were described in a previously published paper (Liu, Y., et al., Nanomedicine: Nanotechnology, Biology, and Medicine 29: 102271 (2020)). Samples were diluted to 1 : 10,000 in PBS, resulting in each view having 20-100 particles. Three sequential standard measurements were performed for 1 minute each at 15.8-15.9 °C (viscosity: 1.108-1.111 cP). All samples used a camera level of 13, a detection threshold of 6, and a screen gain of 10. Measurements were done in triplicate. Z-average size were assayed by dynamic light scattering (DLS, Zen 3600 Zetasizer Malvern Instruments) operating at a wavelength of 633 nm, a scattering angle of 173° Refractive index (RI) was 1.34 and viscosities 1.0568 (25 °C). Measurements were performed 3 times with similar results.

[0275] TEM (Transmission electron microscopy) and NTA (Nanoparticle Tracking Analysis) analysis demonstrated that vesicle number did not change much following exposure to pH=1.5 (37 °C) for 30 min (similar to stomach contents); however, the vesicle number was reduced by -30% when exposed to pH=1.5 followed by pH 7.5 (similar to transit to proximal small bowel) (FIGs. 2A-2D).

[0276] Cryo-EM (cryo-electron microscopy) showed that most PDVs in PBS are intact and round; only 8.9% of PDVs suspended in PBS were not completely sealed (FIG. 2D; n=170). When exposed to 37 °C, pH=1.5 for 30 min, the number of vesicles was reduced by only 8.8%, but the vesicle shape was changed substantially from round to irregular shapes and 36.1% of PDVs appeared broken (FIG. 2C; n=155). TEM images of PDVs following exposure to pepsin (in pH=1.5) showed that particle number was reduced to 21%, and most of the vesicles were broken. The number of intact vesicles was reduced to only 18% upon treatment with pepsin in pH=1.5 followed by exposure to bile extract and pancreatin in pH=7.5; most of the vesicles were digested into small particles (FIG. 2D). These tests show that most of the PDVs are digested in the stomach and proximal small bowel environment.

[0277] To further verify the particle number and size changes after stomach and intestinal pH, various pH treatments were tested with NTA. Cabbage PDVs were primarilydistributed from 100 to 250 nm, and the most abundant size was 177 nm in the control group; after treatment at 37 °C, pH=1.5, for 30 min, the size of the vesicles changed from 120 to 500 nm; the particle number was reduced to 25.9%. With the pH=7.5 treatment, the particle size distribution and number did not change significantly (FIGs. 2B). These results indicate that direct consumption of PDVs is unlikely to be successful due to the low pH.

[0278] To protect PDV compounds, the PDVs were lyophilized with liquid nitrogen (FIGs. 17A-17B) and then loaded into capsules (CapPDVs). Transmission Electron Microscopy (TEM) was used to test CapPDVs without lyophilization suspended in PBS (FIG. 17C) and to test PDVs after lyophilization and suspension in water (FIG. 17D). The results showed that vesicles are recovered successfully after lyophilization. TEM and NTA of PDVs with or without lyophilization demonstrated that vesicle shape and vesicle number are comparable after lyophilization (FIGs. 3 A-3D). Moreover, the contents the vesicles are also largely similar after lyophilization.

[0279] The lyophilized powder was loaded into capsules with use of a capsule filling funnel (FIG. 17E), and the filled capsules were coated with Eudragit S-100 by using an X-HOLD capsule holder (FIG. 17F). Because the coated size M capsule is slightly bigger than the uncoated one, a new device was developed for capsule gavage in mice (FIG. 17G) with a steel tube filled in soft polyurethane feeding tubes (13 ga x 88 mm). The polyurethane tube is for fixing the capsules and the steel tube to provide support during gavage (FIG. 17G). For capsule administration in mice, a steel tube filled in soft polyurethane feeding tubes was used (13 ga x 88 mm; FIG. 13 A). For oral delivery of Cap-PDVs in mice, light anesthesia was used (FIGs. 13B-13F).

[0280] Untargeted and targeted liquid chromatography-mass spectrometry (LC- MS). Untargeted liquid chromatography mass spectrometry analysis was performed on a Q Exactive Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific) coupled to a binary pump HPLC (UltiMate 3000, Thermo Fisher Scientific). Full MS spectra were obtained at 70,000 resolution (200 m / z) with a scan range of 50-750 m / z. Full MS followed by ddMS2 scans were obtained at 35,000 resolution (MSI) and 17,500 resolution (MS2) with a 1.5 m / z isolation window and a stepped NCE (20, 40, 60). Samples were maintained at 4 °C before injection. The injection volume was 10 pL. Chromatographic separation was achieved on a Synergi Fusion 4 pm, 150- x 2 -mm reverse-phase column (Phenomenex) maintained at 30 °C using a solvent gradientmethod. Solvent A was water (0.1% formic acid). Solvent B was methanol (0.1% formic acid). The gradient method used was 0-5 minutes (10% B to 40% B), 5-7 minutes (40% B to 95% B), 7-9 minutes (95% B), 9-9.1 minutes (95% B to 10% B), and 9.1-13 minutes (10% B). The flow rate was 0.4 mL / minute. Sample acquisition was performed using Xcalibur software (Thermo Fisher Scientific). Data analysis was performed with Compound Discoverer 3.1 (Thermo Fisher Scientific). For statistical analysis of LC-MS compounds, three biological replicates were tested and differences in relative abundance between these three replicates were expressed as ranges.

[0281] Mass spectroscopy for proteomics.

[0282] Cabbage PDVs were dissolved in 50 mM ammonium bicarbonate containing 1 mM calcium chloride to prepare lysate. The samples were snap-frozen in liquid nitrogen and thawed twice followed by boiling at 95 °C for 2 min with a short vortex every 30 s. The lysate was digested using LysC+ trypsin proteases at 37 °C overnight. The enzyme reaction was neutralized using 10% formic acid and the peptides were measured using the Pierce Quantitative Colorimetric Peptide Assay (Thermo Scientific). The tryptic peptides were subjected to a simple Cl 8 cleanup using a C18 disk plug (3 M Empore Cl 8) and extracted using 50% acetonitrile containing 0.1% formic acid, then dried in a speed vac (Savant).

[0283] The samples were analyzed as two technical replicates using a nano-LC 1200 system (Thermo Fisher Scientific) coupled to Orbitrap Exploris 480 (Thermo Fisher Scientific) mass spectrometer. Then, 2 pg of peptide was loaded on a pre-column of 2 cm x 100 pm I.D. switched inline with an in-housed 20 cm x 75 pm I D. column (Reprosil- Pur Basic C18, 1.9 pm, Dr. Maisch GmbH, Germany). The peptide elution was done using a 110-min discontinuous gradient of 90% acetonitrile buffer (B) in 0.1% formic acid at 200 nL / min (2-30% B: 86 min, 30-60% B: 6 min, 60-90% B: 8 min, 90-50% B: 10 min). The eluted peptides were directly electro-sprayed into an Orbitrap Exploris 480 (Thermo Fisher Scientific) mass spectrometer operated in the data-dependent acquisition mode acquiring HCD fragmentation spectra of the top 50 strongest ions. The full MS scan was acquired in Orbitrap in the range of 350-1400 m / z at 120,000 resolution followed by MS2 at 15,000 resolution (HCD 32% collision energy) with 15-s dynamic exclusion time.

[0284] The MS raw files were searched using Proteome Discoverer 1.4 software (Thermo Fisher) with the Mascot algorithm (Mascot 2.4, Matrix Science; percolator against the Brassica oleracea var. oleracea protein database from NCBI refseq). The peptidesidentified from the Mascot result file were validated with 5% false discovery rate. Dynamic modification of oxidation, protein N-terminal acetylation, and deamidation on asparagine and glutamine was allowed. The precursor mass tolerance was confined within 20 ppm, with fragment mass tolerance of 0.02 Da, and a maximum of two missed cleavages with trypsin enzyme was allowed.

[0285] The resulting peptides were analyzed by high-sensitivity LC-MS / MS on an Orbitrap-Fusion mass spectrometer (Thermo Scientific, Waltham, MA). Proteins were identified by a database search of the fragment spectra against the SwissProt (EBI) protein database using Mascot software (v 2.6.2, Matrix Science, London, UK). The typical search settings were mass tolerances, a 10-ppm precursor, 0.8-d fragments, variable modifications, methionine sulfoxide, pyro-glutamate formation, and up to two missed cleavages. The FDR estimates were made using Proteome Discoverer software (v 1.4 or 2.2, Thermo Scientific). Two biological replicates of both leaf nanovesicles and sEVs were analyzed, with similar results.

[0286] To identify whether compounds of interest could be detected in PDVs, liquid chromatography-mass spectrometry (LC-MS) was performed on PDVs derived from cabbage, mushrooms, spinach and ginger. From these experiments, 19 anti-inflammatory compounds were found in cabbage PDVs (Table 1), 14 in spinach PDVs (Table 2), 15 in Mushroom PDVs (Table 3), and 11 in ginger PDVs (Table 4). Anti-inflammatory compounds found in cabbage PDVs included the glucosinolate-related metabolites sulforaphane and indole-3 -carbinol, cruciferous vegetable biomarker S-methyl-L- cysteine-S-oxide, jasmonic acid precursor 12-oxo-phytodienoic acid, trigonelline, and many amino acids (Table 1 and FIG. 9A). Indole-3 -carbinol and choline were further verified by standards for comparison, it was about 0.7 mg per 1013PDVs (FIG. 9B).

[0287] The results also showed that the yield of cabbage PDVs were highest among these 4 species. Therefore, cabbage was selected for further investigation. Proteomic test was performed to further characterize cabbage PDVs (Table 1). The result showed cabbage PDV shares protein markers with mammalian extracellular vesicles (FIG. 11) (Thery, C., et al., Journal of extracellular vesicles 7: 1535750 (2018)).Table 1 - Cabbage PDV CompoundsTable 2 - Spinach PDV CompoundsTable 3 - Mushroom PDV CompoundsTable 4 - Ginger PDV Compounds

[0288] Several anti-inflammation compounds were found universally in three species such as Choline, Betaine, Trigonelline, DL-tryptophan, L-Isoleucine and L- Phenylalanine. Indole-3 -Cabinol or similar compounds as 4-Indolecarbaldehyde and trans-3 -Indoleacrylic acid were also commonly found in three species. Some compounds specifically enriched in the plant species such as S-methyl-L-cysteine-S-oxide and 12- OPDA in cabbage and GABA in mushrooms were also found in PDVs.Example 3. Oral formulation of cabbage PDVs

[0289] PDVs were lyophilized and loaded into capsules (Cap-PDVs). TEM and NTA showed that the number and shape of PDVs were similar after lyophilization and resuspension. Resuspension after lyophilization appeared to increase in particle size. This is a similar observation to lyophilized extracellular vesicles (FIGs. 3 A-3B and FIG. 12) (see also Frank, J., et al., Scientific Reports 8: 1-8 (2018)).Example 4. Biodistribution of PDVs

[0290] To assess the biodistribution of orally delivered PDVs, copper-64-labeled cabbage PDVs for Positron Emission Tomography (PET) imaging was used. The PDVs administered by gavage were predominantly detectable in the stomach, colon, and liver (FIG. 14A-14B).

[0291] PET Imaging. PET scanning method followed previous research (Shi, S., et al., Bioconjugate chemistry 30:2675-2683 (2019)). PDVs were reacted with amine-reactive NOTA (p-SCN.Bn.NOTA Fisher) with a weight ratio of 10: 1 (weight of proteins in the PDVs / weight of NOTA) at pH 8.5 for 2 hours. Then purified with PD SpinTrap G-25 desalting column (Cytiva) using PBS (without calcium and magnesium) to remove free NOTA. 64-CuC12 (74 MBq, MD Anderson Cyclotron Radiochemistry Facility) was diluted in 300 pL of 0.1 M sodium acetate buffer (pH 5.5) and mixed with PDV-NOTA. 37 °C for 30 minutes with constant shaking. The resulting 64-Cu.NOTA.PDVs were purified by PD-10 desalting columns (Cytiva) using PBS as the mobile phase. The radio stabilities were determined by incubating each with 25% mouse serum for 24 hours. The radiolabeling yield and radio stability were tested with thin layer chromatography using 50 mM EDTA (pH 5.5) as the mobile phase (n = 3), measured by Bioscan AR-2000 Radio-TLC Imaging Scanner (Eckert & Ziegler). Mice were given oral gavage with 64Cu.NOTA.PDVs (50 pCi / mouse, 200 pL), and then serial PET scans were performed on an Albira PET / SPECT / CT scanner (Bruker) 3 hours and 24 hours post gavage. Quantitative data from region-of-interest analysis on tumor and other organs was presented as percentage dose per gram of tissue (%ID / g). After the final scan at 24 hours after gavage, organs were collected and weighed. The radioactivity was measured using PerkinElmer (Packard) Cobra II Gamma counter (PerkinElmer) (Chen F., et al., Angewandte Chemie International Edition 52: 13319-13323 (2013)).

[0292] Mouse oral gavage with capsules. For capsule administration in mice, a steel tube inside soft polyurethane feeding tube was used for administration (13 Ga x 88 mm; FIG. 13A).

[0293] Capsules were loaded into the tip of a lab-modified capsule gavage needle, and the tip was lubricated with petroleum jelly (FIG. 13 A). The capsule was placed into the esophagus of anesthetized mice by gavage (FIG. 13A-13C), followed by about 0.1 mL of water (FIG. 13D). Each mouse was monitored in a closed container with black tissue paper underneath (FIG. 13E) until the mouse was fully awake (FIG. 13F). If the capsule was spit out, it was administered to the mouse again. The parallel treatment groups have been performed anesthesia to avoid bias.

[0294] PDV Dose. PDVs dosed base on I3C amount according to clinical trials (Naik, R., et al., International Journal of Gynecologic Cancer 16 (2006)). The weight of lyophilizedPDVs per capsule was 1.6 mg, total particle number is about 1012, protein quantity was 0.5 mg.

[0295] PDVs were dosed based on findings that 3.3 mg / kg indole-3 -carbinol showed biological effectiveness in prior clinical and pharmacokinetics studies; 1.6 mg PDVs contain ~70 pg indole-3 -carbinol. Each mouse was dosed once a day with 1.6 mg PDVs at an equivalent indole-3 -carbinol of 3.3 mg / kg.

[0296] The body weight of mice dropped by 7-22%, depending on the dose, by day 8 (FIGs. 15A-15C). Consistent with induction of colitis, the colon length was significantly reduced in all three treatment groups (FIG. 15B). Histological evaluation shows different colitis scores within the different concentrations (FIG. 15C); 2.5% DSS was selected for the subsequent experiments.

[0297] C56BL / 6 background mice were used for these experiments. Four groups of mice were tested (n= 3 per group). Treatment groups were: 1) normal drinking water as a negative control; 2) 2.5% DSS (MP Biomedicals) water and treated with 100 pl PBS as DSS+PBS; 3) 2.5% DSS water and treatment with cabbage PDVs suspended in PBS as DSS+PDVs; 4) 2.5% DSS water and treatment with Cap-PDVs or Cap-cPDVs. In all DSS-treated mice, 2.5% DSS in drinking water was provided for 7 days to establish colitis; medical treatments were started along with DSS treatment (during the 7 days of DSS treatment), and continued for 7 days after DSS treatment was completed for a total of 14 days. For PDV in PBS, Cap-PDVs and Cap-cPDVs, 1.6 mg PDVs was performed for each treatment.

[0298] Biodistribution. C57BL / 6 mice were used for biodistribution experiments. Three conditions were prepared: dyed 1.6 mg PDVs suspended in PBS, 1.6 mg of dyed Cap- PDVs and 1.6 mg of dyed Cap-cPDVs, each condition was tested in 3 mice. Cabbage PDVs were dyed with CellMask Deep Red (Thermo Fisher Scientific). To dye PDVs, CellMask deep red was added to each sample at a 1 : 1,000 dilution, incubated at 37 °C for 30 minutes. The samples were then centrifuged at 4 °C, 100,000 * g for 2 hours. The pellets were resuspended in 4 mL of PBS to wash away residual free CellMask dye and then re-centrifuged at 100,000 x g for 2 hours. The washed pellets were resuspended in 400 pL of PBS or 400 pL of 25 mM D-trehalose. 6 hours after PDV administration the mice were euthanized, and the total intestine was imaged using the Xenogen IVIS-200 in vivo imaging system. The tissues were then placed in optimal cutting temperature embedding media, and frozen sections were generated for microscopy. Tissue sectionswere put in cold acetone for 10 minutes and transferred to PBS for 3 washes. The slice was fixed with 4% paraformaldehyde (Electron Microscopy Science) 15 minutes followed by 3 PBS washes. DAPI (Perkin Elmer) working solution was added on the slice for 15 minutes stain followed by 3 PBS washes. Images were acquired with Zeiss LSM 800 microscope.Example 5. Cabbage PDVs alleviate colitis

[0299] To evaluate the potential therapeutic application of Cap-PDVs, a mouse model of dextran sulfate sodium (DSS)-induced colitis was used. Different concentrations of DSS for inducing colitis were tested, using 2%, 3%, or 4% DSS supplied in drinking water for 7 days.

[0300] Treatment with cabbage PDVs was analyzed to determine if it could reduce colitis in mice subjected to DSS. Mice were divided into four groups: 1) Normal drinking water (control); 2) DSS + PBS (negative control); 3) DSS + PDVs (suspended in PBS); and 4) DSS + Cap-PDVs. Body weight and colon length in the DSS + PDVs and DSS + Cap- PDVs shows alleviated colitis than in the DSS + PBS group (FIGs. 4A-4B and FIGs. 18A-18D). Comparing the treatment groups to the control, histological assessment showed only modest improvement in loss of goblet cells, neutrophil infiltration in lamina propria, mixed leukocytes in submucosa and crypt necrosis with suppurative inflammation. The total histoscore was not significant difference between DSS+PBS, DSS+PDV and DSS+Cap-PDV. Pro-inflammatory cytokines IL1-P, IL-6 and TNF-a levels showed only modest improvement in these treatment groups compared with controls. There is some variability in the cytokine levels between the DSS+PDVs and DSS+Cap-PDVs groups, the difference was not statistically significant (FIGs. 4C-4D). The modest effects of PDV treatment may reflect the high likelihood that some PDVs may not remain intact through the upper gastrointestinal tract.

[0301] To enhance delivery of intact PDVs to the colon, Eudragit L-100 and S-100 coating was tested. The biodistribution of lyophilized PDVs in uncoated capsule was compared with that of in Eudragit L-100 or S-100 coated Capsule. The colonic delivery was significantly higher with Eudragit S-100 coating (FIG. 16A). Therefore, the Eudragit S-100 was selected for further assessment. Because mice have slower gastric emptying (Schwarz, R., et al., Magn Reson Med 48:255-261 (2002)), which makes the transit ofcapsules slower. Therefore, coating the PDVs with Eudragit (cPDVs) was considered to protect them from upper GI contents.

[0302] TEM images of the PDVs revealed that the average size of cPDVs was approximately 100 nm before coating and 110 nm after coating, the vesicle number after coat didn’t change (FIG. 5A). NTA showed similar results (FIG. 5B). This size increase is a similar observation with other Eudragit coated particles (Subudhi, M.B., et al., Materials 8:832-849 (2015)). Total weight of PDVs before and after coating were tested, 55% the total Eudragit S-100 was incorporated as coating on the PDVs. The 81.34% PDVs were coated by Eudragit S-100 (FIG. 5C). TEM images of cPDVs following exposure to pepsin (at pH 1.5) showed that the number of vesicles was reduced to 73% of that in the control group, this indicated cPDVs survive better in upper GI system than PDVs (FIG. 5D).

[0303] Optical coherence tomography slices and the fluorescence intensity was compared with a region-of-interest test of the stomach, small intestine, and colon between PDV in PBS, Cap-PDV and Cap-cPDVs. These results show that Cap-cPDVs had enhanced biodistribution to the colon than PDVs in PBS and Cap-PDVs (FIGs. 6A-6B and FIG. 19A).

[0304] For testing the effects of Cap-cPDVs in the colitis model, Eudragit S-100 coated PDVs were lyophilized and loaded into capsules. Using the DSS-induced colitis model, mice were divided into four groups: 1) Normal drinking water (control); 2) DSS + PBS; 3) DSS + PDVs (suspended in PBS); and 4) DSS + Cap-cPDVs. The DSS + PDV and DSS + Cap-cPDV groups showed alleviate the symptoms of colitis, in body weight and colon length. Comparing the treatment groups to the control, histological assessment showed that mice treated with Cap-cPDVs reached near complete resolution in all the assessed microscopic features of colitis (FIGs. 7A-7D and FIG. 19B). These results were further confirmed through an analysis of the expression of IL1-P, IL-1, and TNF-a cytokines (FIG. 7E).

[0305] Mouse body weight was monitored every day. Mice were euthanized at day 13 for FIG. 4A; day 14 for FIG. 7A, day 8 for FIG. 15 A. Colon length was determined with freshly excised tissue. One-third of each tissue sample was analyzed by hematoxylin and eosin staining and imaging. Another third was used for RNA isolation (RNeasy Mini Kit from Qiagen) to assess levels of mRNA levels of Interleukins -ip and -6, and tumor necrosis factor alpha transcripts with real-time PCR (Applied Biosystems); 36B4 wasused as an endogenous control gene. Relative expression level was compared to samples from control tissue.

[0306] Histological assessment. Histological assessment used the criteria for colitis evaluation from Table 5.Table 5 - Histological Assessment Criteria

[0307] Statistical analysis. The operator was blinded to the experimental groups for the statistics. All results were presented as mean ± standard deviation (SD) values. Data was analyzed by Student’s t test or two-way analysis of variance (ANOVA) with the GraphPad Prism 8. The p values > 0.05 marked as ns, p < 0.05 as *, P < 0.01 as **, P < 0.001 as ***, P < 0.0001 as ****.

[0308] Collectively, the findings indicated that capsules containing coated PDVs (Cap- cPDVs) were effectively delivered to the colon following oral intake and resulted in robust activity in reducing colitis. This method thus represents a potential new approach for effective delivery of PDVs to the colon.Example 6. PDVs treated with GI track-mimicking conditions

[0309] The cabbage PDV stock concentration was 1.0 x 1013particles / mL; 80 pL of 1 : 100 diluted PDVs was added to 3.20 mL of simulated stomach acid (NaCl 2.1 g / L, 0.155 M HC1, pH = 1.0 - 2.0). Samples were then incubated for 30 minutes at 37 °C with light shaking. After the incubation, 1 mL of the PDVs in stomach acid were mixed with 60 mg sodium bicarbonate (NaHCCh) to neutralize pH to 7.0 - 7.4. The same process was performed for cabbage PDVs in the presence of digestive enzymes pepsin (Sigma, 1.0 mg / mL in stomach acid pH = 1.0 - 2.0), pancreatin (Sigma, 2.0 mg / mL in PBS, pH=7.4) or pepsin then pancreatin (Sigma, 2.0 mg / mL in neutralized stomach acid, pH = 7.0 - 7.4) 4. Samples were visualized by TEM and NTA.

[0310] cPDV stock concentration was 1.0 x 1012particles / mL, 80 pL of 1 : 100 diluted PDVs was added to 3.20 mL of simulated stomach acid (NaCl 2.1 g / L, 0.155 M HCL, pH = 1.0 - 2.0) or pepsin (Sigma, 1.0 mg / mL in stomach acid pH = 1.0 - 2.0), Samples were then incubated for 30 minutes at 37 °C with light shaking. Samples were visualized by TEM.

[0311] To assess lyophilized CapPDVs delivery to the colon, the biodistribution of cabbage PDVs suspended in PBS was compared with that of CapPDVs with a protective coat, Eudragit S-100. The polymer assessed for its ability to protect from dissolution in acidic environment in stomach and achieve a better colonic distribution. Cabbage PDVs were dyed with CellMask Deep Red and mice were given two different types of CapPDVs via gavage: uncoated and CapPDVs coated with Eudragit S-100 (FIG. 16A). The results showed that there were more CellMask Deep Red signals in the stomach for uncoated PDVs after Eudragit S-100 coating, and that the colon could uptake even morecabbage PDVs (FIG. 16 A). The total fluorescence intensity was compared with a total region of interest test of the stomach, small intestine, and colon. The results verified the hypothesis and showed that Eudragit S-100-coated CapPDVs had more biodistribution to the colon than uncoated PDVs just suspended in PBS (FIG. 16B). Copper-64 labeled cabbage PDVs were also used to test the biodistribution of orally delivered PDVs. In comparison with other organs, the PDVs were detected in stomach, colon and liver (FIGs. 14A-14B). Additionally, PDVs in PBS had more biodistribution in the stomach and intestine.

[0312] PDVs in the digestive system. A series of experiments was carried out to determine the effects of conditions that mimic some aspects of the gastrointestinal (GI) system on PDVs isolated from cabbage. Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA) showed that the number of PDVs did not change substantially following a 30-minute exposure to 37 °C with simulated stomach acid (pH - 1.5) as compared to 37 °C PBS at pH 7.5; however, the number of PDVs was reduced by -30% when PDVs were incubated in stomach acid that was then neutralized to the pH of the proximal small bowel (FIG. 2A-B). To detect any vesicle shape changes due to the stomach acidic pH, cryogenic electron microscopy (cryo-EM) was performed, the results showed that most PDVs suspended in control PBS (with pH 7; n = 170) were intact and round; only 8.9% of these PDVs were not completely sealed (FIG. 2C). When PDVs were exposed for 30 minutes to 37 °C simulated stomach acid at pH 1.5, the total number of PDVs was reduced by only 8.8%, but PDV shape was changed substantially, from round to more irregular shapes, and 36.1% of PDVs appeared broken (n = 155; FIG. 2C). TEM images of PDVs following exposure to pepsin (at pH 1.5) showed that the number of particles was reduced to 21% of that in the control group, and most of the PDVs were broken (FIG. 2D). The number of intact PDVs was reduced to only 18% of the original number upon treatment with pepsin followed by exposure to pancreatin (in neutralized stomach acid at pH 7.5); most of the PDVs were digested into small particles (FIG. 2D). The number of intact PDVs was reduced to 73% of the original number upon treatment with pancreatin (FIG. 8). These findings strongly suggest that most of the PDVs would be damaged as they pass through the stomach and proximal small bowel environment.

[0313] It was found that about 80% of PDVs were broken down in conditions that mimic the upper GI; this is similar with human salivary extracellular vesicles (Ogawa, Y., et al., Biochemistry and biophysics reports 27: 101034 (2021)). This approach that protectsPDVs from the upper GI system increased their therapeutic effect. Cabbage PDVs in PBS suspension showed modest effect for colitis, but with the Cap-cPDVs surprisingly resulted in effective delivery to the colon, and led to unexpected robust effects in reducing colitis (e.g., improvements in epithelial cell death, goblet cell loss, crypt architecture alterations induced by DSS, body weight, colon length, and cytokine expression). Overall, the DSS + Cap-cPDVs group had better therapeutic effects than the DSS + PDV group; these results suggest that additional benefit may derive from intact cPDVs arriving at damaged sites in the colon.

[0314] This formulation will have broad applications for other diseases, including alcohol-induced liver damage (Zhuang, X. et al., Journal of extracellular vesicles 4:28713 (2019)), wound healing (Sahin, F., et al., Applied biochemistry and biotechnology 188:381-394 (2019)), and anti cancer therapy (Cao, M., et al., Journal of immunotherapy of cancer 7: 1-19 (2019); Chen, Q., et al., Acta Pharmaceutica Sinica B 12:907-923 (2022)).* * *

[0315] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature.

[0316] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A capsule comprising a plurality of plant derived vesicles (PDVs) which are coated with a polymer, wherein the plurality of PDVs are lyophilized and the plurality of PDVs are derived from spinach, cabbage, or a combination thereof.

2. A capsule comprising a plurality of plant derived vesicles (PDVs), wherein the capsule is coated with a polymer, wherein the plurality of PDVs are lyophilized and the plurality of PDVs are derived from spinach, cabbage, or a combination thereof.

3. The capsule of claim 2, wherein the plurality of PDVs are coated with a polymer.

4. The capsule of any one of claims 1-3, wherein the plurality of PDVs is derived from cabbage.

5. The capsule of any one of claims 1-3, wherein the plurality of PDVs is derived from spinach.

6. The capsule of any one of claims 1-5, wherein one or more of the plurality of PDVs comprises a therapeutic agent.

7. The capsule of any one of claims 1-6, wherein the polymer coating comprises a methacrylic acid-methyl methacrylate copolymer.

8. The capsule of claim 7, wherein the polymer coating comprises 1 mg / ml to 4 mg / ml (e.g., 2 mg / ml) methacrylic acid-methyl methacrylate copolymer.

9. The capsule of claim 7, wherein the polymer coating comprises 2% to 20% methacrylic acid-methyl methacrylate copolymer.

10. The capsule of claim 7, wherein the polymer coating comprises 4% methacrylic acid- methyl methacrylate copolymer.

11. The capsule of claim 7, wherein the polymer coating comprises 14% methacrylic acid- methyl methacrylate copolymer.

12. The capsule of claim 7, wherein the plurality of PDVs coated with two or more layers of the polymer.

13. The capsule of claim 12, wherein the polymer coating comprises, a layer of 4% methacrylic acid-m ethyl methacrylate copolymer and a layer of 14% methacrylic acid- methyl methacrylate copolymer.

14. The capsule of any one of claims 1-13, wherein one or more of the plurality of PDVs comprise an anti-inflammatory compound.

15. The capsule of claim 14, wherein the anti-inflammatory compounds comprise choline; betaine; trigonelline; DL-trytophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L- cysteine-S-oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L- glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL-serine; DL-homoserine; DL-arginine; and / or combinations thereof.

16. The capsule of claim 14, wherein the anti-inflammatory compounds comprise choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4- indolecarbaldehyde; trans-3 -indoleacrylic acid; Y-aminobutyric acid (GABA); and / or combinations thereof.

17. The capsule of any one of claims 1-16, wherein the capsule is suitable for oral administration.

18. The capsule of any one of claims 1-17, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the coated plurality of PDVs remain intact at a pH of 1 to 2 for at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes.

19. The capsule of any one of claims 1-17, wherein at least 70% of the plurality of coated PDVs remain intact at a pH of 1 to 2 for at least 30 minutes.

20. The capsule of any one of claims 1-17, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact for at least 30 minutes to 4 hours, at least 1 to 4 hours, at least 1 to 3 hours or at least 1 to 2 hours at a pH of 1 to 2.

21. The capsule of any one of claims 1-17, wherein at least 70% of the plurality of coated PDVs remain intact for at least 30 minutes at a pH of 1 to 2.

22. The capsule of any one of claims 1-17, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.

23. Use of any one of the capsules of claims 1-22 for treating a subject suffering from an inflammatory disease.

24. The use of claim 23, wherein the inflammatory disease is inflammatory bowel disease, colitis, ulcerative colitis, Crohn’s Disease, Rheumatoid arthritis, Psoriasis, Chronic obstructive pulmonary disease (COPD), or vasculitis.

25. The use of claim 23 or 24, wherein the use is for delivery to the colon of the subject, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon of the subject.

26. A method of preparing a capsule for oral delivery comprising (a) isolating a plurality of plant derived vesicles (PDVs) from cabbage or spinach; (b) coating one of more of the plurality of PDVs with a methacrylic acid-methyl methacrylate copolymer (e.g., 2 mg / ml methacrylic acid-methyl methacrylate copolymer); (c) lyophilizing the plurality of coated PDV; (d) filling a capsule with the lyophilized plurality of coated PDV; and optionally, (e) coating the capsule.

27. The method of claim 26, wherein the coated plurality of PDVs remain intact at a pH of 1 to 2 longer than a plurality of uncoated PDVs.

28. The method of claim 26, wherein the coated capsule to remain intact at a pH of 1 to 2 longer than an uncoated capsule.

29. The method of any one of claims 26-28, wherein the plurality of PDVs is derived from cabbage.

30. The method of c any one of claims 26-28, wherein the plurality of PDVs is derived from spinach.

31. The method of any one of claims 26-30, wherein the isolating comprises:(i) disinfecting the cabbage or spinach;(ii) blending the cabbage or spinach;(iii) filtering the blended cabbage or spinach to form a cabbage or spinach juice;(iv) sequentially centrifuging the cabbage or spinach juice to form a supernatant;(v) centrifuging the supernatant into a pellet comprising the plurality of PDVs ; and(vi) suspending the plurality of PDVs in solution, optionally phosphate buffered saline (PBS);(vii) or any combination thereof.

32. The method of any one of claims 26-30, wherein the isolating comprises the isolating comprises(i) blending the cabbage or spinach (e.g., the leaves) to form a juice;(ii) sequentially centrifuging the cabbage or spinach juice to form a supernatant comprising a plurality of PDVs;(iii) centrifuging the supernatant of step (ii) to form a pellet comprising the plurality of PDVs;(iv) suspending the pellet comprising the plurality of PDVs in solution;(v) washing the plurality the suspension of PDVs of step (iv); and(vi) filtering the washed PDVs of step (v), wherein the flow through from the filter comprises the isolated plurality of PDVs.

33. The method of any one of claims 26-32, wherein the lyophilizing comprises:(i) mixing the plurality of PDVs to create a mixture;(ii) freezing the mixture in liquid nitrogen; and(iii) lyophilizing the frozen mixture to obtain the lyophilized plurality of PDVs.

34. The method of any one of claims 26-33, wherein the coating comprises a methacrylic acid-methyl methacrylate copolymer.

35. The method of any one of claims 26-33, wherein the coating comprises between about 1 mg / mL to about 4 mg / ml methacrylic acid-methyl methacrylate copolymer.

36. The method of any one of claims 26-33, wherein the coating comprises 2% to 20% methacrylic acid-methyl methacrylate copolymer.

37. The method of any one of claims 26-33, wherein the coating comprises 4% methacrylic acid-methyl methacrylate copolymer.

38. The method of any one of claims 26-33, wherein the coating comprises 14% methacrylic acid-methyl methacrylate copolymer.

39. The method of any one of claims 26-33, wherein the coating comprises a first coat with 4% methacrylic acid-methyl methacrylate copolymer and a second coat with 14% methacrylic acid-methyl methacrylate copolymer.

40. The method of any one of claims 26-33, wherein the coating comprises a first coat with 4% methacrylic acid-methyl methacrylate copolymer, a second coat with 4% methacrylic acid-methyl methacrylate copolymer, a third coat with 14% methacrylic acid-methyl methacrylate copolymer, and a fourth coat with 14% methacrylic acid-methyl methacrylate copolymer.

41. The method of any one of claims 26-40, wherein one or more of the plurality of PDVs comprises an anti-inflammatory compound.

42. The method of claim 41, wherein the anti-inflammatory compound is selected from one or more compounds in Table 2, Table 3, and / or Table 4.

43. The method of claim 41, wherein the anti-inflammatory compound comprises choline; betaine; trigonelline; DL-trytophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L- cysteine-S-oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L- glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL-serine; DL-homoserine; DL-arginine; or any combination thereof.

44. The method of claim 41, wherein the anti-inflammatory compound comprises choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4- indolecarbaldehyde; trans-3 -indoleacrylic acid; Y-aminobutyric acid (GABA); or any combination thereof.

45. The method of any one of claims 26-44, wherein one or more of the plurality of PDVs comprises a therapeutic agent.

46. A composition comprising a plurality of PDVs (e.g., coated PDVs), a composition comprising a plurality of PDVs (e.g., coated PDVs), or a capsule comprising a plurality of PDVs (e.g., coated PDVs) produced by the method of any one of claims 26-45.

47. A method of treating inflammation in the intestine of a subject in need thereof comprising administering to the subject a plurality of PDVs, a composition comprising a plurality of PDVs, or a capsule comprising a plurality of PDVs of any one of claims 1-23.

48. The method of claim 47, wherein the plurality of PDVs are coated PDVs.

49. The method of claim 48, wherein at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the plurality of coated PDVs remain intact in the colon when administered orally to a subject.

50. The method of any one of claims 47-49, wherein the plurality of PDVs is derived from a cabbage.

51. The method of any one of claims 47-49, wherein the plurality of PDVs is derived from a spinach.

52. The method of any one of claims 48-51, wherein the coating comprises a methacrylic acid-methyl methacrylate copolymer.

53. The method of any one of claims 48-51, wherein the coating comprises 4% methacrylic acid-methyl methacrylate copolymer.

54. The method of any one of claims 48-51, wherein the coating comprises 14% methacrylic acid-methyl methacrylate copolymer.

55. The method of claim 48, wherein one or more of the plurality of PDVs comprises an antiinflammatory compound.

56. The method of claim 55, wherein the anti-inflammatory compounds comprises choline; betaine; trigonelline; DL-trytophan; L-isoleucine; L-phenylalanine; I3C; S-methyl-L- cysteine-S-oxide; 9S,13R-12-oxophytodienoic acid (12-OPDA); sulforaphane; L- glutamic acid; valine; L-(+)-citrulline; D-(+)-proline; D-(+)-pyroglutamic acid; L-aspartic acid; DL-serine; DL-homoserine; DL-arginine; or any combination thereof.

57. The method of claim 55, wherein the anti-inflammatory compounds comprises choline; betaine; trigonelline; hypoxanthine; D-(+)-tryptophan; L-isoleucine; 4- indolecarbaldehyde; trans-3 -indoleacrylic acid; Y-aminobutyric acid (GABA); or any combination thereof.

58. The method of any one of claims 47-57, wherein one or more of the plurality of PDVs comprises a therapeutic agent.

59. The method of any one of claims 47-58, wherein the inflammation is caused by an inflammatory disease.

60. The method of claim 59, wherein the inflammatory disease is an inflammatory bowel disease, colitis, ulcerative colitis, Crohn’s Disease, Rheumatoid arthritis, Psoriasis, Chronic obstructive pulmonary disease (COPD), or vasculitis.

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