Methods for generating extracellular vesicles loaded with resolvins

Abstract

Generally, the present disclosure is directed to methods of producing Resolvin-loaded extracellular vesicles, compositions comprising Resolvin-loaded extracellular vesicles, and methods of treating diseases using such compositions. In some embodiments, methods described herein comprise providing a population of cells, transfecting the population of cells with at least one plasmid vector encoding human cyclooxygenase-2 and 5-lipoxygenase, applying DHA, EP A, and ASA to the transfected population of cells, and isolating the Resolvin-loaded extracellular vesicles from culture media of the transfected population of cells. In some cases, a composition described herein comprises a plurality of extracellular vesicles comprising at least one Resolvin packaged within an interior of the EVs. In some embodiments, upon disposing a plurality of Resolvin-loaded extracellular vesicles within a biological compartment of the patient, inflammation from an inflammatory condition or disease, such as acute lung respiratory distress syndrome, is mitigated or resolved.

Description

METHODS FOR GENERATING EXTRACELLULAR VESICLES LOADED WITHRESOLVINSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 596,393, filed November 6, 2023, which is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING OR TABLE

[0002] The material in the accompanying sequence listing is hereby incorporated by reference in its entirety into this application. The accompanying file, named 069596-00015, was created on October 30, 2024 and is 14 kB.FIELD

[0003] The present disclosure is directed to compositions and methods for treating and / or diagnosing a condition or disease in a patient in need thereof. In particular, extracellular vesicles for enhanced cellular delivery of Resolvin are described.BACKGROUND

[0004] Extracellular vesicles (EVs) are lipid bilayer membrane-bound vesicles that are secreted by many cell types into the extracellular space and transport cargoes, including proteins, nucleic acids, and lipids, from the parent cell. In lung cells, released EVs have been shown to transport biologically active molecules that mediate intercellular communications. However, the efficient generation of pro-resolution lipid mediators in cell culture has proven challenging.SUMMARY

[0005] The present application is directed to Resolvin-loaded extracellular vesicle (RvEVs) compositions, their methods of making, and their use for treating and / or diagnosing a condition or disease in a human patient and / or other subject.

[0006] In one aspect, methods described herein comprise methods of producing Resolvin- loaded extracellular vesicles. In some embodiments, a method described herein comprisesproviding a population of cells and transfecting the population cells with at least one plasmid vector encoding cyclooxygenase-2 (C0X2) and encoding 5 -lipoxygenase (5-LOX). In some implementations, docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A), and acetylsalicylic acid (ASA) are applied to the transfected population of cells. In some embodiments, RvEVs are isolated from culture media of the transfected population of cells.

[0007] In some implementations, Resolvin comprises Resolvin D or Resolvin E. In some instances, Resolvin D comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, or a combination thereof. In some cases, Resolvin E comprises Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof. Many combinations of Resolvins or Resolvin molecules may be contemplated.

[0008] The identity of the population of cells is not limited. The cells and / or cell line used may be any that is not inconsistent with the technical objectives of this disclosure. In some cases, the population of cells may be primary cells. In some embodiments, the population of cells may be an immortalized cell line. In some implementations, the population of cells comprises HEK293 cells. In some cases, HEK293 cells comprise HEK 293T cells or HEK293sus cells. In some cases, the population of cells comprises CHO cells. In some embodiments, the population of cells comprises A549 cells.

[0009] Moreover, in some embodiments, the at least one plasmid further encodes 15- lipoxygenase (15-LOX). In some instances, transfecting includes applying 5-20 pg of the at least one plasmid vector encoding cyclooxygenase-2 (COX2) and encoding 5 -lipoxygenase (5-LOX). Additionally, in some instances, the transfecting includes the use of a non-lipid transfection reagent. Further, in some embodiments, 3-5 pM each of DHA and EPA and 0.1-1 mM of ASA are applied to the transfected cells. In some cases, isolating the RvEVs includes filtering the culture media through a 0.22 pm filter to remove microvesicles. In some implementations, filtering comprises passing the microvesicle depleted culture media through 100 kDa centrifugal filters to isolate EVs.

[0010] In another aspect, compositions comprising a plurality of extracellular vesicles (EVs) comprising at least one Resolvin disposed within an interior of the EVs are described. In some embodiments, the EVs are 100 nm to 200 nm in average size (e.g., as measured by dynamic light scattering). In some instances, the Resolvin of the EVs comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, Resolvin El, Resolvin E2, Resolvin E3,Resolvin E4, Resolvin E5, or a combination thereof. In some embodiments, the EVs comprise Resolvin DI and Resolvin El. In some cases, the EVs contain at least 60,000 pg of RvDl per mg of EV protein and at least 12,000 pg of RvEl per mg of EV protein. In some embodiments, prostaglandin F2a (PGF2a), prostaglandin D2 (PGD2), 12-hydroxyeicosatetraenoic acid (12- HETE), 15-hydroxy eicosatetraenoic acid (15-HETE), 13 -hydroxy octadecadienoic acid (13- HODE), leukotriene E4 (LTE4), and lipoxin A4 (LXA4) are not detectable in the EVs.

[0011] In some instances, the plurality of EVs further comprise a cargo. Moreover, in some cases, the cargo is operable to diffuse out of the interior of the EVs when the composition is delivered into recipient cells by membrane fusion, endocytic pathways (clathrin-dependent or clathrin-independent endocytosis), macropinocytosis, phagocytosis, and lipid raft-mediated internalization. In some instances, it is also possible that EVs may enter cells by more than one mechanism.

[0012] In another aspect, a method described herein of treating and / or diagnosing a condition or disease in a human patient and / or subject in need thereof, in some embodiments, comprises disposing a composition described herein within a biological compartment of the patient and / or subject, such as lungs or another pulmonary site of a patient. Further, in some cases, a method described herein comprises penetrating a membrane of a cell or population of cells within the biological compartment with the Resolvin-loaded extracellular vesicles (RvEVs) of the composition, and subsequently releasing at least a portion of the Resolvin, Resolvin content, and / or the cargo of the composition within a cytosol of the cell or population of cells after penetrating the membrane of the cell or population of cells. Additionally, in some embodiments, a method described herein further comprises biologically delivering the RvEVs of the composition after penetrating the membrane of the cell or population of cells.

[0013] In some cases, a composition described herein is disposed within the biological compartment of the patient through intranasal or intravenous administration. In addition, in some embodiments of methods described herein, the treated and / or diagnosed condition or disease comprises an inflammatory condition or disease. Further, in some such cases, the biological compartment (to which the composition is delivered) is a lung site.

[0014] In some embodiments, a method of treating an inflammatory condition or disease in a patient and / or subject in need thereof is provided, wherein the method includes disposing a plurality of RvEVs within a biological compartment of the patient. As such, in someimplementations, inflammation from the inflammatory condition or disease is mitigated or resolved. Additionally, in some cases, the inflammatory condition or disease is acute lung respiratory distress syndrome (ARDS). In some embodiments, the method further comprises penetrating a membrane of a cell or population of cells within the biological compartment with the plurality of RvEVs. In some instances, at least a portion of the Resolvin, Resolvin content, and / or the cargo of the RvEVs is released within the cytosol of the cell or population of cells after penetrating the membrane of the cell or population of cells. Further, in some embodiments, the RvEVs are disposed within the biological compartment of the patient through intranasal or intravenous administration.

[0015] In addition, in some embodiments, methods of restoring tissue homeostasis in a patient having an inflammatory condition or disease are provided. The method includes disposing a plurality of RvEVs within a biological compartment of the patient so that tissue homeostasis is restored in that patient.

[0016] The foregoing embodiments and other embodiments are further described in the detailed description which follows.BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1A is a schematic of the release of co-6 polyunsaturated fatty acid metabolites as a result of inflammation caused by ALI.

[0018] FIG. IB is a schematic of the release of ro-3 polyunsaturated fatty acid metabolites as a result of inflammation caused by ALI.

[0019] FIG. 2 is a volcano plot of the altered protein expression in BALF EVs isolated from mice with ALI compared to saline controls.

[0020] FIG. 3A illustrates a schematic of a method of transfecting HEK293T cells with two plasmid vectors, one plasmid vector encoding COX2 and one plasmid vector encoding 5-LOX, and treating the cells with DHA, EP A, and ASA, according to an embodiment described herein.

[0021] FIG. 3B illustrates an image of an immunoblot of the cell lysates of cells transfected with two plasmid vectors, one plasmid vector encoding COX2 and one plasmid vector encoding 5-LOX (pCDNA3.1-COX2-5-LOX), and treated with ethanol or DHA, EP A, and ASA as compared to cells transfected with a control plasmid (pCDNA3.1) and treated with ethanol or DHA, EP A, and ASA, according to some embodiments described herein.

[0022] FIG. 4A illustrates a plot of the size of particles as related to the concentration in the cell supernatant before filtering from cells transfected with vectors comprising COX2 and 5- LOX (pCDNA3.1-COX2-5-LOX), according to some embodiments described herein.

[0023] FIG. 4B illustrates a plot of the size of particles as related to concentration in the cell supernatant filtrate of cells transfected with pCDNA3.1, according to some embodiments described herein.

[0024] FIG. 4C illustrates a plot of the size of particles as related to concentration in the cell supernatant filtrate of cells transfected with vectors comprising COX2 and 5-LOX (pCDNA3.1- COX2-5-LOX), according to some embodiments described herein.

[0025] FIG. 4D illustrates an image of an immunoblot of COX2, 5-LOX, Calnexin, CD9, and HSP90 for the EVs and the cell lysates from cells transfected with a control plasmid and with vectors comprising COX2 and 5-LOX (pCDNA3.1-COX2-5-LOX), according to some embodiments described herein.

[0026] FIG. 4E illustrates a bar graph of the EV number from cells transfected with the control vector pCDNA3.1 and cells transfected with vectors comprising COX2 and 5-LOX (pCDNA3.1-COX2-5-LOX), according to some embodiments described herein.

[0027] FIG. 4F illustrates a bar graph of the size of EVs from cells transfected with the control vector pCDNA3.1 and cells transfected with vectors comprising COX2 and 5-LOX (pCDNA3.1-COX2-5-LOX), according to some embodiments described herein.

[0028] FIG. 5 A illustrates a plot of the expression of RvDl in pg / mg protein for HEK293 cells transfected with pCDNA3.1 treated with ethanol at 0.001%, HEK293 cells transfected with pCDNA3.1 treated with DHA, EP A, and ASA, HEK293 cells transfected with pCDNA3.1 - COX2 and pCDNA3.1 -5-LOX (pCDNA3.1-COX2-5-LOX) treated with ethanol at 0.001%, and HEK293 cells transfected with pCDNA3.1 -COX2 and pCDNA3.1 -5-LOX (pCDNA3.1-COX2- 5-LOX) and treated with DHA, EP A, and ASA, according to some embodiments described herein.

[0029] FIG. 5B illustrates a plot of the expression of RvEl in pg / mg protein for HEK293 cells transfected with pCDNA3.1 treated with ethanol at 0.001%, HEK293 cells transfected with pCDNA3.1 treated with DHA, EP A, and ASA, HEK293 cells transfected with pCDNA3.1 - COX2 and pCDNA3.1 -5-LOX (pCDNA3.1-COX2-5-LOX) treated with ethanol at 0.001%, and HEK293 cells transfected with pCDNA3.1 -COX2 and pCDNA3.1 -5-LOX (pCDNA3.1-COX2-5-LOX) and treated with DHA, EP A, and ASA, according to some embodiments described herein.

[0030] FIG. 6A is a box and whisker plot of the relative fluorescence (RFU) of TNFa-treated EAhy926 cells-PKH26 labeled DdHL60 (neutrophil) cells treated with EVs from cells transfected with pCDNA3.1, EVs from cells transfected with pCDNA3.1-COX2 and pCDNA3.1-5-LOX (pCDNA3.1-COX2-5-LOX EVs) and grown in medium with ethanol or DHA, EP A, and ASA, or some combination thereof, according to some embodiments described herein.

[0031] FIG. 6B is a box and whisker plot of the RFU of EAhy926 cells-DdHL60 cells treated with TNFa and nystatin or cytochalasin D, followed by treatment with EVs from cells transfected with pCDNA3.1-COX2 and pCDNA3.1 -5-LOX (RvDIEl EVs) and treated with DHA, EP A, and ASA, or some combination thereof, according to some embodiments described herein.

[0032] FIG. 6C is a box and whisker plot of the NF-KB reporter activity (relative light unit, RLU) of macrophage cells stimulated with LPS, treated with EVs produced from pCDNA3.1 or pCDNA3.1-COX2 and pCDNA3.1-5-LOX (pCDNA3.1-COX2-5-LOX) and grown in presence of ethanol or DHA, EP A, and ASA, according to some embodiments described herein.

[0033] FIG. 6D is a box and whisker plot of the NF-KB reporter activity (RLU) of macrophage cells treated with LPS, nystatin, and cytochalasin D, then followed by RvDIEl EVs, or some combination thereof, according to some embodiments described herein.

[0034] FIG. 7A is a plot of the normalized frequency as compared to the intensity of isolated pulmonary cells stained for CD45-APC (leukocytes CD45+) and lung structural cells (negative for CD45-APC (CD45')), according to some embodiments described herein.

[0035] FIG. 7B is a flow cytometry plot indicating CD45+, F4 / 80+(BV421), and EV+(PE) cells, according to some embodiments described herein.

[0036] FIG. 7C is a flow cytometry plot indicating CD45', CD326+, and EV+cells, according to some embodiments described herein.

[0037] FIG. 7D is a flow cytometry plot indicating CD45', CD3 U, and EV+cells, according to some embodiments described herein.

[0038] FIG. 7E is a bar graph of the percentage of EV+cells of the macrophages, epithelial cells, and endothelial cells of the isolated pulmonary cells, according to some embodiments described herein.

[0039] FIG. 8A is a box and whisker plot of BALF cells in control EV-treated and RvDIEl EV-treated mice with or without treatment with LPS, according to some embodiments described herein.

[0040] FIG. 8B is images of neutrophil staining in control EV-treated and RvDIEl EV- treated mice with or without treatment with LPS, according to some embodiments described herein.

[0041] FIG. 8C is a box and whisker plot of neutrophils per field in control EV-treated and RvDIEl EV-treated mice with or without treatment with LPS, according to some embodiments described herein.

[0042] FIG. 8D is a box and whisker plot of BALF protein in pg / pL in control EV-treated and RvDIEl EV-treated mice with or without treatment with LPS, according to some embodiments described herein.

[0043] FIG. 8E is a box and whisker plot of IL-6 in pg / mL in control EV-treated and RvDIEl EV-treated mice with or without treatment with LPS, according to some embodiments described herein.

[0044] FIG. 8F is a box and whisker plot of BALF TNFa in pg / mL in control EV-treated and RvDIEl EV-treated mice with or without treatment with LPS, according to some embodiments described herein.

[0045] FIG. 9 is a schematic of the generation of bioactive lipids from co-3 polyunsaturated fatty acids.

[0046] FIG. 10A is a plot of the particle size in nm as compared to the concentration of the particles for microvesicles (MVs) in mice BALF, according to some embodiment described herein.

[0047] FIG. 10B is a plot of the particle size in nm as compared to the concentration of the particles for EVs in mice BALF, according to some embodiment described herein.

[0048] FIG. 10C is a bar graph of the number of EVs and MVs in particles / mL for control mice and mice treated with LPS, according to some embodiment described herein.

[0049] FIG. 10D is a TEM image of an EV, according to some embodiment described herein.

[0050] FIG. 10E is an image of an immunoblot for CD9 and CD63 of cell lysates and EVs, according to some embodiment described herein.

[0051] FIG. 10F is a bar graph of the amount of phospholipids in nmole present in MVs, EVs, and EV-depleted BALF, according to some embodiment described herein.

[0052] FIG. 11 A is a flow cytometry plot for CD31+(x-axis) and CD326+(y-axis) EVs from unstained BALF EVs attached to CD9 Exo-Flow capture beads, according to some embodiment described herein.

[0053] FIG. 1 IB is a flow cytometry plot for CD31+(x-axis) and CD326+(y-axis) EVs from BALF EVs attached to CD9 Exo-Flow capture beads with saline treatment, according to some embodiment described herein.

[0054] FIG. 11C is a flow cytometry plot for CD31+(x-axis) and CD326+(y-axis) EVs from BALF EVs attached to CD9 Exo-Flow capture beads with LPS treatment, according to some embodiment described herein.

[0055] FIG. 1 ID is a flow cytometry plot for F4 / 80+(x-axis) and Ly6G+(y-axis) EVs from unstained BALF EVs attached to CD9 Exo-Flow capture beads, according to some embodiment described herein.

[0056] FIG. 1 IE is a flow cytometry plot for F4 / 80+(x-axis) and Ly6G+(y-axis) EVs from BALF EVs attached to CD9 Exo-Flow capture beads with saline treatment, according to some embodiment described herein.

[0057] FIG. 1 IF is a flow cytometry plot for F4 / 80+(x-axis) and Ly6G+(y-axis) EVs from BALF EVs attached to CD9 Exo-Flow capture beads with LPS treatment, according to some embodiment described herein.

[0058] FIG. 12A is a heat map of the detection of co-6 and co-3 PUFA metabolites in BALF EVs from LPS-treated mice, according to some embodiment described herein.

[0059] FIG. 12B is a box and whisker plot of the relative amount of AA in mole percent in LPS-treated mice as compared to control mice, according to some embodiment described herein.

[0060] FIG. 12C is a box and whisker plot of the specific activity of cPLA2 in nmol / mg protein in LPS-treated mice as compared to control mice, according to some embodiment described herein.

[0061] FIG. 12D illustrates an image of an immunoblot of cPLA2, COXI, COX2, 5-LOX, 12-LOX, CYP450, and |3-actin in LPS-treated mice as compared to control mice, according to some embodiment described herein.

[0062] FIG. 13 is a bar graph of the relative in vivo uptake of PKH-labeled EVs by pulmonary cells, according to some embodiment described herein.

[0063] FIG. 14 is a plot of the RFU of FITC-dextran flux for PMVECs grown on transwells treated with control EVs, EVs from LPS-treated mice (LPS-ALI EV), nystatin, control EVs and nystatin (Nystatin + Control EV), and EVs from LPS-treated mice and nystatin (Nystatin + LPS ALI EV), according to some embodiment described herein.

[0064] FIG. 15 is a bar graph of the RFU of FITC-dextran flux for PMVECs grown on transwells treated with thrombin at 0.2 units / mL, RvDIEl EVs, nystatin at 50 ug / mL, or some combination thereof, according to some embodiment described herein.

[0065] FIG. 16A is a schematic illustrating the multigene expression plasmid comprising the genes COX2, LOX-5, and 15-LOX being transfected into HEK293T cells that were grown in presence of DHA, EP A, and ASA to produce EVs loaded with Resolvins D1-D5 and E1-E5, according to some embodiment described herein.

[0066] FIG. 16B illustrates an image of an immunoblot of the EVs and the cell lysates for COX2, 5-LOX, 15-LOX, CD9, and Calnexin for HEK293T cells transfected with a control plasmid vector and the plasmid vector shown in FIG. 16A, according to some embodiments described herein.

[0067] FIG. 17A illustrates a schematic of a neutrophil adhesion assay, according to an embodiment described herein.

[0068] FIG. 17B is a bar graph of the RFU of TNFa-treated EAhy926 cells-DdHL60 cells treated with control EVs or and Resolvin D1-D5 and El-E5-loaded extracellular vesicles, according to some embodiment described herein.

[0069] FIG. 17C is a bar graph of the RFU of TNFa-treated EAhy926 cells-DdHL60 cells treated with nystatin, cytochalsin D, Resolvin D1-D5 and El-E5-loaded extracellular vesicles, or some combination thereof, according to some embodiments described herein.

[0070] FIG. 18A is a bar graph of the RFU of FITC-dextran flux through PMVEC monolayers grown on transwells treated with control EVs, Resolvin D1-D5 and El-E5-loadedextracellular vesicles, thrombin, or some combination thereof, according to some embodiment described herein.

[0071] FIG. 18B is an immunoblot for VE-cadherin and [3-actin of PMVECs treated with control EVs and Resolvin D1-D5 and El-E5-loaded extracellular vesicles, according to some embodiment described herein.

[0072] FIG. 19 is a bar graph of the NF -KB reporter activity of macrophage cells (RLU) treated with control EVs or with Resolvin D1-D5 and El-E5-loaded EVs with or without treatment with LPS, according to some embodiment described herein.

[0073] FIG. 20A is a bar graph of BALF cells / mL in control EV-treated mice and mice treated with Resolvin D1-D5 and El-E5-loaded extracellular vesicles with or without treatment with LPS, according to some embodiment described herein.

[0074] FIG. 20B is a bar graph of TNFa in pg / mL in control EV-treated mice and mice treated with Resolvin D1-D5 and El-E5-loaded extracellular vesicles with or without treatment with LPS, according to some embodiment described herein.

[0075] FIG. 20C is a bar graph of IL-6 in pg / mL in control EV-treated mice and mice treated with Resolvin D1-D5 and El-E5-loaded extracellular vesicles with or without treatment with LPS, according to some embodiment described herein.DETAILED DESCRIPTION

[0076] Embodiments described herein can be understood more readily by reference to the following detailed description, figures, and examples and their previous and following descriptions. Elements, apparatus, and methods described herein, however, are not limited to the specific embodiments presented in the detailed description, figures, and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

[0077] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

[0078] All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10” should generally be considered to include the end points 5 and 10.

[0079] It is also to be understood that the article “a” or “an” refers to “at least one,” unless the context of a particular use requires otherwise.

[0080] In general, the present disclosure is directed to compositions such as Resolvin-loaded extracellular vesicles (RvEVs), and methods of making and using such compositions, including for the diagnosis and / or treatment of a condition or disease in a patient and / or subject in need thereof.I. Methods of Producing RvEVs

[0081] In one aspect, methods of producing Resolvin-loaded extracellular vesicles (RvEVs) are described herein. In some embodiments, a method described herein comprises providing a population of cells and transfecting the population of cells with at least one plasmid vector encoding cyclooxygenase-2 (COX2) and encoding 5-lipoxygenase (5-LOX). In some instances, the method further comprises applying docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A), and acetylsalicylic acid (ASA) to the transfected population of cells. In some implementations, the method further comprises isolating RvEVs from culture media of the transfected population of cells.

[0082] In some implementations, methods of producing Resolvin-loaded extracellular vesicles (RvEVs) described herein may form RvEVs in which Resolvin is contained, encapsulated, located, and / or disposed in the interior of EVs. In some embodiments, Resolvin may comprise one or more Resolvins. In some instances, Resolvin comprises Resolvin D or Resolvin E. For reference purposes herein, Resolvin Ds are polyhydroxyl metabolites of DHA. Moreover, it is to be understood that Resolvin Es are di- or tri-hydroxyl metabolites of EPA. In some instances, Resolvin D comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, or a combination thereof. In some implementations, Resolvin E comprises Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof. Many combinations of Resolvins and / or Resolvin molecules may be contemplated. For example, in some preferred embodiments, Resolvin DI (RvDl) and Resolvin El (RvEl) may be disposed in the interior of the extracellular vesicles. In some other preferred embodiments,Resolvin DI (RvDl), Resolvin D2 (RvD2), Resolvin D3 (RvD3), Resolvin D4 (RvD4), and Resolvin D5 (RvD5), Resolvin El (RvEl), Resolvin E2 (RvE2), Resolvin E3 (RvE3), Resolvin E4 (RvE4), and Resolvin E5 (RvE5) may be disposed in the interior of the extracellular vesicles.

[0083] In some instances of methods described herein, at least one plasmid vector encodes cyclooxygenase-2 (COX2) and encodes 5 -lipoxygenase (5-LOX). Thus, in some embodiments, a single plasmid vector may be used to encode cyclooxygenase-2 (COX2) and encode 5- lipoxygenase (5-LOX). However, in some implementations, multiple plasmid vectors may be used to encode cyclooxygenase-2 (COX2) and encode 5 -lipoxygenase (5-LOX). For example, in some implementations, one plasmid vector may be used to encode cyclooxygenase-2 (COX2), and another separate plasmid vector may be used to encode 5 -lipoxygenase (5-LOX).

[0084] Moreover, in some cases of methods described herein, an at least one plasmid vector may comprise additional genes. For example, in some cases, an at least one plasmid vector may further encode 15 -lipoxygenase (15-LOX). Many plasmid vector designs and / or configurations for the arrangement of COX2, 5-LOX, and 15-LOX may be contemplated. For example, in some implementations, the gene for 15 -lipoxygenase (15-LOX), along with the gene for cyclooxygenase-2 (COX2) and the gene for 5 -lipoxygenase (5-LOX), may be encoded and / or present on a single plasmid vector. Stated differently, in some cases, a single plasmid vector may comprise or encode COX2, 5-LOX, and 15-LOX. In some instances, the gene for 15-LOX may be on a particular plasmid vector, while the gene for cyclooxygenase-2 (COX2) and the gene for 5-lipoxygenase (5-LOX) may be present on an additional vector. Moreover, in some instances, the gene for 15-LOX may be on a plasmid vector along with the gene for COX2, and the gene for 5-lipoxygenase (5-LOX) may be present on an additional vector. In some implementations, the gene for 15-LOX may be on a plasmid vector along with the gene for 5-LOX, and the gene for COX2 may be present on an additional vector. In some cases, the gene for 15-LOX, the gene for 5-LOX, and the gene for COX2 may each be on separate plasmid vectors.

[0085] Additionally, in some embodiments, the nucleotide sequence encoding COX2 may be derived from any organism not inconsistent with the technical objectives of the current disclosure. In some instances, the nucleotide sequence for COX2 may be derived from a mammalian species. Non-limiting examples of organisms wherein the nucleotide sequence of COX2 may be derived from include but are not limited to humans, mice, rats, monkeys, dogs, cats, and horses. Moreover, in some implementations, the nucleotide sequence encoding COX2may be subjected to codon optimization, dependent upon the cell type and / or cell line selected for the population of cells for transfection. Additionally, in some cases, the nucleotide sequence encoding C0X2 may comprise SEQ. ID NO. 1. In some implementations, SEQ. ID NO. 2 is the translated protein sequence for C0X2.

[0086] Further, in some embodiments, the nucleotide sequence encoding 5-LOX may be derived from any organism not inconsistent with the technical objectives of the current disclosure. In some instances, the nucleotide sequence for 5-LOX may be derived from a mammalian species. Non-limiting examples of organisms wherein the nucleotide sequence of 5- LOX may be derived from include but are not limited to humans, mice, rats, monkeys, dogs, cats, and horses. Moreover, in some implementations, the nucleotide sequence encoding 5-LOX may be subjected to codon optimization, dependent upon the cell type and / or cell line selected for the population of cells for transfection. Additionally, in some cases, the nucleotide sequence encoding 5-LOX may comprise SEQ. ID NO. 3. In some cases, SEQ. ID NO. 4 is the translated protein sequence for 5-LOX.

[0087] Additionally, in some embodiments, the nucleotide sequence encoding 15-LOX may be derived from any organism not inconsistent with the technical objectives of the current disclosure. In some instances, the nucleotide sequence for 15-LOX may be derived from a mammalian species. Non-limiting examples of organisms wherein the nucleotide sequence of 15- LOX may be derived from include but are not limited to humans, mice, rats, monkeys, dogs, cats, and horses. Moreover, in some implementations, the nucleotide sequence encoding 15-LOX may be subjected to codon optimization, dependent upon the cell type and / or cell line selected for the population of cells for transfection. Additionally, in some cases, the nucleotide sequence encoding 15-LOX may comprise SEQ. ID NO. 5. In some cases, SEQ. ID NO. 6 is the translated protein sequence for 15-LOX.

[0088] Moreover, it is also to be understood that in some embodiments, the at least one plasmid vector may encode or comprise further additional genes and / or additional nucleotide sequences. For example, in some cases, the at least one plasmid vector may comprise one or more promoter sequences. In some implementations, the at least one plasmid vector may comprise one or more restriction sites. In some embodiments, the at least one plasmid vector may comprise an antibiotic resistance gene. Non-limiting examples of an antibiotic resistancegene include but are not limited to an ampicillin resistance gene, a chloramphenicol resistance gene, or a kanamycin resistance gene.

[0089] In some embodiments, transfecting the population of cells comprises or includes transfecting the population of cells with a non-lipid and / or non-liposomal transfection reagent. Any non-lipid and / or non-liposomal transfection reagent not inconsistent with the technical objectives of the present disclosure may be used. Non-limiting examples of non-lipid and / or liposomal transfection reagents include but are not limited to FuGENE® HD Transfection Reagent, FuGENE® 6 Transfection Reagent, FuGENE® 4K Transfection Reagent, and GeneJuice® Transfection Reagent. Moreover, it is to be understood that the transfection conditions (e.g., volume of transfection agent or ratio of amount of transfection agent to amount of plasmid vectors(s) transfected) generally varies and does vary based upon the identity of the non-lipid and / or non-liposomal transfection reagent and the selected population of cells. In some embodiments, the ratio of the amount of non-lipid and / or non-liposomal transfection reagent to the amount of at least one plasmid vector is in the range of 1: 1-8:1, 1 : 1-6: 1, 1:1-4:1, 1:1-3:I, 1:1- 3:2, 3:2-8: 1, 3:2-6:l, 3:2-4: 1, 3:2-3: 1, 3:1-8:1, 3: 1-6: 1, 3:1-4: 1, 4: 1-8:1, 4: 1-6: 1, or 6: 1-8: 1.

[0090] In some instances of methods described herein, transfecting the population of cells comprises or includes applying the at least one plasmid vector encoding COX2 and encoding 5- LOX. As previously described, in some cases, the at least one plasmid vector may comprise additional genes. The amount of the at least one plasmid vector is not necessarily limited. In some implementations, 0.01-1000 pg, 0.04-1000 pg, 1-1000 pg, 5-1000 pg, 20-1000 pg, 50- 1000 pg, 100-1000 pg, 200-1000 pg, 500-1000 pg, 600-1000 pg, 700-1000 pg, 800-1000 pg, 900-1000 pg, 0.01-900 pg, 0.04-900 pg, 1-900 pg, 5-900 pg, 20-900 pg, 50-900 pg, 100-900 pg, 200-900 pg, 500-900 pg, 600-900 pg, 700-900 pg, 800-900 pg, 0.01-800 pg, 0.04-800 pg, 1-800 pg, 5-800 pg, 20-800 pg, 50-800 pg, 100-800 pg, 200-800 pg, 500-800 pg, 600-800 pg, 700-800 pg, 0.01-700 pg, 0.04-700 pg, 1-700 pg, 5-700 pg, 20-700 pg, 50-700 pg, 100-700 pg, 200-700 pg, 500-700 pg, 600-700 pg, 0.01-600 pg, 0.04-600 pg, 1-600 pg, 5-600 pg, 20-600 pg, 50-600 pg, 100-600 pg, 200-600 pg, 500-600 pg, 0.01-500 pg, 0.04-500 pg, 1-500 pg, 5- 500 pg, 20-500 pg, 50-500 pg, 100-500 pg, 200-500 pg, 0.01-200 pg, 0.04-200 pg, 1-200 pg, 5-200 pg, 20-200 pg, 50-200 pg, 100-200 pg, 0.01-100 pg, 0.04-100 pg, 1-100 pg, 5-100 pg, 20-100 pg, 50-100 pg, 0.01-50 pg, 0.04-50 pg, 1-50 pg, 5-50 pg, 20-50 pg, 0.01-20 pg, 0.04-20 pg,1-20 pg, 5-20 pg, 0.01-5 pg, 0.04-5 pg, 1-5 pg, 0.01-1 pg, 0.04-1 pg, or 0.01-0.04 pg of the atleast one plasmid vector encoding cyclooxygenase-2 (C0X2) and encoding 5 -lipoxygenase may be applied. However, it is to be understood that in some embodiments, the amount of the at least one plasmid vector and / or any other additional vector or vectors that may be transfected into the population of cells generally varies and does vary based upon the identity of the non-lipid and / or non-liposomal transfection reagent, the transfection conditions, and the selected population of cells. It is also to be understood that in some implementations, the amount of the at least one plasmid vector and / or any other additional vector or vectors that may be transfected into the population of cells generally varies and does vary based upon the number of cells in the population of cells.

[0091] The identity of the population of cells may be any cell type not inconsistent with the technical objectives of the present disclosure. The identity of the population of cells is not limited. In some cases, the population of cells are primary cells. In some embodiments, the population of cells are an immortalized cell line. In some implementations, the population of cells comprises HEK293 cells. In some cases, HEK293 cells comprise HEK293T, HEK293-H, HEK293-F, HEK293sus, and HT1080 cells. In some cases, the population of cells comprises CHO cells. In some embodiments, the population of cells comprises A549 cells. Other cell lines and / or cell types that may be used include but are not limited to HeLa cells, MCF-7 cells, PC-12 cells, MRC-5 cells, Vero cells, NIH3T3 cells, and HepG2 cells.

[0092] The amount of cells in the population of cells (i.e., the amount of cells used for transfection) may be any amount not inconsistent with the technical objectives of the present disclosure. In some embodiments, the amount of cells in the population of cells (i.e., the amount of cells used for transfection) may be in the range of 1 x 104cells to 1 x 109cells, 1 x 104cells to 1 x 108cells, 1 x 104cells to 1 x 107cells, 1 x 104cells to 1 x 106cells, 1 x 104cells to 1 x 105cells, 1 x 105cells to 1 x 109cells, 1 x 105cells to 1 x 108cells, 1 x 105cells to 1 x 107cells, 1 x105cells to 1 x 106cells, 1 x 106cells to 1 x 109cells, 1 x 106cells to 1 x 108cells, 1 x 106cells to1 x 107cells, 1 x 107cells to 1 x 109cells, 1 x 107cells to 1 x 108cells, or 1 x 108cells to 1 x 109cells.

[0093] In some implementations, methods described herein comprise applying DHA to the transfected population of cells. In some cases, DHA may be a free molecule. Moreover, in some embodiments, DHA may be conjugated to another molecule. For example, in some instances, DHA may be conjugated to serum albumin. Further, in some cases, DHA may be applied directlyto the transfected cells or as a component of culture media applied to the transfected population of cells. Additionally, the amount of DHA applied to the transfected population of cells may be any amount not inconsistent with the technical objectives of the present disclosure. In some instances, DHA may be applied to the transfected cells in the amount of 1-10 pM, 1-8 pM, 1-6 pM, 1-5 pM, 1-3 pM, 3-10 pM, 3-8 pM, 3-6 pM, 3-5 pM, 5-10 pM, 5-8 pM, 5-6 pM, 6-10 pM, 6-8 pM, or 8-10 pM.

[0094] Additionally, DHA may be applied to the transfected cells for any amount of time not inconsistent with the technical objectives of the present disclosure. For example, in some cases, DHA may be applied to the cells in the range of 12-18 hours, 12-24 hours, 12-30 hours, 12-36 hours, 12-42 hours, 12-48 hours, 12-54 hours, 12-60 hours, 12-72 hours, 18-24 hours, 18-30 hours, 18-36 hours, 18-42 hours, 18-48 hours, 18-54 hours, 18-60 hours, 18-72 hours, 24-30 hours, 24-36 hours, 24-42 hours, 24-48 hours, 24-54 hours, 24-60 hours, 24-72 hours, 30-36 hours, 30-42 hours, 30-48 hours, 30-54 hours, 30-60 hours, 30-72 hours, 36-42 hours, 36-48 hours, 36-54 hours, 36-60 hours, 36-72 hours, 42-48 hours, 42-54 hours, 42-60 hours, 42-72 hours, 48-54 hours, 48-60 hours, 48-72 hours, 54-60 hours, 54-72 hours, or 60-72 hours.Moreover, in some embodiments, the application of DHA may be continuous (i.e., without stopping or reapplication). In some instances, the application of DHA may be discontinuous. For example, in some cases, DHA may be reapplied in fresh culture media at the same concentration on the cells without the treatment time starting over.

[0095] In some implementations of methods described herein, methods comprise applying EPA to the transfected population of cells. In some cases, EPA may be a free molecule.Moreover, in some cases, EPA may be conjugated to another molecule. For example, in some embodiments, EPA may be conjugated to serum albumin. Additionally, in some instances, EPA may be applied directly to the transfected cells or as a component of culture media applied to the transfected population of cells. Further, the amount of EPA applied to the transfected population of cells may be any amount not inconsistent with the technical objectives of the present disclosure. In some implementations, EPA may be applied to the transfected cells in the amount of 1-10 pM, 1-8 pM, 1-6 pM, 1-5 pM, 1-3 pM, 3-10 pM, 3-8 pM, 3-6 pM, 3-5 pM, 5-10 pM, 5- 8 pM, 5-6 pM, 6-10 pM, 6-8 pM, or 8-10 pM.

[0096] Further, EPA may be applied to the transfected cells for any amount of time not inconsistent with the technical objectives of the present disclosure. For example, in some cases,EP A may be applied to the cells in the range of 12-18 hours, 12-24 hours, 12-30 hours, 12-36 hours, 12-42 hours, 12-48 hours, 12-54 hours, 12-60 hours, 12-72 hours, 18-24 hours, 18-30 hours, 18-36 hours, 18-42 hours, 18-48 hours, 18-54 hours, 18-60 hours, 18-72 hours, 24-30 hours, 24-36 hours, 24-42 hours, 24-48 hours, 24-54 hours, 24-60 hours, 24-72 hours, 30-36 hours, 30-42 hours, 30-48 hours, 30-54 hours, 30-60 hours, 30-72 hours, 36-42 hours, 36-48 hours, 36-54 hours, 36-60 hours, 36-72 hours, 42-48 hours, 42-54 hours, 42-60 hours, 42-72 hours, 48-54 hours, 48-60 hours, 48-72 hours, 54-60 hours, 54-72 hours, or 60-72 hours. Moreover, in some embodiments, the application of EPA may be continuous (i.e., without stopping or reapplication). In some instances, the application of EPA may be discontinuous. For example, in some cases, EPA may be reapplied at the same concentration in fresh culture media on the cells without the treatment time starting over.

[0097] In some implementations of methods described herein, methods comprise applying ASA to the transfected population of cells. In some cases, ASA may be a free molecule.Moreover, in some embodiments, ASA may be conjugated to another molecule. For example, in some instances, ASA may be conjugated to serum albumin. Additionally, in some embodiments, ASA may be applied directly to the transfected cells or as a component of culture media to the transfected population of cells. Further, the amount of ASA applied to the transfected population of cells may be any amount not inconsistent with the technical objectives of the present disclosure. In some embodiments, ASA may be applied to the transfected cells in the amount of 0.01-10 mM, 0.01-8 mM, 0.01-6 mM, 0.01-4 mM, 0.01-2 mM, 0.01-1 mM, 0.01-0.1 mM, 0.01- 0.05 mM, 0.05-10 mM, 0.05-8 mM, 0.05-6 mM, 0.05-4 mM, 0.05-2 mM, 0.05-1 mM, 0.05-0.1 mM, 0.1-10 mM, 0.1-8 mM, 0.1-6 mM, 0.1-4 mM, 0.1-2 mM, 0.1-1 mM, 1-10 mM, 1-8 mM, 1-6 mM, 1-4 mM, 1-2 mM, 2-10 mM, 2-8 mM, 2-6 mM, 2-4 mM, 4-10 mM, 4-8 mM, 4-6 mM, 6-10 mM, 6-8 mM, or 8-10 mM.

[0098] Additionally, ASA may be applied to the transfected cells for any amount of time not inconsistent with the technical objectives of the present disclosure. For example, in some cases, ASA may be applied to the cells in the range of 12-18 hours, 12-24 hours, 12-30 hours, 12-36 hours, 12-42 hours, 12-48 hours, 12-54 hours, 12-60 hours, 12-72 hours, 18-24 hours, 18-30 hours, 18-36 hours, 18-42 hours, 18-48 hours, 18-54 hours, 18-60 hours, 18-72 hours, 24-30 hours, 24-36 hours, 24-42 hours, 24-48 hours, 24-54 hours, 24-60 hours, 24-72 hours, 30-36 hours, 30-42 hours, 30-48 hours, 30-54 hours, 30-60 hours, 30-72 hours, 36-42 hours, 36-48hours, 36-54 hours, 36-60 hours, 36-72 hours, 42-48 hours, 42-54 hours, 42-60 hours, 42-72 hours, 48-54 hours, 48-60 hours, 48-72 hours, 54-60 hours, 54-72 hours, or 60-72 hours. Moreover, in some embodiments, the application of ASA may be continuous (i.e., without stopping or reapplication). In some instances, the application of ASA may be discontinuous. For example, in some cases, ASA may be reapplied at the same concentration in fresh culture media on the cells without the treatment time starting over.

[0099] Moreover, in some implementations of methods described herein, DHA, EP A, and ASA are applied to the transfected populations of cells. In some implementations, DHA, EP A, and ASA may be applied as separate steps and / or solutions to the cells. However, in some embodiments, DHA, EP A, and ASA may be applied together, that is, as a single solution or as a single solution in culture media.

[0100] In some embodiments of methods described herein, isolating RvEVs from culture media of the transfected population of cells comprises or includes filtering the culture media to remove microvesicles. Any method of filtering not inconsistent with the technical objectives of the current disclosure may be used to remove microvesicles (MVs). In some implementations, filtering the culture media to remove MVs comprises or includes sterile filtration. For example, in some cases, filtering the culture media may comprise passing the media through a 0.22 pm filter. Non-limiting examples of methods of filtering may include but are not limited to syringe filtering, vacuuming filtering, and tank vent filtration.

[0101] In some implementations, methods described herein may further comprise concentrating isolated RvEVs. Any method of concentrating isolated RvEVs not inconsistent with the technical objectives of the present disclosure may be used. For example, in some cases, concentrating isolated RvEVs comprises concentrating isolated RvEVs with a centrifugal concentrator. Non-limiting examples of concentrating isolated RvEVs include but are not limited to ultracentrifugation of RvEVs, lyophilizing RvEVs, and vacuum concentrating RvEVs.II. Compositions Comprising a Plurality of EVs

[0102] In one aspect, compositions are particularly described herein. In some embodiments, compositions described herein comprise a plurality of EVs comprising at least one Resolvin disposed within an interior of the EVs. Any method described herein above in Section I may be used to form a composition described herein.

[0103] An EV described herein may have any shape not inconsistent with the technical objectives of the present disclosure. The shape of the EV is not particularly limited. In some cases, an EV described herein may be round, spherical, or substantially spherical. In some instances, an EV described herein may have a measured sphericity. Sphericity may be measured or determined in any manner known to one of ordinary skill in the art. In some embodiments, sphericity may be measured using dynamic light scattering (e.g., size analysis using a NanoSight particle analyzer). Thus, in some cases, an EV described herein may have an average sphericity of 0.49-1.0, 0.7-1.0, 0.8-1.0, 0.9-1.0, 0.49-0.9, 0.7-0.9, or 0.49-0.7. However, in some instances, an EV described herein may also be oblate. Thus, in some cases, an EV described herein may have a measured aspect ratio. The aspect ratio may be measured or determined in any manner known to one of ordinary skill in the art. In some embodiments, the aspect ratio may be measured using dynamic light scattering. In some implementations, an EV may have an aspect ratio greater than 1.0, 1.1, or 1.2. In some embodiments, an EV may have an aspect ratio between 1.1 and 1.5, between 1.1 and 1.3, or between 1.3 and 1.5.

[0104] Moreover, in some implementations, the EVs have an average size in three dimensions. For example, in some instances, an EV can have an average size in a first dimension or the x dimension. In some cases, an EV can have an average size in a second dimension or in the y dimension. In some embodiments, an EV can have an average size in a third dimension or in the z dimension. It is to be understood that the average size of the EVs can be considered, for size comparison purposes, as the diameter of a round, spherical, or substantially spherical EV having an equal volume of the EV of the composition. The EVs of a composition described herein can have any size not inconsistent with the technical objectives of the present disclosure. For example, in some implementations, the average size of the EVs in three dimensions is between 50 nm and 300 nm, 50 nm and 250 nm, 50 nm and 200 nm, 50 nm and 150 nm, 50 nm and 100 nm, 50 nm and 75 nm, 75 nm and 300 nm, 75 nm and 250 nm, 75 nm and 200 nm, 75 nm and 150 nm, 75 nm and 100 nm, 100 nm and 300 nm, 100 nm and 250 nm, 100 nm and 200 nm, 100 nm and 150 nm, 150 nm and 300 nm, 150 nm and 250 nm, 150 nm and 200 nm, 200 nm and 300 nm, 200 nm and 250 nm, or 250 nm and 300 nm. In preferred embodiments, the average size of EVs is 200 nm or less. In some cases, the average size may be greater than 300 nm or less than 50 nm. Moreover, it is to be understood that in some instances, the average size of each of the first, second, and third dimension may be in these ranges. In some implementations, theaverage size of EVs described herein may be determined using dynamic light scattering (e g., size analysis using a NanoSight particle analyzer).

[0105] EVs described herein may also be formed using any material not inconsistent with the technical objectives of the present disclosure. In some cases, EV membranes may have a higher content of sphingomyelin, cholesterol, and glycosphingolipids compared to their parent cells (i.e., the cells the EVs are formed from or the transfected cells). For example, in some embodiments, the content of sphingomyelin, cholesterol, and glycosphingolipids of the EV membranes is at least 10%, at least 20%, at least 30%, at least 50%, or at least 80% higher than the content of sphingomyelin, cholesterol, and glycosphingolipids of the membranes of the cells they are formed from.

[0106] In some embodiments described herein, EV membranes are suitable to fuse with recipient cell membranes and deliver their luminal contents. EVs, in some cases, permit delivery of a Resolvin, a plurality of Resolvins, a cargo, and / or solvent into and / or out of the interior of the EVs. In this manner, an EV can release its contents (i.e., a Resolvin, a plurality of Resolvins, a cargo, and / or solvent) into a biological compartment or into the cytosol of a cell over a desired time period. In some embodiments, the contents of an EV may be operable to diffuse out of the interior of the EVs when the composition is delivered into a biological environment, such as recipient cells, or a lipophilic environment. Moreover, in some cases, the contents of an EV or the EV itself may be delivered into recipient cells through other processes. Non-limiting examples of these processes include but are not limited to membrane fusion, endocytic pathways (clathrin-dependent or clathrin-independent endocytosis), macropinocytosis, phagocytosis, lipid raft-mediated internalization, and combinations thereof.

[0107] In some instances, at least one Resolvin is disposed within an interior of the EVs. It is to be understood that in some embodiments, the at least one Resolvin may exit the EV and may be delivered to a biological compartment of a patient and / or subject as described herein below. However, in some cases, the at least one Resolvin may remain encapsulated within the EV. Moreover, in some cases, the at least one Resolvin is operable to diffuse out of the interior of the EV when the composition is delivered into a biological environment, as described further herein below.

[0108] In some cases, a Resolvin comprises one or more Resolvins. In some implementations, a Resolvin may comprise a plurality of Resolvins. In some instances, Resolvincomprises Resolvin D or Resolvin E. In some instances, Resolvin D comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, or a combination thereof. In some implementations, Resolvin E comprises Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof. In some preferred embodiments, Resolvin comprises Resolvin DI (RvDl) and / or Resolvin El (RvEl). In some other preferred embodiments, Resolvin comprises Resolvin DI (RvDl), Resolvin D2 (RvD2), Resolvin D3 (RvD3), Resolvin D4 (RvD4), Resolvin D5 (RvD5), Resolvin El (RvEl), Resolvin E2 (RvE2), Resolvin E3 (RvE3), Resolvin E4 (RvE4), and Resolvin E5 (RvE5). However, many combinations of Resolvins may be contemplated.

[0109] The amount of Resolvin and / or the content of Resolvin in the EVs that may be present in a composition described herein may be any amount not inconsistent with the technical objectives of the present disclosure. Stated differently, the content and / or the amount of Resolvin disposed in the interior of the EVs may not be necessarily limited. For example, the content of a Resolvin may be present in the range of 500-100,000 pg / mg of EV protein, 750-100,000 pg / mg of EV protein, 1,000-100,000 pg / mg of EV protein, 5,000-100,000 pg / mg of EV protein, 8,000- 100,000 pg / mg of EV protein, 10,000-100,000 pg / mg of EV protein, 12,000-100,000 pg / mg of EV protein, 15,000-100,000 pg / mg of EV protein, 20,000-100,000 pg / mg of EV protein, 50,000- 100,000 pg / mg of EV protein, 55,000-100,000 pg / mg of EV protein, 60,000-100,000 pg / mg of EV protein, 65,000-100,000 pg / mg of EV protein, 75,000-100,000 pg / mg of EV protein, 80,000- 100,000 pg / mg of EV protein, 90,000-100,000 pg / mg of EV protein, 500-90,000 pg / mg of EV protein, 750-90,000 pg / mg of EV protein, 1,000-90,000 pg / mg of EV protein, 5,000-90,000 pg / mg of EV protein, 8,000-90,000 pg / mg of EV protein, 10,000-90,000 pg / mg of EV protein, 12,000-90,000 pg / mg of EV protein, 15,000-90,000 pg / mg of EV protein, 20,000-90,000 pg / mg of EV protein, 50,000-90,000 pg / mg of EV protein, 55,000-90,000 pg / mg of EV protein, 60,000- 90,000 pg / mg of EV protein, 65,000-90,000 pg / mg of EV protein, 75,000-90,000 pg / mg of EV protein, 80,000-90,000 pg / mg of EV protein, 500-80,000 pg / mg of EV protein, 750-80,000 pg / mg of EV protein, 1,000-80,000 pg / mg of EV protein, 5,000-80,000 pg / mg of EV protein, 8,000-80,000 pg / mg of EV protein, 10,000-80,000 pg / mg of EV protein, 12,000-80,000 pg / mg of EV protein, 15,000-80,000 pg / mg of EV protein, 20,000-80,000 pg / mg of EV protein, 50,000- 80,000 pg / mg of EV protein, 55,000-80,000 pg / mg of EV protein, 60,000-80,000 pg / mg of EV protein, 65,000-80,000 pg / mg of EV protein, 75,000-80,000 pg / mg of EV protein, 500-75,000pg / mg of EV protein, 750-75,000 pg / mg of EV protein, 1,000-75,000 pg / mg of EV protein, 5,000-75,000 pg / mg of EV protein, 8,000-75,000 pg / mg of EV protein, 10,000-75,000 pg / mg of EV protein, 12,000-75,000 pg / mg of EV protein, 15,000-75,000 pg / mg of EV protein, 20, GOO- 75, 000 pg / mg of EV protein, 50,000-75,000 pg / mg of EV protein, 55,000-75,000 pg / mg of EV protein, 60,000-75,000 pg / mg of EV protein, 65,000-75,000 pg / mg of EV protein, 500-65,000 pg / mg of EV protein, 750-65,000 pg / mg of EV protein, 1,000-65,000 pg / mg of EV protein, 5,000-65,000 pg / mg of EV protein, 8,000-65,000 pg / mg of EV protein, 10,000-65,000 pg / mg of EV protein, 12,000-65,000 pg / mg of EV protein, 15,000-65,000 pg / mg of EV protein, 20, GOO- 65, 000 pg / mg of EV protein, 50,000-65,000 pg / mg of EV protein, 55,000-65,000 pg / mg of EV protein, 60,000-65,000 pg / mg of EV protein, 500-60,000 pg / mg of EV protein, 750-60,000 pg / mg of EV protein, 1,000-60,000 pg / mg of EV protein, 5,000-60,000 pg / mg of EV protein, 8,000-60,000 pg / mg of EV protein, 10,000-60,000 pg / mg of EV protein, 12,000-60,000 pg / mg of EV protein, 15,000-60,000 pg / mg of EV protein, 20,000-60,000 pg / mg of EV protein, 50,000- 60,000 pg / mg of EV protein, 55,000-60,000 pg / mg of EV protein, 500-55,000 pg / mg of EV protein, 750-55,000 pg / mg of EV protein, 1,000-55,000 pg / mg of EV protein, 5,000-55,000 pg / mg of EV protein, 8,000-55,000 pg / mg of EV protein, 10,000-55,000 pg / mg of EV protein, 12,000-55,000 pg / mg of EV protein, 15,000-55,000 pg / mg of EV protein, 20,000-55,000 pg / mg of EV protein, 50,000-55,000 pg / mg of EV protein, 500-50,000 pg / mg of EV protein, 750- 50,000 pg / mg of EV protein, 1,000-50,000 pg / mg of EV protein, 5,000-50,000 pg / mg of EV protein, 8,000-50,000 pg / mg of EV protein, 10,000-50,000 pg / mg of EV protein, 12,000-50,000 pg / mg of EV protein, 15,000-50,000 pg / mg of EV protein, 20,000-50,000 pg / mg of EV protein, 500-20,000 pg / mg of EV protein, 750-20,000 pg / mg of EV protein, 1,000-20,000 pg / mg of EV protein, 5,000-20,000 pg / mg of EV protein, 8,000-20,000 pg / mg of EV protein, 10,000-20,000 pg / mg of EV protein, 12,000-20,000 pg / mg of EV protein, 15,000-20,000 pg / mg of EV protein, 500-15,000 pg / mg of EV protein, 750-15,000 pg / mg of EV protein, 1,000-15,000 pg / mg of EV protein, 5,000-15,000 pg / mg of EV protein, 8,000-15,000 pg / mg of EV protein, 10,000-15,000 pg / mg of EV protein, 12,000-15,000 pg / mg of EV protein, 500-12,000 pg / mg of EV protein, 750-12,000 pg / mg of EV protein, 1,000-12,000 pg / mg of EV protein, 5,000-12,000 pg / mg of EV protein, 8,000-12,000 pg / mg of EV protein, 10,000-12,000 pg / mg of EV protein, 500-10,000 pg / mg of EV protein, 750-10,000 pg / mg of EV protein, 1,000-10,000 pg / mg of EV protein, 5,000-10,000 pg / mg of EV protein, 8,000-10,000 pg / mg of EV protein, 500-8,000 pg / mg of EVprotein, 750-8,000 pg / mg of EV protein, 1,000-8,000 pg / mg of EV protein, 5,000-8,000 pg / mg of EV protein, 500-5,000 pg / mg of EV protein, 750-5,000 pg / mg of EV protein, 1,000-5,000 pg / mg of EV protein, 500-1,000 pg / mg of EV protein, 750-1,000 pg / mg of EV protein, or 500- 750 pg / mg of EV protein.

[0110] Moreover, in some implementations, an amount of Resolvin and / or content of Resolvin disposed in the interior of the EVs may be present for a particular Resolvin or for a plurality of Resolvins in particular combinations. For example, in some cases, the RvDl content of a composition is at least 60,000 pg / mg of EV protein. Moreover, in some such instances, the RvDl content of a composition is at least 50,000 pg / mg of EV protein, at least 55,000 pg / mg of EV protein, at least 60,000 pg / mg of EV protein, or at least 65,000 pg / mg of EV protein. In some cases, the RvD2 content of a composition is at least 12,000 pg / mg of EV protein. Moreover, in some such instances, the RvD2 content is at least 750 pg / mg of EV protein, at least 1,000 pg / mg of EV protein, at least 5,000 pg / mg of EV protein, at least 8,000 pg / mg of EV protein, at least 10,000 pg / mg of EV protein, at least 12,000 pg / mg of EV protein, or at least 15,000 pg / mg of EV protein.

[0111] Turning to compositions with particular combinations, in some implementations, an EV of a plurality of EVs may comprise Resolvin DI and Resolvin El. In some such instances, the RvDl content of a composition is at least 60,000 pg / mg of EV protein, and the RvEl content of a composition is at least 12,000 pg / mg of EV protein. Moreover, in some cases, an EV of a plurality of EVs comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, and Resolvin E5. In some embodiments, the content of Resolvin DI is in the range of 16,000 pg / mg of EV protein to 24,000 pg / mg EV protein or 10,000 pg / mg of EV protein to 50,000 pg / mg EV. In some instances, the content of Resolvin D2 is in the range of 16,000 pg / mg of EV protein to 24,000 pg / mg EV protein or 10,000 pg / mg of EV protein to 50,000 pg / mg EV. In some implementations, the content of Resolvin El is in the range of 16,000 pg / mg of EV protein to 24,000 pg / mg EV protein or 10,000 pg / mg of EV protein to 50,000 pg / mg EV. In some embodiments, the content of Resolvin E2 is in the range of 16,000 pg / mg of EV protein to 24,000 pg / mg EV protein or 10,000 pg / mg of EV protein to 50,000 pg / mg EV.

[0112] In some cases, EVs described herein may further comprise a cargo. It is to be understood that such a “cargo” can comprise a chemical species, component, or agent (orcombination of two or more such species, components, or agents) that exits the EV and is delivered to a biological compartment of a patient and / or subject as described herein, or that remains encapsulated within the EV but provides functionality (e.g., fluorescence) to the overall composition or EV. In some embodiments, the cargo is physically entrapped within the interior of the EV. Moreover, in some cases, the cargo is operable to diffuse out of the interior of the EV when the composition is delivered into a biological environment, such as recipient cells, or a lipophilic environment. In some implementations, the cargo is delivered into recipient cells by membrane fusion, endocytic pathways (clathrin-dependent or clathrin-independent endocytosis), macropinocytosis, phagocytosis, or lipid raft-mediated internalization. Additionally, in some cases, a cargo may enter a cell by more than one mechanism.

[0113] Any cargo not inconsistent with the technical objectives of the present disclosure may be used in a composition described herein. In some embodiments, the cargo comprises an imaging agent, a therapeutic agent, a theranostic agent, or a combination of two or more of the foregoing. In some instances, for example, the cargo comprises an imaging agent, and the imaging agent is luminescent (e.g., fluorescent or phosphorescent). Any luminescent imaging agent not inconsistent with the technical objectives of the present disclosure may be used. In some embodiments, an imaging agent comprises a molecular dye having a luminescence emission in the visible or infrared (IR) region of the electromagnetic spectrum (e.g., having a peak emission wavelength between 400 nm and 800 nm, or between 840 nm and 1500 nm), such as an indocyanine dye (e.g., indocyanine green), a rhodamine dye (such as rhodamine B), a coumarin dye, fluorescein, or methylene blue. In some instances, an imaging agent comprises a luminescent biomolecule, such as green fluorescent protein (GFP). Additionally, in some cases, an imaging agent comprises a luminescent quantum dot or other luminescent nanoparticle, such as a quantum dot or other luminescent nanoparticle having an average size in three dimensions of less than 15 nm or less than 10 nm. An imaging agent may also comprise a contrast agent (e.g., an MRI contrast agent), such as a lanthanide compound or complex. Other imaging agents may also be used, and the imaging agent is not particularly limited.

[0114] A therapeutic agent used in a composition described herein, in some implementations, comprises a small molecule drug or other molecular drug (e.g., vancomycin), which may be hydrophobic or hydrophilic or amphiphilic. In some instances, a therapeutic agent comprises anucleic acid, such as a small interfering ribonucleic acid (siRNA). Other therapeutic agents may also be used, and the therapeutic agent is not particularly limited.

[0115] Similarly, a variety of theranostic agents (agents that can provide both diagnosis, such as by luminescence or magnetic resonance imaging (MRI), and also treatment, such as by hyperthermia, chemotherapy, or gene therapy) may be used in a composition described here. The theranostic agent is not particularly limited.

[0116] The cargo of a composition described herein can be present in any amount not inconsistent with the technical objectives of the present disclosure. In some embodiments, for example, the cargo is present in the composition in an amount of 1-80 wt. %, based on the total weight of the composition. In some cases, the cargo is present in an amount of 1-70 wt. %, 5-80 wt. %, 5-70 wt. %, 5-50 wt. %, 5-40 wt. %, 5-30 wt. %, 5-25 wt. %, 5-20 wt. %, 5-15 wt. %, 10- 80 wt. %, 10-70 wt. %, 10-60 wt. %, 10-50 wt. %, 20-80 wt. %, 20-70 wt. %, 20-60 wt. %, 20-50 wt. %, 20-40 wt. %, 30-80 wt. %, 30-70 wt. %, 40-80 wt. %, 40-70 wt. %, or 40-60 wt. %, based on the total weight of the composition.

[0117] In some cases, compositions described herein exclude, lack detectable amounts, or substantially lack detectable amounts of one or more lipid mediators. In some instances, one or more lipid mediators may be present at less than 50 pg / mg EV protein, less than 40 pg / mg EV protein, less than 30 pg / mg EV protein, less than 20 pg / mg EV protein, or less than 10 pg / mg EV protein. In some embodiments, prostaglandin F2ot (PGF2a) is not detectable in a composition described herein. In some embodiments, prostaglandin D2 (PGD2) is not detectable in a composition described herein. In some implementations, 12-hydroxy eicosatetraenoic acid (12- HETE) is not detectable in a composition described herein. Moreover, in some instances, 15- hydroxy eicosatetraenoic acid (15-HETE) is not detectable in a composition described herein. In some embodiments, 13 -hydroxy octadecadienoic acid (13-HODE) is not detectable in a composition described herein. Additionally, in some cases, leukotriene E4 (LTE4) is not detectable in a composition described herein. Further, in some embodiments, lipoxin A4 (LXA4) is not detectable in a composition described herein.III. Methods of Treating a Condition

[0118] In another aspect, methods of treating and / or diagnosing a condition or disease in a human patient and / or subject in need thereof are described herein. In some embodiments, such amethod comprises disposing a composition described herein within a biological compartment of the patient and / or subject. Any composition described herein in Section II may be used. Moreover, any composition produced by any method described in Section I may be used.

[0119] Additionally, the biological compartment can be any suitable biological compartment of the patient and / or subject. In some instances, a biological compartment comprises an internal organ of a patient and / or subject. In some embodiments, a composition described herein may also be disposed in or delivered to the bloodstream of the patient or in or to a blood vessel of the patient. Disposing or delivering the composition can be carried out in any manner not inconsistent with the objectives of the present disclosure. In some cases, for example, the composition is injected into the biological compartment. In some preferred embodiments, the composition is disposed within the biological compartment of the patient through intranasal administration. In some such embodiments, injection is avoided.

[0120] In some cases, a method described herein further comprises fusing with a membrane of a cell or population of cells or penetrating a membrane of a cell or population of cells within the biological compartment with the EVs of the composition. That is, in some embodiments, the EVs are uptaken within a cell or population of cells (e.g., within the lungs of a patient or from blood when administered intravenously). Additionally, in some implementations, a method described herein further comprises releasing at least a portion of the Resolvin, Resolvin content, and / or cargo of the composition within a cytosol of the cell or population of cells after penetrating the membrane of the cell or population of cells. Further, in some cases, a method described herein also comprises biologically delivering the contents of the EVs and / or the EVs themselves of the composition after penetrating the membrane of the cell or population of cells. Moreover, in some cases, the cargo of a composition used in a method described herein comprises an imaging agent or a theranostic agent, and the method further comprises imaging the cell or population of cells with the imaging agent or theranostic agent, which may occur before, during, or after release of a cargo or penetration within a cell or population of cells.

[0121] In some exemplary embodiments of a method, the condition or disease comprises an inflammatory condition or disease. In some instances, the biological compartment is a pulmonary site. Additionally, in some such cases, the disease comprises an inflammatory, degenerative or immune disease. In some embodiments, the inflammatory condition or disease comprises acute lung respiratory distress syndrome (ARDS). Moreover, in some implementations, theinflammatory condition or disease causes inflammation. Moreover, in some cases, the cargo comprises a therapeutic agent effective for the treatment of the inflammation caused by the condition or disease. In some implementations, the inflammation from the inflammatory condition or disease is mitigated or resolved. In some embodiments, tissue homeostasis is restored.EXAMPLES

[0122] The Examples further describe various aspects of embodiments of the present disclosure. These Examples are not meant to limit embodiments solely to such Examples herein but rather to illustrate some possible implementations.EXAMPLE 1

[0123] EVs are small, lipid bilayer membrane-bound vesicles released by most living cells. EVs have been shown to transport a variety of biomolecules, including DNA, RNA, proteins, and lipids. The EV-transported biomolecular cargo mediates intercellular communication, both under physiological and disease conditions. Herein, the total proteome and lipidome of bronchoalveolar lavage fluid (BALF) EVs from mouse acute lung injury (AL1) models was analyzed by tandem liquid chromatography and mass spectrometry. It was demonstrated that ALI causes an increased release of EVs into the alveolar space, which are packaged with a diverse array of bioactive lipid mediators (FIGS. 1A-B). Moreover, pro-inflammatory regulators 5-HPETE, 5-HETE, 11-HETE, 12-HETE, 15-HETE, 9-HODE / 9-oxoODE, 13-HODE / 13- oxoODE, PGE2, PGD2, and TXB2 were detected. LTA4, PGH2, PGE3, LTB5, RvEl, RvE2, PD1, RvDl-D5, and 14-HDHA were not detected. Additionally, pro-resolution mediators LXA4, 18-HEPE, 17-HDHA, RvD6, and MaRl were detected.

[0124] These lipid mediators are released from both co-6 (e.g., arachidonic acid (AA)) and co- 3 polyunsaturated fatty acids (e.g., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EP A)) by the enzymatic action of cyclooxygenases (COXI and COX2), lipoxygenases (5-LOX, 12-LOX, and 15-LOX) and cytochrome p450 (CYP450). Apart from the dietary route of linoleic acid to AA conversion, inflammatory stimuli activate cytoplasmic phospholipase A2 (cPLA2) that releases AA from cell membrane phospholipids (as indicated by the lightning bolt in FIG.1 A). AA further generates eicosanoids that regulate inflammatory signaling at multiple levels.Notably, eicosanoids and lipid mediators from co-6 and co-3 polyunsaturated fatty acids regulate both the initiation and resolution of inflammation during ALI and can act at nanomolar concentrations on target cells. AA metabolites, particularly prostaglandins (PGs) and leukotrienes (LTs), are generally pro-inflammatory, promote vasodilation, increased vascular permeability, and chemotaxis of immune cells. Excessive production of pro-inflammatory AA metabolites can contribute to inflammation and lung tissue damage during ALI and certain other inflammatory diseases. However, the lipid mediators released from co-3 polyunsaturated fatty acids actively resolve inflammation and are known as specialized pro-resolution lipid mediators (SPMs).

[0125] SPMs encompass lipoxins, resolvins, protectins, and maresins, which exert potent anti-inflammatory and pro-resolving effects by orchestrating a coordinated shutdown of inflammatory responses (by decreasing the pro-inflammatory cytokines and leukocyte infiltration) and restoring cellular repair and tissue homeostasis. Not intending to be bound by theory, it is believed that SPMs can be important regulators of inflammation resolution and tissue repair.

[0126] As described herein, EVs from bronchoalveolar lavage fluid (BALF) of mice with ALI are packaged with significantly higher amounts of pro-inflammatory lipid mediators and trace amounts of SPMs. SPMs packaged in BALF EVs have been identified herein. Not intending to be bound by theory, it is believed that SPMs may reduce inflammation and promote tissue repair in several inflammatory diseases. Notably, in this Example, it is demonstrated that SPMs are not generated by a single type of cell but rather result from the coordinated interaction of different cell types, such as immune cells, vascular cells and platelets, known as transcellular synthesis. It is to be understood that SPMs may be generated from populations of human cells. This intricate process poses challenges in replicating their synthesis within cell cultures.

[0127] In addition, lipid mediators synthesized in vivo are very short-lived, and synthetic methods are not widely established for large-scale production. Dietary intake of co-3 polyunsaturated fatty acids to increase the endogenous synthesis of SPMs is not an efficient approach, as the conversion of a-linoleic acid to DHA and EPA is a very low-efficiency process (FIGS. 1A-B). Furthermore, metabolic proteins of lipid mediator biosynthesis, including COX2 and 5-LOX were not detected in both healthy and ALI mouse BALF EVs (FIG. 2).

[0128] Not intending to be bound by theory, it is believed that while EVs do not inherently carry enzymes responsible for generating lipid mediators, they do serve as carriers for various types of lipid mediators. Not intending to be bound by theory, it is also believed that the use of EVs encapsulating SPMs for the treatment of inflammatory disorders is therapeutically beneficial. EV membranes are enriched in saturated fats and are relatively stable which helps promote the stability of the lipid mediator cargo. Unlike proteins, lipid mediators and SPMs are not species-specific. Further, EVs are not species-specific and are easily taken up by recipient cells from a different species. EVs can also be less immunogenic compared to cell therapy strategies. Finally, EVs can counteract excessive inflammation without suppressing the immune system of the host.

[0129] Despite medical advancements, acute lung respiratory distress syndrome (ARDS) remains associated with high mortality and morbidity and previously has lacked effective targeted treatments, relying mainly on supportive care options, like the use of antibiotics, fluid management and ventilator support. The economic burden of ARDS is substantial due to prolonged hospital stays, intensive care, and the need for mechanical ventilation. Thus, there has been an unmet need for therapies that effectively treat ARDS and improve patient outcomes.

[0130] Disclosed herein are EVs containing Resolvin DI (RvDl) and Resolvin El (RvEl), with demonstrated efficacy in pre-clinical mouse models of ALI. The present disclosure also describes the preclinical efficacy of RvD IE 1 -loaded EVs in mitigating and resolving ARDS- associated lung inflammation and restoring tissue homeostasis. Not intending to be bound by theory, it is believed that RvDIEl-loaded EVs may modulate the inflammatory response, promote tissue repair, and improve the clinical outcomes of patients with ARDS.

[0131] As illustrated in FIG. 3 A, 2 x 106HEK293T cells grown overnight in Opti-MEM were transfected with 10 pg each of pCDNA3.1-COX2 and pCDNA3.1-5-LOX plasmid vectors (GenScript, Piscataway NJ) using a non-lipid transfection reagent FuGENE 6 (Promega, Madison, WI). It is understood that other human cell lines are compatible with the present transfection steps. Further, in some cases, transfections of 5-20 pg each of pCDNA3.1-COX2 and pCDNA3.1-5-LOX plasmid vectors are possible. After 24 hours of transfection, the medium was removed and supplemented with fresh Opti-MEM containing 5 pM each of DHA and EPA and 1 mM ASA (Cayman, Detroit MI). The cells were grown for another 24 hours. After 24 hours, the culture medium was used for EV isolation, and the cells were lysed in IX RIPA bufferto analyze the over-expression of human COX2 and 5-LOX proteins. Both COX2 and 5-LOX were over-expressed in cell lysates, whereas the control plasmid (pCDNA3.1) transfected cell lysates showed no detectable COX2 and 5-LOX proteins (FIG. 3B, lanes 1-2 vs. 3-6).

[0132] The cell culture medium was filtered through 0.22 pm syringe filters to remove microvesicles. The resulting filtrate was loaded on 20 mL Pierce protein concentrators with a 100 kDa cut off (ThermoFisher) and centrifuged at 4000g for 30 minutes at 4°C. The concentrate was washed in IX DPBS and resuspended in 100 pL of IX DPBS or saline. Based on the plasmid vectors transfection and medium supplementation, 4 different groups of EVs were isolated: 1) pCDNA3.1 EVs, which were vector control EVs from pCDNA3.1 transfected cells grown in the presence of a) 0.001% ethanol (solvent control) or b) 5 pM DHA, 5 pM EP A, and 1 mM ASA; and 2) c) pCDNA3.1-COX2-5-LOX control EVs, which were EVs from cells transfected with pCDNA3.1-COX2 and pCDNA3.1 -5-LOX (pCDNA3.1-COX2-5-LOX) and grown in the presence of 0.001% ethanol or d) RvDIEl EVs, which were EVs from cells transfected with pCDNA3.1-COX2 and pCDNA3.1 -5-LOX (pCDNA3.1-COX2-5-LOX) and grown in the presence of 5 pM DHA, 5 pM EP A, and 1 mM ASA. The size of the EVs in the cell culture filtrate was confirmed with the nanoparticle size analyzer Nanosight (FIGS. 4A-C, and FIGS. 4E-F). Over-expression of COX2 and 5-LOX was confirmed in the HEK293 cell lysates, which was absent for the EV lysates, and the absence of Calnexin and enrichment of CD9 was confirmed in the EVs, confirming the purity of the EV preparation (FIG. 4D).

[0133] The EVs were screened, and the presence of RvDl (-1200 pg / mg) and RvEl (-750 pg / mg) was confirmed using Cayman ELISA kits (Cayman, Detroit, MI) (FIGS. 5A-B, ****p < 0.0001 for EVs from pCDNA3. l-COX2-5-LOX-transfected cells grown in the presence of DHA, EP A, and ASA vs. pCDNA3.1 or pCDNA3.1-COX2-5-LOX control EVs). The protein from the EVs were precipitated by ethanol and extracted in PBS.

[0134] This observation was further confirmed by LC-MS / MS. The levels of PGE2, PGD2, 12-HETE, 15-HETE, 13-HODE, LTE4, LXA4, RvDl, and RvEl were assessed in HEK293 cells transfected with a pCDNA3.1 control vector and treated with ethanol at 0.001% or DHA, EP A, and ASA as previously indicated (Exp. 1 and 2, respectively, in Table 1), HEK293 cells transfected with pCDNA3.1-COX2 and pCDNA3.1 -5-LOX expression vectors and then treated with ethanol at 0.001% or DHA, EP A, and ASA as previously indicated (Exp. 3 and 4, respectively, in Table 1), and in HEK293 cells treated with an Opti-MEM control (Exp. 1 inTable 5). It was found that significant amounts of RvDl (61.87 x 103pg / mg) and RvEl (12.97 x 103pg / mg) were detected in RvDIEl EVs generated from HEK293T cells transfected with pCDNA3.1-COX2 and pCDNA3.1-5-LOX expression vectors and treated with DHA, EP A, and ASA (Exp. 4, Table 1). These quantities are nearly a thousand fold higher than the levels of Resolvin D6 that was detected in BALF EVs. Notably, AA-released lipid mediators PGD2, 12- HETE, 15-HETE, 13-HODE, LTE4 and LXA4 were not detectable in these EVs, but -6.96 x 103pg / mg of AA and 10.92 x 103pg / mg of EPA were detected (data not shown).Table 1. Lipid mediators (LMs) detected in EVs isolated from HEK293 cells transfected with pCDNA3.1 control or pCDNA3.1 -C0X2 and pCDNA3.1 -5-L0X expression vectors and grown in presence of ethanol or DHA, EP A, and ASA.

[0135] The anti-inflammatory activity of RvDIEl EVs was evaluated in in vitro cellular assays (neutrophil adhesion activity and NF-KB reporter activity) and in an in vivo LPS-induced ALI mouse model. The ability of RvDIEl EVs to alter in vitro neutrophil adhesion activity was measured using EAhy926 cells grown in 96 well-black polystyrene culture plates (Perkin Elmer, Shelton, CT). EAhy926 cells were activated by TNFa (50 ng / mL, 16 hours) and washed 3 times with DPBS. An equal number of PKH26-labeled DMSO-differentiated HL60 neutrophil cells were added to EAhy926 cells, and after 1 hour, control EVs and RvDIEl EVs were added separately to the cells and incubated for 15 hours. After 16 hours, the cells were washed 3 times with DPBS, and the relative fluorescence (RFU) was measured on a plate reader. Adherence of PKH26-labeled neutrophils to activated EAhy926 cells increased for the pCDNA3.1 andpCDNA3.1-COX2-5-LOX EV treatments (FIG. 6A; ***p<0.001 TNFa vs. PBS or RvDIEl EVs vs. pCDNA3.1 or pCDNA3.1-COX2-5-LOX vector control or ethanol controls, p<0.01 RvDIEl EVs vs. TNFa). In contrast, RvDIEl EVs significantly decreased the neutrophil adherence to EAhy926 cells. Not intending to be bound by theory, it is believed this is due to their anti-inflammatory effect.

[0136] Using the same experimental design, NF-KB reporter macrophage cells were stimulated with LPS (50 ng / mL for 30 minutes) and treated with pCDNA3.1, pCDNA3.1-COX2- 5-LOX control EVs, or RvDIEl EVs and incubated for another 3 hours. After 3 hours, the cells were washed and lysed in cell lysis buffer. Then, the NF-KB driven luciferase reporter activity was measured using a luciferin substrate on a luminometer (Promega, Madison, WI). It was found that the LPS increased NF-KB luciferase reporter activity, which is a measure of inflammation (FIGS. 6C-D; ***p<0.001 RvDIEl EVs vs. pCDNA3 1 or pCDNA3.1-COX2-5- LOX vector control or ethanol controls,#p<0.01 RvDIEl EVs vs. TNFa or LPS or nystatin or cytochalasin D or RvDIEl EVs and nystatin or RvDIEl EVs and cytochalasin D). In contrast, NF-KB reporter activity (relative light unit, RLU) significantly decreased with RvDIEl EV treatment. The biological function of EVs was further confirmed by including EV uptake inhibitors nystatin and cytochalasin D in both these assays. Inhibition of EV uptake by nystatin and cytochalasin D restored the neutrophil adhesion and NF-KB luciferase reporter activity, confirming the anti-inflammatory activity of RvDIEl EVs (FIG. 6B, FIG. 6D;#p<0.001 TNFa vs. PBS / LPS vs. PBS, ** / ## p<0.01 RvDIEl EVs vs. TNFa or nystatin or cytochalasin D or RvDIEl EVs and nystatin or RvDIEl EVs and cytochalasin D).

[0137] The ability of RvDIEl EVs to decrease pulmonary inflammation and ALI was measured in a mouse model of ALI. The EVs were labeled with PKH26, purified by density gradient centrifugation, and delivered into the lungs of aestheticized C57BL6 mice. 1 hour after the EV delivery, the mice were euthanized. The total lung cells were obtained via collagenase and DNase digestion. The single cell lung suspension was analyzed for the uptake of PKH26- labeled EVs by different lung cells using an AMNIS Imagestream Imaging Flow Cytometer.

[0138] PKH26-labeled EVs delivered into mice (50 pg / 25 g body weight in 25 pL saline, intranasal) were taken up more efficiently by macrophages (CD45+F4 / 80+EV(PE)+, 20-23%), followed by microvascular endothelial cells (CD45' F4 / 80’ CD311PE1, 8-12%), and alveolar epithelial cells (CD45-F4 / 8Q- CD326+PE+, 4-6.5%) (FIGS. 7A-E).

[0139] To investigate the in vivo efficacy of RvDIEl EVs in reducing lung injury, mice were treated with either RvDIEl EVs or control EVs (50 pg / 25 g body weight in 25 pL saline, intranasal, n=6-10 mice in each group) one hour after a high-dose LPS treatment (4 mg / kg in 25 pL saline, intranasal). All the mice were euthanized after 16 hours of EV treatment, and ALI severity was assessed. Intranasal delivery of the high dose of LPS caused severe ALI in pCDNA3.1 or pCDNA3.1-COX2-5-LOX control EV-treated mice, reflected by an increase in immune cell / neutrophil cell infiltration (FIGS. 8A-C), an increase of protein leakage into the alveolar space (FIG. 8D), and an increase of inflammatory cytokines IL-6 and TNFa (FIGS. 8E- F). Treating ALI mice 1 hour after the LPS challenge with RvDIEl EVs drastically decreased all the lung injury parameters, demonstrating their therapeutic efficacy in treating ALI (FIGS. 8A- F).EXAMPLE 2

[0140] Extracellular vesicles (EVs) released by lung cells have been shown to transport biologically active molecules that mediate intercellular communications. It has been shown that EVs in bronchoalveolar lavage fluid (BALF) carry multiple lipid mediators that regulate alveolar epithelial cell barrier function during acute lung injury / acute respiratory distress syndrome (ALI / ARDS). Blocking the activation of the nuclear factor of activated T-cells cytoplasmic member 3 (NFATc3) with a cell-permeable peptide (CNI-103) inhibitor decreased ALI severity in a mouse model of ARDS. Described herein is experimental data related to whether inhibition of NFATc3 alters the EV-transported biological cargo and pulmonary microvascular endothelial cell (PMVEC) function in mouse models of ALI.

[0141] Extracellular vesicles (EVs) from control, LPS-, and LPS / CNI- 103 -treated mouse BALF were isolated by ultracentrifugation. EVs were characterized by size, transmission electron microscopy, cell surface receptors, and expression of endosomal marker proteins. The protein and lipid cargo carried by EVs were analyzed by liquid chromatography in conjunction with mass spectrometry (LC-MS). Mouse PMVECs were co-cultured in presence of EVs from the control or calcineurin inhibitor CNI 103 -treated mice subjected to LPS-induced ALI and assayed for PMVEC monolayer barrier function. EVs were selectively enriched in protein components of the complement system, histones, and other cell surface proteins.

[0142] Lipidomic analyses identified that EVs released into the BALF were enriched with cyclooxygenase, lipoxygenase, and cytochrome p450 metabolites that increased during the acute inflammatory phase of LPS-induced ALI. EVs from CNI103 pre-treated mice subjected to LPS- induced ALI were shown to harbor decreased amounts of cyclooxygenase and lipoxygenase metabolites, and lower expression levels of cPLA2 and COX2 were detected in pulmonary macrophages. The co-culture of the mouse PMVEC monolayer with BALF EVs from LPS- treated mice disrupted the PMVEC barrier integrity. In contrast, EVs from CNI103 / LPS treated mice have not altered PMVEC monolayer barrier integrity.

[0143] This non-limiting Example indicates that BALF EVs comprise NF ATc3 -dependent biologically active lipids and proteins that disrupt the integrity of the monolayer barrier of PMVECs, leading to ALI in mice. Not intending to be bound by theory, it is believed that CNI103, a novel protease-resistant calcineurin peptide blocker that prevents the activation of NFATc3 in macrophages, prevents PMVEC barrier disruption and the development of ARDS in mice by altering the lipid mediators found in BALF EVs.EXAMPLE 3

[0144] ALI / ARDS is characterized by an over-activated innate immune system that produces significant amounts of pro-inflammatory cytokines and chemokines. Not intending to be bound by theory, it is believed that the chemokine increase further recruits neutrophils that secrete proteases, platelet-activating factors, and oxygen free radicals, which damage the surrounding alveolar epithelial cells (AECs) and pulmonary microvascular endothelial cells (PMVECs). Proteins leak in to the interstitium because of the damaged PMVEC / AEC barrier and impaired ion channel activity (EnaC and Na+ / K+ATPase), leading to pulmonary edema and impaired gaseous exchange. As described herein, one paradigm of ALI / ARDS is to target the hyperinflammation, dysregulated lung AEC type I and type II / PMVEC function leading to the accumulation of protein-rich fluid in the alveolar space, impaired fluid removal, and hypoxemic respiratory failure. Different pharmacologic agents have been evaluated for treating ARDS. However, clinical trials on 2-adrenergic receptor agonist, statins, early neuromuscular blockade agents, the most recent vitamin D3, and mesenchymal stem cell therapy have not been effective in Phase III trials. Efforts to target single receptor / protein / cell type in ALI / ARDS have alsoproven unsuccessful in addressing the intricate interplay between lung innate immune and structural cells.

[0145] Lipid mediators (LMs) derived from co-6 and co-3 polyunsaturated fatty acids (PUFAs) are involved in both triggering and resolving inflammation. ALI / ARDS causes increased release of arachidonic acid (AA) from cell membrane phospholipids by phospholipase A2 (PLA2). While different classes of PLA2 operate in lipid metabolism, class IV cPLA2 is major source of cellular free AA content. AA is further metabolized by the enzymatic action of different members of cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450 (CYP 450) epoxygenases / co-hydroxylase pathways or via non-enzymatic free radical mechanisms. The COX, LOX, and CYP450 enzymes generate bioactive lipids from both co-6 (AA) and co-3 (docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A)) PUFAs that act as signaling molecules (FIG. 9, which depicts only some enzymes and LMs of particular relevance to this non-limiting Example). AAs generated during inflammation are catalyzed by the COX pathway enzymes COXI and COX2, generating prostanoids, such as PGE2, which is a vasodilator;PGD2, which is involved in vascular permeability and chemotaxis; PGF2a, which is a biomarker for oxidative stress; prostacyclins, such as PGI2, a vasodilator that inhibits platelet aggregation; and thromboxanes, such as TXA2, a vasoconstrictor that aids in platelet aggregation. The LOX pathway includes 5-LOX, 8-LOX, 12-LOX, and 15-LOX (12 / 15-LOX in mice), producing leukotrienes (LTA4, LTB4, LTC4, LTD4, LTE4), lipoxins (LXA4, LXB4), and 8, 12, or 15- HPETE compounds. In some cases, such as in instances of inflammation, LTC4, LTD4, and LTE4 may cause vascular permeability and bronchospasm. The CYP450 pathway involves CYP450 epoxygenase and CYP450 co-hydroxylase, generating epoxy eicosatri enoic acid (EETs) and hydroxy eicosatetraenoic acid compounds (HETEs).

[0146] Pro-resolution lipoxins released from AA, resolvins, protectins and maresins released from co-3 PUFAs are collectively known as SPMs, and not intending to be bound by theory, it is believed that SPMs are involved in the resolution of inflammation (IL 10 increase) and the restoration of tissue homeostasis through processes such as efferocytosis (FIG. 9). There are few reports on LM measurements in ARDS patients. However, several classes of SPMs have been detected in plasma, cerebrospinal fluid, synovial fluid, and several tissues, including brain and spleen, in healthy volunteers and type 2 diabetes and rheumatoid arthritis patients. Notably, plasma from ARDS patients indicate that non-survivors had significantly higher levels of AAmetabolites, like prostaglandin F2a and LTB4. It has been shown that non-survivors of ARDS have significantly higher levels of PGF2, PGF2a, and LTB4. Elevated levels of the pro-resolving mediators Resolvin El, Resolvin D5, and 17r-protectin DI persisted along with pro- inflammatory mediators until 7 days in these patients.

[0147] ProstaglandinF2a, hydroxyeicosatetraenoic acids (HETEs), and leukotriene B4 (LTB4) were shown to induce oxidative stress and act as neutrophilic / leukocyte chemo attractants, whereas the pro-inflammatory effects of 9-HODE and 13-HODE were reported in atherosclerosis and other inflammatory conditions. Furthermore, trauma patients with uncomplicated recoveries have had higher Resolvin pathway gene expression and lower gene expression ratios of leukotriene to Resolvin pathways. However, the conventional approach of dietary supplementation with co-3 fatty acids had no positive impact on the clinical parameters, hospital stay, or mortality computed in a meta-analysis of adults with ARDS. Not intending to be bound by theory, it is believed that this is possibly because only 5-10% of dietary alpha-linoleic acid (ALA) gets converted to DHA and EPA by different fatty acid chain elongase and desaturase enzymes in humans.

[0148] EVs are lipid bilayer membrane-bound vesicles released extracellularly, which originate from the cellular endosomal system (small EVs) or plasma membrane (microvesicles, MV). EVs transfer bioactive cargo including, nucleic acids, lipids, peptides and other membrane proteins, and receptors to the recipient cells. Interestingly, bilayer lipid membranes of EVs are enriched in sphingomyelin, cholesterol, and glycosphingolipids compared to their parent cells, offering them more structural stability. As described herein, not intending to be bound by theory, it is believed that EVs increase the stability of their biological cargo that mediate intercellular communication.

[0149] It is demonstrated herein that the functional role of LMs packaged in 50-150 nm small EVs from mouse BALF and EVs from injured mice lungs alone could elicit barrier dysfunction in the alveolar epithelial cell monolayer. Previous research has suggested that LMs are short-lived biomolecules that act close to the site of synthesis or in circulation / pulmonary edema fluid after their generation. In contrast, in this non-limiting Example, not intending to be bound by theory, it is believed that LMs transported by small EVs are stable by virtue of their saturated lipid rich bilayer membrane. Furthermore, not intending to be bound by theory, it is believed that during inflammatory conditions, the disrupted intercellular barrier increases thereach of EVs to distant cells. In this context, disclosed herein is an innovative approach, utilizing EVs as carriers of Resolvin, particularly EVs loaded with Resolvin DI and Resolvin El, to actively reprogram inflammation and cellular injury towards resolution and homeostasis.

[0150] The total proteome of BALF EVs was analyzed by mass spectrometry. Although a wide array of LMs were detected, the COX and LOX catalytic enzymes that generate these LM were not detectable in both healthy and lung injured mouse BALF EVs (FIG. 2). Not intending to be bound by theory, it is believed that the PUFA catalytic enzymes COX2 and 5-LOX are intrinsically not destined for packaging in small EVs. EVs from LPS-treated mice contained higher levels of antimicrobial or bactericidal proteins like lysozyme and lipid signaling / transport related proteins (e.g., glycerol-3 -phosphate dehydrogenase 1 (Gpdl), which regulates cellular NAD / NADH potential and glycerophospholipid biosynthesis; acetyl-CoA acyltransferase- (ACAA2), which regulates linoleic acid and alpha-linolenic acid metabolism; and low-density lipoprotein receptor protein 1 (Lrpl), which plays a role in lipid homeostasis and the clearance of apoptotic cells) (FIG. 2).

[0151] The Resolvin D and E series of molecules are synthesized by the sequential action of the acetylated COX2 and 5-LOX enzymes that utilize the coordinated action of different cell types, called transcellular synthesis. The leukocytes, endothelial and epithelial cells and monocytes / macrophages act together. In vascular endothelial cells, aspirin-acetylated COX2 converts EPA to 18 R-hydroperoxyeicosapentaenoic acid, 18 R-HEPE, which is then released from the endothelium and rapidly transformed by activated human polymorphonuclear leukocytes (PMN) to bioactive Resolvin El (RvEl). Similarly, the intermediate 17-HDHA is converted to Resolvin DI (RvDl). However, COX2 and 5-LOX may individually generate prostanoids and leukotrienes in vivo from cellular AA.

[0152] Herein, an innovative strategy for loading EVs with Resolvin DI and El completely excluding leukotrienes is disclosed. In some embodiments described herein, this is achieved by co-expressing human COX2 and 5-LOX proteins (pCDNA3.1-COX2 and pCDNA3.1 -5-LOX expression vectors) in HEK293T cells, grown in serum-free medium supplemented with 5 pM each of DHA and EPA and 1 mM acetylsalicylic acid (i.e., aspirin) to generate EVs loaded with Resolvin DI and Resolvin El, each at approximately 1000 times the levels found in mouse BALF EVs. RvDIEl EVs demonstrated significant pro-resolution activity in vitro in PMVEC / AEC cellular assays and mouse post-injury ALI models.

[0153] Moreover, RvDIEl EVs containing Resolvin DI and El levels 1000 times higher than mouse BALF EVs showed robust pro-resolution effects in post-injury mouse ALI models. Further, not intending to be bound by theory, it is believed that pro-resolution mediator packaged EVs can be used in other inflammatory pathologies.

[0154] EVs from mice BALF were characterized. Intranasal (i.n.) delivery of LPS (4 mg / kg) in mice increased the release of EVs into the alveolar space (confirmed by particle size, FIGS. 10A-B, no. of vesicles / mL; FIG. 10C, * / #p<0.05 LPS vs. saline). The size (100-140 nm) and endosomal origin of BALF EVs was confirmed by transmission electron microscope (TEM) (FIG. 10D) and immunoblotting with CD9 and CD63 antibodies (FIG. 10E). The amount of phospholipid protein for EVs and MVs from mice BALF was significantly higher than that of EV-depleted BALF, indicating that EVs transport considerable amount of phospholipids to recipient cells (FIG. 10F, modified from Nirujogi et al, 2022, ***p<0.001 MV / EV vs. EV- depleted BALF).

[0155] The cellular source of BALF EVs was determined by cell-specific receptor staining. EVs were attached to 9-pm CD9 Exo-Flow capture beads (System Biosciences, Palo Alto, CA) as per manufacturer’s instructions and stained with F4 / 80-APC, CD31-FITC, CD326-PE, and Ly6G-BV421 at 4°C for 16 hours. EVs released from epithelial cells (CD326+) and neutrophils (Ly6G+) were increased during ALI compared to saline (FIGS. 11 A-F). In contrast, the EVs released from macrophages (F4 / 80+) remained unchanged, whereas EVs from PMVECs (CD31+) were negligible during LPS-induced ALI (FIGS. 11 A-F).

[0156] The BALF EV lipidome was characterized in LPS-treated ALI mice using high throughput tandem LC-MS / MS. BALF EVs from control and LPS-treated mice (i.e., ALI mice) (6 hours, 3 days, 15 days) were analyzed to determine the amount of AA, EP A, DHA, and LA- derived metabolites from the enzymatic action of COX, LOX, CYP450, and nonenzymatic lipid peroxidation. The total lipids were extracted using chloroforrmmethanol (2: 1 vol / vol Folch method) and analyzed by untargeted lipidomics. BALF EVs from mice carried a wide array of PUFA metabolites released from the enzymatic and non-enzymatic conversion of co -6 (AA) and ro-3 (DHA and EPA) PUFAs (FIG. 12A). COX metabolites from AA (PGE2, PGD2, PGF2a, and TXB2), LOX metabolites from the essential fatty acid linoleic acid (LA) 9- and 13- hydroxyoctadecadienoic acids (9-HODE and 13-HODE), and LOX metabolites from AA (LTB4, 5-HETE, 12-HETE, 15-HETE) were preferentially packaged in BALF EVs from LPS-ALI micecompared to saline treated controls (FIG. 12A). In addition, derivatives of 9- and 13-HODE, 9- and 13-oxo-octadecadienoic acid (9-oxoODE and 13-oxoODE), generated by NAD+dependent dehydrogenase, were significantly elevated in LPS-EVs. As the content of total esterified FAs in the membrane phospholipids correlate with downstream metabolism in cells, the molar concentration of the AA increased with ALI (FIG. 12B; *** p< 0.001 LPS vs. saline control). The increase in eicosanoids in the EVs correlated with an increase in cPLA2 catalytic activity (FIG. 12C; *** p< 0.001 LPS vs. saline control; assayed by pmol of arachidonoyl thio-PC hydrolyzed per min per mg cellular protein in the presence of sPLA2 and iPLA2 inhibitors; Cayman Chemical, Ann Arbor, MI) and protein expression in pulmonary macrophages (FIG. 12D). Similarly, the expression of AA catabolic proteins COX1 / 2 and 5-LOX, 12-LOX, and CYP450 increased during LPS-induced ALI (FIG. 12D). The increases in PUFA metabolizing proteins correlated with increased AA and LA-derived LMs in EVs from LPS-induced ALI mice.

[0157] BALF EVs were labeled with PKH26 (Millipore Sigma) and purified by sucrose density gradient ultracentrifugation. PKH26-labeled EVs were taken up by macrophages and AEC cells, and EV uptake inhibitors nystatin (50 pg / mL) and cytochalasin D (10 pg / mL) significantly decreased the uptake by more than 60%, as described previously. To determine the in vivo uptake of EVs, PKH26 (PE+)-labeled EVs were delivered intranasally 2-4 mg / kg body weight into saline- and LPS-treated mice. Lung cells were isolated by collagenase digestion, stained with cell-specific markers, and analyzed with an AMNIS Imagestream Flow Cytometer. Different pulmonary cells showed differences in uptake efficiency. EV uptake in saline-treated mouse lungs was maximum in macrophages (CD45+PE+F / 480+, 20-23%), followed by PMVECs (CD45 PE+CD325 CD31+, 8-12%) and AECs (CD45 PE+CD31 CD326+, 4.6-5%). The EV uptake doubled in LPS-lung injured mouse pulmonary cells (40% macrophages; 35-42% in neutrophils (CD45+PE+F / 480’Grl+); 19-21% PMVECs; and 16-20% AECs; FIGS. 7A-D gating strategy, FIG. 13 (% of PE1cell population)).

[0158] It was previously demonstrated that BALF EVs from LPS-treated mice (i.e., ALI mice) disrupt the AEC monolater barrier integrity and induce TNFa release by macrophages. Herein, the impact of EVs on the PMVEC monolayer barrier function was analyzed. PMVECs were grown to 100% confluence on collagen-coated transwells and were co-cultured in the presence of unlabeled EVs (109, 1012 / well) from saline and mouse LPS induced-ALI. 10,000 Da FITC-dextran was added to transwells at the start of co-culture. PMVEC monolayer barrierdisruption was determined by measuring the FITC-dextran flux from the transwell chamber to the bottom chamber at 8 hour intervals, as described previously. EVs (1012 / well) from LPS- treated mice disrupted the PMVEC monolayer barrier integrity, resulting in a significant increase in FITC-dextran flux compared to control EVs (FIG. 14). In contrast, EVs from LPS-treated mice in the presence of EV uptake inhibitor nystatin at 50 pg / mL showed only -50-60% of FITC-dextran flux compared to that observed in ALI EVs (FIG. 14; *** / 7spf 0.001, ** p< 0.01).

[0159] The generation of engineered EVs loaded with RvDl and RvEl may occur by transiently overexpressing COX2 and 5-LOX in HEK293T cells (FIG. 3A). 2 x 106HEK293T cells grown overnight in Opti-MEM were transfected with 10 pg each of human pCDNA3.1- COX2 and pCDNA3.1 -5-LOX plasmid vectors (GenScript, Piscataway NJ) using a nonlipid transfection reagent, FuGENE 6 (Promega, Madison, WI). After 24 hours of transfection, the medium was removed and supplemented with fresh Opti-MEM containing 5 pM each of DHA and EPA and 1 mM aspirin or acetyl salicylic acid (ASA) (Cayman, Detroit MI). The cells were grown for another 24 hours. After 24 hours, the culture medium was used for EV isolation, and the cells were lysed in IX RIP A buffer for analyzing the overexpression of human COX2 and 5- LOX proteins. Both COX2 and 5-LOX were overexpressed in cell lysates, whereas the control plasmid (pCDNA3.1)-transfected cell lysates showed no detectable COX2 and 5-LOX proteins (FIG. 3B, lanes 1-2 vs 3-6).

[0160] To isolate the EVs, the cell culture medium was filtered through 0.22 pm syringe filters to remove microvesicles. The resulting filtrate was loaded on 20 mL Pierce 100 kDa-cut off centrifugal filters (ThermoFisher) and centrifuged at 4°C and 4000g for 30 minutes. The concentrate was washed in IX DPBS and resuspended in 100 pL of DPBS or saline and analyzed by nanoparticle tracking for size and numbers. Based on the plasmid vector transfection and medium supplementation, 4 different groups of EVs were evaluated: 1) pCDNA3.1 -transfected cells grown in presence of 0.001% ethanol, which yielded vector control EVs and these EVs are not included in assays in FIG. 5, FIG. 6, and FIG. 15; 2) pCDNA3.1 transfected cells grown in presence of DHA, EPA, and ASA, which yielded vector / DHA+EPA+ASA control EVs labeled as pCDNA3.1 control EVs; 3) pCDNA3.1-COX2 and pCDNA3.1 -5-LOX transfected cells grown in the presence of 0.001% ethanol referred as pCDN A3.1-C 0X2-5 -LOX EVs; 4) pCDNA3.1-COX2 and pCDNA3.1-5-LOX transfected cells grown in presence of DHA, EPA, and ASA, which are referred to as RvDIEl EVs and contain both Resolvin DI and Resolvin ELThe size of the EVs (nanoparticle size analysis in FIGS. 4A-C and FIGS. 4E-F), enrichment of CD9, and absence of Calnexin proteins (FIG. 4D) confirmed the purity of the EV preparation. CD9 was unable to be detected in the cell lysates because the amount of protein loaded was minimal to be able to match the low protein levels present in EVs. Initial screening of EVs was done by Cayman ELISA kits (Cayman, Detroit, MI). RvDIEl EVs contained RvDl (-1200 pg / mg) and El (-750 pg / mg) measured by ELISA (FIGS. 5A-B). The LC-MS measurements showed 62 ng RvDl / mg of EV protein, 12.9 ng RvEl / mg of EV protein, and 6.9 ng PGE2 / mg of EV protein. These EVs showed no detectable AA-released LMs: PGF2, PGD2, 12-HETE, 15- HETE, 13HODE, LTE4, and LXA4 (Table 1).

[0161] The anti-inflammatory activity of RvDIEl EVs was evaluated in in vitro cellular assays (neutrophil adhesion activity, AEC / PMVEC barrier function, and NF -KB reporter activity) and in an in vivo LPS-induced mouse ALI model. The ability of RvDIEl EVs to alter in vitro neutrophil adhesion activity was measured using EAhy926 cells grown in 96 well-black polystyrene culture plates (Perkin Elmer), activated with 50 ng / mL of TNFa for 8 hours, and added to PKH26-labeled neutrophils differentiated from HL60 cells. After 1 hour, pCDNA- control EVs, pCDNA3.1-COX2-5-LOX EVs, and RvDIEl EVs were added separately to the cells and incubated for a further 15 hours. After 16 hours, the cells were washed 3 times with DPBS, and the relative fluorescence (RFU) was measured on a plate reader. The adherence of the PKH26-labeled neutrophils to activate EAhy926 cells increased for the pCDNA3.1 control vector and pCDNA3.1-COX2-5-LOX EV treatments (FIG. 6A). In contrast, RvDIEl EVs significantly decreased the neutrophil adherence to EAhy926 cells. Not intending to be bound by theory, it is believed that this is due to their anti-inflammatory effect.

[0162] Using the same experimental design, NF-KB reporter macrophage cells (Blackwell, TS 2003) were stimulated with LPS (50 ng / mL for 30 min) and added to pCDNA3.1 EVs, or pCDNA3.1-COX2-5-LOX control EVs, or RvDIEl EVs and incubated for another 3 hours. After 3 hours, the cells were washed and lysed in cell lysis buffer. Then, the NF-KB driven luciferase reporter activity was measured using Luciferin substrate on a luminometer (Promega, Madison, WI). LPS increased NF-KB luciferase reporter activity, which is a measure of inflammation (FIG. 6C). In contrast, NF-KB reporter activity significantly decreased with RvDIEl EV treatment. The biological function of EVs was further confirmed by including EV uptake inhibitors nystatin and cytochalasin D in both these assays. Inhibition of EV uptake bynystatin and cytochalasin D restored the neutrophil adhesion and NF -KB luciferase reporter activity, confirming the anti-inflammatory activity of RvDIEl EVs (FIG. 6B, FIG. 6D). Similarly, thrombin (0.2 units / mL)-induced PMVEC barrier dysfunction was repaired by RvDIEl EVs (FIG. 15; *** / # / $ p< 0.001 control vs. thrombin; RvDIEl EVs vs. RvDIEl EVs with nystatin) as measured by FITC-dextran, as described previously. Under similar experimental conditions, inhibition of RvDIEl EV uptake by nystatin failed to decrease the FITC-dextran flux caused by barrier dysfunction.

[0163] The ability of RvDIEl EVs to decrease pulmonary inflammation was measured in a mouse model of ALL To investigate the in vivo efficacy of RvDIEl EVs in reducing lung injury, mice were treated with either RvDIEl EVs or control EVs (50 pg / 25 g body weight in 25 pL saline, intranasal, n=6-10 mice in each group) one hour after a high-dose LPS treatment (4 mg / kg in 25 pL saline, intranasal). All the mice were euthanized after 16 hours of EV treatment, and ALI severity was assessed. Intranasal delivery of the high dose LPS caused severe ALI in pCDNA3. 1 control EV-treated mice or pCDNA3.1-COX2-5-LOX control EV-treated mice, reflected by an increase in BALF cell numbers (FIG. 8A), infiltration of neutrophils into alveolar space (FIGS. 8B-C), protein leakage into BALF (FIG. 8D), and an increase in inflammatory cytokines (TNFot, IL-6, FIGS. 8E-F). 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[0164] In this non-limiting Example, the pro-resolution activity of a plurality of extracellular vesicles comprising a plurality of Resolvins disposed on an interior of the EVs were further improved by a multigene expression vector. Moreover, herein the inclusion of 15-LOX and 5- LOX generated EVs loaded with Resol vin DI, D2, D3, D4 and D5 and Resol vin El, E2, E3, E4, and E5.

[0165] The vector or plasmid used encoded human COX2, 5-LOX and 15-LOX and simultaneously expressed COX2, 5-LOX, and 15-LOX in significant quantities (FIG. 16A-B). COX2, 5-LOX and 15-LOX were separated by P2A and T2A sequences to generate the three proteins independently. A control vector (pRP-control) that did not have any cloned genes was used to generate control EVs (control EVs). In addition to the aspirin-triggered resolvins, 15- LOX and 5-LOX generated Resolvin DI, D2, D3, D4, and D5 from DHA and Resolvin El, E2, E3, E4, and E5 from EPA (FIG. 16A).

[0166] HEK293T cells were transfected with the multigene vector, grown for 24 hours, and washed with fresh Opti-MEM. The cells were grown for another 48 hours in fresh Opti-MEM supplemented with fatty acid-free BSA conjugated DHA, EPA, and acetylsalicylic acid (ASA). EVs loaded with Resolvin D1-D5 and E1-E5 (Resolvin EVs for the purpose of this non-limiting Example) and control EVs were isolated from cell culture supernatants and assayed for their biological activity.

[0167] The Resolvin EVs from the multigene expression vector contained Resolvin DI, Resolvin D2, Resolvin El, and Resolvin E2 at concentrations ranging from 16-24 ng / mg EV protein as assayed by ELISA. Resolvin EVs and control EVs had no detectable COX2, 5- LOX, 15-LOX, and Calnexin proteins but expressed higher EV marker protein CD9 (FIG. 16B).

[0168] In a neutrophil adhesion assay, EAhy926 cells were activated by TNFa (8 hours), washed three times, and added to an equal number of PKH26-positive HL60 neutrophil cells(FIG. 17A). After 1 hour, Resolvin EVs were added separately to the cells and incubated for 15 hours. Then, the cells were washed with DPBS, and the relative fluorescence (RFU) was measured on a plate reader. Resolvin EVs decreased neutrophil adhesion to TNFa-activated EAhy926 cells, confirming their pro-resolution activity (FIG. 17B). Inhibition of Resolvin EV uptake by nystatin and cytochalasin D restored the TNFa-induced adhesion of neutrophils to endothelial cells (FIG. 17C). Similarly, Resolvin EVs mitigated thrombin-induced PMVEC monolayer barrier dysfunction (FIG. 18A), restored the VE-cadherin expression (FIG. 18B), and restored NF-kB activation in macrophages (FIG. 19). More importantly, when administered in mice that were pretreated with LPS, the Resolvin EVs decreased pulmonary inflammation markers measured in terms of immune cell infiltration, TNFa, and IL6 (FIGS. 20A-C).

[0169] Additional exemplary embodiments contemplated herein are as follows:

[0170] Embodiment 1. A method of producing Resolvin-loaded extracellular vesicles (RvEVs), the method comprising: providing a population of cells; transfecting the population of cells with at least one plasmid vector encoding cyclooxygenase-2 (COX2) and encoding 5-lipoxygenase (5-LOX); applying docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A), and acetylsalicylic acid (ASA) to the transfected population of cells; and isolating RvEVs from culture media of the transfected population of cells.

[0171] Embodiment 2. The method of Embodiment 1, wherein Resolvin comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof.

[0172] Embodiment 3. The method of Embodiment 1 or Embodiment 2, wherein the at least one plasmid vector further encodes 15-lipoxygenase (15-LOX).

[0173] Embodiment 4. The method of any of Embodiments 1-3, wherein the population of cells comprises HEK293T cells, CHO cells, or A549 cells.

[0174] Embodiment 5. The method of any of Embodiments 1-4, wherein transfecting includes applying 5-20 pg of the at least one plasmid vector encoding cyclooxygenase-2 (COX2) and encoding 5-lipoxygenase (5-LOX).

[0175] Embodiment 6. The method of any of Embodiments 1-5, wherein the transfecting includes use of a non-lipid transfection reagent.

[0176] Embodiment 7. The method of any of Embodiments 1-6 wherein 3-5 pM each of DHA and EPA and 0.1-1 mM of ASA are applied.

[0177] Embodiment 8. The method of any of Embodiments 1-7, wherein the isolating includes filtering the culture media to remove microvesicles.

[0178] Embodiment 9. The method of Embodiment 8, wherein the filtering includes passing the culture media through a 0.22 pm filter.

[0179] Embodiment 10. The method of any of Embodiments 1-9, wherein the RvEVs are 100 nm to 200 nm in average size.

[0180] Embodiment 11. The method of any of Embodiments 1-10, wherein the RvEVs contain at least 60,000 pg of RvDl per mg of EV protein and at least 12,000 pg of RvEl per mg of EV protein.

[0181] Embodiment 12. The method of any of Embodiments 1-11, wherein prostaglandin D2 (PGD2), 12-hydroxy eicosatetraenoic acid (12-HETE), 15-hydroxy eicosatetraenoic acid (15- HETE), 13 -hydroxy octadecadienoic acid (13-HODE), leukotriene E4 (LTE4), and lipoxin A4 (LXA4) are not detectable in the RvEVs.

[0182] Embodiment 13. A method of treating an inflammatory condition or disease in a patient in need thereof, the method comprising: disposing a plurality of Resolvin-loaded extracellular vesicles (RvEVs) within a biological compartment of the patient; wherein inflammation from the inflammatory condition or disease is mitigated or resolved.

[0183] Embodiment 14. The method of Embodiment 13, wherein the inflammatory condition or disease is acute lung respiratory distress syndrome (ARDS).

[0184] Embodiment 15. The method of Embodiment 13 or Embodiment 14, further comprising penetrating a membrane of a cell or population of cells within the biological compartment with the plurality of RvEVs.

[0185] Embodiment 16. The method of Embodiment 15, further comprising releasing at least a portion of the Resolvin of the RvEVs within a cytosol of the cell or population of cells after penetrating the membrane of the cell or population of cells.

[0186] Embodiment 17. The method of any of Embodiments 13-16, wherein the RvEVs are disposed within the biological compartment of the patient through intranasal or intravenous administration.

[0187] Embodiment 18. A method of restoring tissue homeostasis in a patient having an inflammatory condition or disease, the method comprising: disposing a plurality of Resolvin-loaded extracellular vesicles (RvEVs) within a biological compartment of the patient; wherein tissue homeostasis is restored.

[0188] Embodiment 19. A composition comprising: a plurality of extracellular vesicles (EVs) comprising at least one Resolvin disposed within an interior of the EVs.

[0189] Embodiment 20. The composition of Embodiment 19, wherein an average size of the plurality of EVs is 100 nm to 200 nm.

[0190] Embodiment 21. The composition of Embodiment 19 or Embodiment 20, wherein Resolvin comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof.

[0191] Embodiment 22. The composition of any of Embodiments 19-21, wherein the plurality of EVs contain at least 60,000 pg of RvDl per mg of EV protein and at least 12,000 pg of RvEl per mg of EV protein.

[0192] Embodiment 23. The composition of any of Embodiments 19-22, wherein prostaglandin D2 (PGD2), 12-hydroxy eicosatetraenoic acid (12-HETE), 15- hydroxyeicosatetraenoic acid (15-HETE), 13 -hydroxy octadecadienoic acid (13-HODE), leukotrieneE4 (LTE4), and lipoxin A4 (LXA4) are not detectable therein.

[0193] Embodiment 24. The composition of any of Embodiments 19-23, wherein the plurality of extracellular vesicles further comprises a cargo.

[0194] Embodiment 25. The composition of Embodiment 24, wherein the cargo is operable to diffuse out of the interior of the EVs when the composition is delivered into a biological or lipophilic environment.

[0195] All patent documents referred to herein are incorporated by reference in their entireties. Various embodiments of the invention have been described in fulfdlment of thevarious objectives of the invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations thereof will be readily apparent to those skilled in the art without departing from the spirit and scope of the invention.

Claims

CLAIMS1. A method of producing Resolvin-loaded extracellular vesicles (RvEVs), the method comprising: providing a population of cells; transfecting the population of cells with at least one plasmid vector encoding cyclooxygenase-2 (C0X2) and encoding 5-lipoxygenase (5-LOX); applying docosahexaenoic acid (DHA), eicosapentaenoic acid (EP A), and acetylsalicylic acid (ASA) to the transfected population of cells; and isolating RvEVs from culture media of the transfected population of cells.

2. The method of claim 1, wherein Resolvin comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof.

3. The method of claim 1, wherein the at least one plasmid vector further encodes 15- lipoxygenase (15-LOX).

4. The method of claim 1, wherein the population of cells comprises HEK293T cells, CHO cells, or A549 cells.

5. The method of claim 1, wherein transfecting includes applying 5-20 pg of the at least one plasmid vector encoding cyclooxygenase-2 (COX2) and encoding 5-lipoxygenase (5-LOX).

6. The method of claim 1, wherein the transfecting includes use of a non-lipid transfection reagent.

7. The method of claim 1, wherein 3-5 pM each of DHA and EPA and 0.1-1 mM of ASA are applied.

8. The method of claim 1, wherein the isolating includes filtering the culture media to remove microvesicles.

9. The method of claim 8, wherein the filtering includes passing the culture media through a 0.22 pm filter.

10. The method of claim 1, wherein the RvEVs are 100 nm to 200 nm in average size.

11. The method of claim 1, wherein the RvEVs contain at least 60,000 pg of RvDl per mg of EV protein and at least 12,000 pg RvEl per mg of EV protein.

12. The method of claim 1, wherein prostaglandin D2 (PGD2), 12-hydroxy eicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), 13 -hydroxy octadecadienoic acid (13-HODE), leukotriene E4 (LTE4), and lipoxin A4 (LXA4) are not detectable in the RvEVs.

13. A method of treating an inflammatory condition or disease in a patient in need thereof, the method comprising: disposing a plurality of Resolvin-loaded extracellular vesicles (RvEVs) within a biological compartment of the patient; wherein inflammation from the inflammatory condition or disease is mitigated or resolved.

14. The method of claim 13, wherein the inflammatory condition or disease is acute lung respiratory distress syndrome (ARDS).

15. The method of claim 13, further comprising penetrating a membrane or membrane fusion of a cell or population of cells within the biological compartment with the plurality of RvEVs.

16. The method of claim 15, further comprising releasing at least a portion of the Resolvin of the RvEVs within a cytosol of the cell or population of cells after penetrating the membrane of the cell or population of cells.

17. The method of claim 13, wherein the RvEVs are disposed within the biological compartment of the patient through intranasal or intravenous administration.

18. A method of restoring tissue homeostasis in a patient having an inflammatory condition or disease, the method comprising: disposing a plurality of Resolvin-loaded extracellular vesicles (RvEVs) within a biological compartment of the patient; wherein tissue homeostasis is restored.

19. A composition comprising: a plurality of extracellular vesicles (EVs) comprising at least one Resolvin disposed within an interior of the EVs.

20. The composition of claim 19, wherein an average size of the plurality of EVs is 100 nm to 200 nm.

21. The composition of claim 19, wherein Resolvin comprises Resolvin DI, Resolvin D2, Resolvin D3, Resolvin D4, Resolvin D5, Resolvin D6, Resolvin El, Resolvin E2, Resolvin E3, Resolvin E4, Resolvin E5, or a combination thereof.

22. The composition of claim 19, wherein the plurality of EVs contain at least 60,000 pg of RvDl per mg of EV protein and at least 12,000 pg of RvEl per mg of EV protein.

23. The composition of claim 19, wherein prostaglandin D2 (PGD2), 12- hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxy eicosatetraenoic acid (15-HETE), 13- hydroxyoctadecadienoic acid (13-HODE), leukotriene E4 (LTE4) and lipoxin A4 (LXA4) are not detectable therein.

24. The composition of claim 19, wherein the plurality of extracellular vesicles further comprises a cargo.

25. The composition of claim 24, wherein the cargo is operable to diffuse out of the interior of the EVs when the composition is delivered into a biological or lipophilic environment.

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