Use of blood shadow nanovesicles as therapeutic agents

Abstract

The present disclosure provides ghost nanovesicles (gNVs) that lack cytosolic components. Also provided are methods of making such vesicles and therapeutic uses of such vesicles. The gNVs can be used to prevent or treat a disorder that can benefit from administration of gNVs. Such disorders include disorders involving inflammation.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 899,931, filed September 13, 2019, which is incorporated herein by reference in its entirety.

[0003] introduction

[0004] Vesicles, including microvesicles and nanovesicles, have been described. These vesicles have been used as vaccines or for delivering therapeutic agents. However, these vesicles have several drawbacks, such as a lack of consistent size, non-specific immune responses, and the fact that they are produced by methods that lack consistency and reproducibility.

[0005] This disclosure provides several aspects that address these shortcomings and offers numerous other advantages and therapeutic applications for nanovesicles. Summary of the Invention

[0006] This disclosure provides ghost nanovesicles (gNVs) lacking cytoplasmic components. Methods for preparing such vesicles and their therapeutic uses are also provided. gNVs can be used to prevent or treat conditions from which treatment of gNVs may be beneficial. Such conditions include those involving inflammation. Attached Figure Description

[0007] Figure 1 The steps for generating blood shadow nanovesicles (gNVs) by extrusion and exposure to alkaline conditions according to one embodiment of the present disclosure are described.

[0008] Figure 2A and 2B This indicates fewer contaminants in gNV, as evidenced by an increase in the number of gNV particles per μg of protein. Figure 2A ) and the reduction in the number of other fragments in the formulation as compared by imaging ( Figure 2B It's obvious.

[0009] Figure 3A and 3B RNA in gNV is shown. Figure 3A ) and DNA ( Figure 3B The amount of ) was significantly lower than the amount of RNA and DNA in NV.

[0010] Figure 4A and 4B Comparative proteomics between NV and gNV was depicted, showing that NV and gNV have unique quantitative spectra.

[0011] Figure 5A and Figure 5BThe proteomic profiles of NV and gNV were depicted. Based on changes of 1.5 fold or higher, there were three groups: 1) NV-rich proteins, 2) gNV-rich proteins, and 3) ordinary proteins.

[0012] Figure 6 The proteomic profiles of NV and gNV were depicted. Based on changes of 1.5 fold or higher, there were three groups: 1) NV-rich proteins, 2) gNV-rich proteins, and 3) ordinary proteins.

[0013] Figure 7 It was shown that gNV contains fewer nuclear and cytoplasmic proteins compared to NV.

[0014] Figure 8 The study showed that, compared to NV, transport proteins were rich in gNV, and metabolic pathway proteins were rich in NV.

[0015] Figure 9 The results showed that gNV inhibited the inflammatory response induced by outer membrane vesicles to a similar degree as NV.

[0016] Figure 10 The study showed that gNV inhibited OMV-induced decreases in body weight and body temperature in vivo, comparable to NV.

[0017] Figure 11 The study showed that gNV inhibited OMV-induced peritoneal inflammation in vivo, comparable to NV.

[0018] Figure 12 gNV was shown to suppress OMV-induced systemic inflammation in vivo, comparable to NV.

[0019] definition

[0020] As used herein, the term "outer membrane vesicle" or "OMV" refers to a vesicle containing an outer membrane surrounding the periplasmic contents, cytoplasmic contents, and inner membrane components. OMVs include vesicles produced by outer membrane budding of organisms such as Gram-negative bacteria. Such OMVs may also be referred to as natural OMVs. OMVs can also be generated by forcing cells to form vesicles by disrupting Gram-negative bacteria in hydrophilic solutions (e.g., by squeezing, acoustic treatment, detergents, or osmotic shock).

[0021] As used in this article, the term "vesicle" refers to a spherical structure containing an internal volume separated from the external environment by a lipid bilayer membrane. Vesicles can be secreted by cells or synthesized artificially by cells. Vesicles are typically smaller than the cells from which they originate.

[0022] As used in this article, the term "revesicle formation" and its grammatical equivalents refer to the process of opening a vesicle (e.g., a cell-derived vesicle) to release its internal contents, then separating the open lipid bilayer membrane, and closing the open lipid bilayer membrane to reform the vesicle. Such a vesicle is called a ghost vesicle.

[0023] As used in this article, the term "non-revesicalization" and its grammatical equivalents refer to vesicles, such as those not of the cell origin of the blood shadow vesicle, that have not undergone the following processes: opening the vesicle to release its internal contents, then separating the open lipid bilayer membrane, and closing the open lipid bilayer membrane to reform the vesicle. Therefore, non-revesicalized vesicles contain significantly more internal contents derived from the cells of their origin, as compared to blood shadow vesicles prepared from the same cell type.

[0024] As used herein in the context of the components present in cell-derived blood shadow nanovesicles (gNVs), the term "lacking" means having at least 50% less of the components present in non-blood shadow nanovesicles derived from the same cells, for example, 60%, 70%, 80%, 90%, or 99%. Vesicles that have not yet been prepared by opening and closing vesicles are referred to as nanovesicles.

[0025] As disclosed herein, the term "enrichment" in the context of proteins (e.g., membrane proteins) present in cell-derived gNVs means that the component constitutes a larger fraction of the total protein in the gNV compared to the fraction of the same protein in an NV produced from the same cell type. For example, the enriched protein may constitute at least 25% or more of the total protein in the gNV, while the same protein may constitute at most 20% of the total protein in the NV. The enriched component may be present in the gNV at a higher concentration by weight than the concentration of that component by weight in an NV produced from the same cell type from which the gNV originates, for example, at least three times higher, such as at least five times higher, at least 10 times higher, at least 30 times higher, at least 50 times higher, or at least 100 times higher by weight.

[0026] As used in this article, the term "extracellular vesicle" refers to a vesicle released by eukaryotic cells (e.g., mammalian cells). Examples of "extracellular vesicles" include exosomes, ectosomes, microvesicles, prostasomes, oncosomes, and apoptotic bodies. As used in this article, the term "tumor vesicle" refers to an extracellular vesicle present in tumor tissue (e.g., released by tumor cells). Tumor vesicles can be opened and closed to generate gTVs, such as g-shadow tumor microvesicles or g-shadow tumor nanovesicles (gTMV or gTNV). In some respects, gNVs are not generated by extracellular vesicles or tumor vesicles.

[0027] Therefore, if the concentration of a particular component is 1 microgram per gram of total cell preparation (or total cell protein), the enrichment formulation will contain more, for example, at least 3 micrograms of that component per gram of total cell preparation (or total cell protein).

[0028] As used in this article, the term "inflammatory response" refers to the secretion of pro-inflammatory cytokines, activation of toll-like receptors (TLRs), and / or systemic inflammation. Examples of pro-inflammatory cytokines include IL-6, IL-4, IL-6, IL-12, IL-12p70, IL-17, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ).

[0029] "Separated" means the target entity in an environment different from the environment in which it can naturally exist. "Separated" is intended to include entities in samples that are substantially enriched with the target entity and / or in which the target entity is partially or substantially purified.

[0030] The terms “object” and “patient” refer to an animal that is the subject of treatment, observation, or experimentation. By way of example only, an object includes, but is not limited to, mammals, including but not limited to, humans or non-human mammals such as non-human primates, cattle, horses, dogs, sheep, or cats.

[0031] As used herein, the term "treatment" encompasses any treatment of a disease or symptom in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or symptom in subjects susceptible to it but not yet diagnosed with it; (b) suppressing a disease or symptom, i.e., halting its development; (c) alleviating and / or improving a disease or symptom, i.e., causing it to subside; or (d) curing a disease or symptom, i.e., stopping its development or progression. The target group for treatment by the methods of the present invention includes subjects suffering from an undesirable symptom or disease, and subjects at risk of developing such a symptom or disease.

[0032] The term "therapeutic effect" refers to some degree of relief of symptoms of one or more diseases (e.g., infection, tumor formation, or tumor) or their associated pathological conditions. As used herein, "therapeutic effective amount" refers to the amount of a pharmaceutical agent that is effective after administration of a single or multiple doses to cells or a subject in: prolonging the survival of a patient with such a disease, reducing one or more signs or symptoms of the disease, preventing or delaying, and exceeding the similarity expected without such treatment. "Therapeutic effective amount" is intended to define the amount required to achieve a therapeutic effect. A physician or veterinarian with ordinary skill in the art can readily determine and prescribe a desired "therapeutic effective amount" (e.g., ED50) of a pharmaceutical composition. For example, a physician or veterinarian may begin with a dose of the vesicles of this disclosure used in a pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0033] Before further describing the invention, it should be understood that the invention is not limited to the specific embodiments described, as they are of course subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0034] When a range of values ​​is provided, it should be understood that, unless the context explicitly indicates otherwise, every intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other specified value or intermediate value within the specified range, is covered by this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and are also covered by this invention, subject to any explicit exclusions within the specified range. When a specified range contains one or both limitations, the range excluding one or both of those included limitations is also included in this invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, some preferred methods and materials are described hereafter. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials relating to the cited publications.

[0036] It must be noted that, unless the context clearly indicates otherwise, nouns without quantifiers as used herein and in the appended claims include one / more / a type. Thus, for example, a reference to “vesicle” includes a plurality of such vesicles, and a reference to “vesicle” includes a reference to one or more vesicles and their equivalents known to those skilled in the art, etc. It should also be noted that the claims may be drafted to exclude any optional elements. Similarly, this statement is intended to serve as a prior basis for the use of exclusive terms such as “merely,” “only,” etc., or for the use of “negative” limiting terms associated with the description of the claimed element.

[0037] It should be understood that, for clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, multiple features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments belonging to the invention are expressly included in the invention and disclosed herein, just as each and every combination is separately and expressly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are also expressly included in the invention and disclosed herein, just as each and every such sub-combination is separately and expressly disclosed herein.

[0038] The publications discussed herein are provided only for their disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precede such publications. Furthermore, the provided publication date may differ from the actual publication date, which may need to be determined independently. Detailed Implementation

[0039] Blood shadow nanovesicles (gNVs) derived from mammalian cells, wherein the gNVs lack cytoplasmic proteins and nucleic acids, are disclosed; and a method for reducing inflammation in subjects who require it by administering gNVs.

[0040] Blood Shadow Nanovesicles and Their Compositions

[0041] Blood shadow nanovesicles (gNVs) derived from mammalian cells are disclosed. These gNVs lack cytoplasmic proteins and nucleic acids and reduce the level of at least one pro-inflammatory cytokine when administered to subjects in need of them.

[0042] In some respects, mammalian cells can be autologous mammalian cells. Autologous cells can be derived from the tissues or organs of a recipient. In some respects, mammalian cells can be xenologous mammalian cells. Xenologous mammalian cells can be derived from the tissues or organs of a donor, or from a cell line. The tissues or organs from which mammalian cells are derived can be bone marrow, blood, blood products, adipose tissue, umbilical cord blood, fallopian tubes, liver, fetal liver, or fetal lungs, etc. In some respects, mammalian cells can be monocytes, macrophages, or dendritic cells. In some respects, mammalian cells may not be cancer cells or cells derived from tumors.

[0043] In some respects, mammalian cells can be stem cells. In some respects, mammalian cells can be cell lines, such as embryonic stem cell lines, induced pluripotent stem cell lines, or any other cell line. In some respects, stem cells can be embryonic stem cells or somatic stem cells, such as those found in children and adults. In some respects, mammalian cells can be hematopoietic stem cells, breast stem cells, intestinal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells, neural crest stem cells, or testicular stem cells. In some respects, mammalian cells can be mesenchymal stem cells. In other respects, mammalian cells can be cell lines genetically modified to alter the content and carrier of gNV.

[0044] In some respects, mammalian cells can be genetically modified. Mammalian cells can be genetically modified to express therapeutic agents, such as membrane proteins or lipids located on the plasma membrane. In some respects, mammalian cells can be naturally occurring or genetically modified to produce one or more therapeutic agents, such as: adhesion molecules, such as integrins; protein kinases, such as tyrosine kinases, serine / threonine kinases; transcription factors; ion channels, such as calcium channels, potassium channels, sodium channels; growth factors; interleukins; neurotrophic factors, etc.

[0045] In some respects, mammalian cells can be naturally occurring or genetically modified to produce one or more therapeutic agents, such as: trophic factors, such as CDNF, GDNF, neurturin, IGF1, VEGF, HGF; molecular chaperones, such as HSP104, HSP70; ephA4; ephA4 ligands; Poly(A) binding protein nuclear 1 (PABPN1); matrin ubiquilin 2; zinc finger protein 106 (ZFP106); IRE1α kinase / RNase; ubiquitin; TANK-binding kinase 1 (TBK1); Muskk agonist antibodies; and ankyrin repeat and KH domain-containing 1. 1, ANKHD1); affitin; glycerophosphodiester phosphodiesterase 2 (GDE2); MMIF; SRSF1 nuclear transport; anti-mir155; miRNA 125b; miRNA 31; miRNA-206; miRNA 133b; TREM2 activating antibody; SARM1 inhibitor; macrophage migration inhibitory factor (MIF); dominant negative NFkB; muscle-specific kinase; siRNA targeting tristetraprolin; PPARγ coactivator 1α; Ret receptor; notch intracellular domain; TGFβ; INFγ, etc.

[0046] The gNVs presented herein retain membrane proteins in a substantially native conformation. For example, during gNV generation, the gNVs are not exposed to denaturing agents used as vesiculation agents. For example, methods for preparing gNVs do not include the step of exposing mammalian cells to vesiculation agents to form vesicles. In other words, during or after gNV generation, the gNVs are not exposed to vesiculation agents (e.g., thiol blocking agents), allowing the gNVs to maintain the membrane protein in its native conformation. The gNVs are not exposed to vesication agents, such as formaldehyde and dithiothreitol, during or after formation. Thiol-based blocking agents include formaldehyde, acetaldehyde, acetaldehyde, glyoxal, glutaraldehyde, acrolein, methacrolein, pyridoxal, N-ethylmaleimide (NEM), maleimide, chloromercuric benzoate, iodoacetate, potassium arsenite, sodium selenite, thimerosal, benzoyl peroxide, cadmium chloride, hydrogen peroxide, iodobenzoic acid, and meralluride. Sodium, mercuric chloride, mercurous chloride, neohydrin, phenylhydrazine, potassium tellurite, sodium malonate, p-arsenobenzoic acid, 5,5'-diamino-2,2'-dimethylarsene, disodium N,N'-dimethylenesulfonate, iodoacetamide, mapharsen, gold chloride, p-chloromercuric benzoic acid, p-chloromercuric benzenesulfonic acid, copper chloride, iodomercuric bromoerythromycin (red mercury)porphyrin, potassium permanganate, mercsalyl (salyrgan), silver nitrate, protargol, uranyl acetate, etc. Other examples of vesicle-forming agents include cytotoxins such as cytochalasin B or melitin.

[0047] As used herein, the phrase “not exposed to” in the context of vesicating agents means that gNVs are not exposed to a significant amount of vesicating agent sufficient to induce vesicle formation. In other words, gNVs may be exposed to trace amounts of vesicating agent during or after their formation that do not cause denaturation of membrane proteins and do not induce vesicle formation.

[0048] In some respects, gNVs presented herein can be distinguished from gNVs generated using vesicle-forming agents by measuring vesicles. Assays such as immunoassays or functional assays can be used. In some respects, antibodies that bind to membrane proteins in their native conformation but do not bind to them when the proteins are denatured can be used for immunoassays to distinguish gNVs from gNVs prepared using vesicle-forming agents. Functional assays may include measuring the membrane protein activity of gNVs, such as ligand binding, ligand uptake, the ability to deliver or pump drugs or uptake molecules, etc.

[0049] The gNV can be generally spherical in shape and can have a smaller diameter compared to the cell that produces the gNV. In some aspects, the gNV can be relatively large, with a diameter ranging from 100 nm to 900 nm, such as 100 nm to 800 nm, 100 nm to 700 nm, 100 nm to 600 nm, 100 nm to 500 nm, 100 nm to 400 nm, 100 nm to 300 nm, or 100 nm to 200 nm. In some aspects, the gNV can be relatively small, with a diameter ranging from 10 nm to 100 nm, such as 20 nm to 100 nm, 30 nm to 100 nm, or 40 nm to 100 nm. In some aspects, the preparation of the gNV, such as a composition of gNVs, can include both large and small gNVs.

[0050] GNVs can be formed by opening nanovesicles (NVs), for example by exposing NVs to a high pH, ​​separating open sheets of the cell membrane, and closing open sheets of the cell membrane to produce gNVs. In some respects, gNVs can be formed by: disrupting mammalian cells to produce vesicles; separating vesicles and nanovesicles using a density gradient; exposing the separated nanovesicles to an alkaline pH to open them, thereby producing plasma membrane sheets; purifying the plasma membrane sheets; and applying sufficient energy to the purified plasma membrane sheets to convert them into gNVs. NVs formed by such methods include cytoplasmic components such as organelles, cytoplasmic proteins, the nucleus, and nucleic acids (e.g., RNA, such as mRNA, miRNA, etc.). gNVs lack components that are present in amounts at least 50% less than those present in NVs that are not ghosts, for example, 60%, 70%, 80%, 90%, or 99% less.

[0051] In some respects, gNVs are prepared by adding a therapeutic agent to a composition comprising a purified membrane sheet, and applying energy to the composition sufficient to convert the membrane sheet into gNVs containing the therapeutic agent. In some respects, the therapeutic agent does not contain an anti-inflammatory agent.

[0052] In some respects, gNVs can be loaded with therapeutic agents, which are nucleic acids, peptides, or proteins. These therapeutic agents can be antibodies, growth factors (e.g., EGF, FGF, VEGF, etc.), siRNA, miRNA, shRNA, etc. In some respects, the therapeutic agents can be anticancer agents or angiogenesis inhibitors. In some respects, anticancer agents can be: DNA alkylating agents, such as nitrogen mustard, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; anticancer antibiotics, such as actinomycin D, doxorubicin, epirubicin, idarubicin, mitoxantrone, procainoxine, mitomycin, and C-bleomycin; and plant alkaloids, such as vincristine, vinblastine, paclitaxel, docetaxel, daunorubicin, paclitaxel, oncovin, prednisone, cisplatin, Herceptin, rituximab, etoposide, teniposide, topotecan, and irinotecan.

[0053] In some respects, gNVs may be enriched with membrane proteins, such as proteins located in the plasma membrane, such as transport proteins. In the context of the components enriched in gNVs disclosed herein, “enriched” means that the enriched component is present in the gNV at a higher concentration by weight than the concentration of that component in the NV from which the gNV originates, for example, at least three times higher, for example, at least five times higher, at least 10 times higher, at least 30 times higher, at least 50 times higher, or at least 100 times higher.

[0054] In some aspects, compositions comprising gNVs are provided. The compositions may comprise gNVs and carriers, diluents, loading agents, excipients, etc. In some aspects, the compositions of this disclosure may comprise gNVs and pharmaceutically acceptable carriers, diluents, loading agents, excipients, etc. In some aspects, the compositions may also comprise additional prophylactic or therapeutic agents. In some aspects, the compositions may contain gNVs that effectively reduce inflammation in subjects with this need. In some aspects, the compositions may comprise gNVs derived from different cells and / or loaded with different therapeutic agents. For example, gNVs may be derived from two, three, four, or more different types of cells. In some aspects, the compositions may comprise a first type of gNV containing a first therapeutic agent and a second type of gNV containing a second therapeutic agent, etc.

[0055] Carriers, diluents, loading agents, excipients, etc., can be salts, buffers, antioxidants (e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzyl alcohol, methylparaben, ethylparaben, or n-propylparaben), emulsifiers, suspending agents, dispersants, solvents, fillers, fillers, detergents, and / or excipients. For example, a suitable loading agent can be an aqueous solution of physiological saline or a buffered saline solution, possibly supplemented with other materials commonly found in pharmaceutical compositions for, for example, parenteral administration. Neutral buffered saline solutions or saline solutions mixed with serum albumin are further exemplary loading agents. Those skilled in the art will readily recognize a variety of buffers that can be used in compositions. Typical buffers include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. For example, the buffer component can be a water-soluble material such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and their salts. Acceptable buffers include, for example, Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), sodium 2-(N-morpholino)ethanesulfonate (MES), 3-(N-morpholino)propanesulfonic acid (MOPS) and N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS). In some respects, the excipients included in the disclosed compositions may be poly-ICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, carrier systems, PLGA microparticles, resiquimod, SRL172, viral microsomes and other virus-like particles, YF-17D, VEGF Traps, R848, β-glucan, Pam3Cys, acrylic or methacrylic acid polymers, or copolymers of maleic anhydride and Aquila's QS21 stimulant.

[0056] In some respects, gNVs may be loaded with therapeutic agents, such as chemotherapy agents, anticancer antibodies, etc. In some respects, the gNVs disclosed herein may not be loaded with therapeutic agents. In some respects, the gNVs disclosed herein may not be loaded with anti-inflammatory agents. In some respects, the gNVs disclosed herein may not be administered in conjunction with anti-inflammatory agents.

[0057] In some respects, compositions containing the gNVs disclosed herein may not contain an effective amount of adjuvants, such as those used to enhance the immunogenicity of the composition.

[0058] In some respects, gNV and its compositions can be used in methods for reducing at least one pro-inflammatory cytokine in subjects with this need, the method comprising administering the composition to the subject. Such methods will be described in detail in the next section.

[0059] method

[0060] In some aspects, methods are provided for treating subjects in need. In other aspects, methods are provided for reducing inflammation in subjects in need. These methods may include administering an effective amount of blood shadow nanovesicles (gNVs) derived from mammalian cells to the subject, wherein the gNVs lack cytoplasmic proteins and nucleic acids, and wherein the gNVs reduce the level of at least one pro-inflammatory cytokine in the subject.

[0061] In the context of an inflammatory response, the term "reduced" means that the level of pro-inflammatory cytokines produced in the presence of gNV is lower than the level produced in the absence of gNV. In some embodiments, the production of cytokines is reduced by at least 5%, such as at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or more, compared to the production without gNV administration. At least one pro-inflammatory cytokine may include one or more of IL-2, IL-4, IL-6, IL-12, IL-12p70, IL-17, tumor necrosis factor-α (TNF-α), or interferon-γ (IFN-γ). The mammalian cells from which gNV is derived may be as described in the preceding sections of the description of gNV that provide for this disclosure.

[0062] In some respects, gNVs can be prepared by methods including: disrupting mammalian cells to generate vesicles; separating vesicles based on density and isolating nanovesicles; exposing the isolated nanovesicles to an alkaline pH to open the nanovesicles to generate plasma membrane sheets; purifying the plasma membrane sheets; and applying energy to the purified plasma membrane sheets sufficient to convert the plasma membrane sheets into gNVs.

[0063] In some aspects, the method may include adding a therapeutic agent to a composition comprising a purified membrane sheet, and applying energy to the composition sufficient to convert the membrane sheet into a gNV containing the therapeutic agent. In some aspects, the therapeutic agent does not contain an anti-inflammatory agent. In some aspects, the gNV does not contain a significant amount of an anti-inflammatory agent; for example, the gNV may contain trace amounts of an anti-inflammatory agent. In some aspects, the composition containing the gNV is substantially free of an anti-inflammatory agent.

[0064] In some aspects, disrupting mammalian cells to generate vesicles can involve mechanical, electrical, or chemical methods for cell lysis. Examples of techniques for cell lysis include, but are not limited to, osmosis, electroporation, acoustic treatment, homogenization, detergent treatment, freeze-thaw cycles, extrusion, mechanical degradation, and chemical treatment. In some aspects, mammalian cells are not disrupted by detergent treatment. In mechanical degradation methods, the mammalian cell solution is agitated with metal, ceramic, or sufficiently rigid plastic spheres. In some aspects, disrupting mammalian cells may include applying shear forces to the mammalian cells. Shear forces can be applied by extruding the mammalian cells. Extrusion may include forcing the mammalian cells through pores smaller than the size of the mammalian cells. In the case of extrusion, the mammalian cells may be forced sequentially through a series of filters with decreasing pore sizes. For example, mammalian cells may sequentially pass through three filters with pore sizes of 10 μm → 5 μm → 1 μm to form vesicles.

[0065] In some respects, destroying mammalian cells may include applying acoustic energy to mammalian cells. The acoustic energy can be applied using an acoustic treatment device. The acoustic treatment conditions can be adjusted for the desired destructive energy. For example, when destroying protoplasts to generate vesicles, acoustic treatment can be performed at low temperatures, low energy, and / or short durations. Acoustic treatment can be performed at varying intensities, including low-energy acoustic treatment lasting from 1 minute to 3 hours. In some respects, acoustic treatment can be performed using an ultrasound probe-type device. In some respects, an ultrasound bath can be used for acoustic treatment. The duration of acoustic treatment can be adjusted depending on the type of device used to perform the acoustic treatment. For example, an ultrasound probe-type device can provide approximately 1000 times the energy of an ultrasound bath. In some respects, an ultrasound probe-type device can be used to destroy mammalian cells.

[0066] After mammalian cells are disrupted to produce vesicles (e.g., vesicles with a plasma membrane surrounding cytoplasmic contents), these vesicles can be separated from any remaining mammalian cells. These vesicles can be separated from mammalian cells using differences in size, density, buoyancy, etc. In some respects, centrifugation (e.g., density gradient centrifugation or density gradient ultracentrifugation) or filtration can be performed to separate the vesicles. In some respects, density gradient ultracentrifugation can be used to purify vesicles, where vesicles present in a density gradient of 10% to 50% can be separated. Vesicles present in a density gradient of 10% to 50% are mostly nanoscale vesicles or nanovesicles.

[0067] The isolated nanovesicles can then be exposed to an alkaline solution to open them, allowing them to expel their cytoplasmic contents. In some respects, the pH of the alkaline solution used to open the nanovesicles can be 11 to 14. The alkaline solution used to open the nanovesicles can be prepared as a sodium carbonate (Na₂CO₃), sodium hydroxide (NaOH), ammonia (NH₃), calcium hydroxide (Ca(OH)₂), potassium hydroxide (KOH), sodium bicarbonate (NaHCO₃), or magnesium hydroxide (Mg(OH)₂) solution. The duration of incubation of the nanovesicles in the alkaline solution can be adjusted based on the number of nanovesicles, the total volume of the solution, etc. As used herein, the step of incubating or exposing the vesicles to an alkaline pH may include using an alkaline solution with a pH of 9 to 14, such as pH 10 to 14, pH 11 to 14, pH 12 to 14, or pH 13 to 14.

[0068] The plasma membrane sheet resulting from the opening of nanovesicles can be separated from the whole nanovesicle (i.e., unopened) using any suitable separation method. In some aspects, purification of the plasma membrane sheet may include centrifugation, such as centrifugation (e.g., density gradient centrifugation or density gradient ultracentrifugation), filtration, or another suitable method, such as size exclusion, dialysis, tangential flow filtration, etc. In some aspects, density gradient ultracentrifugation can be used to purify the plasma membrane sheet, where a plasma membrane sheet present in a density gradient of 10% to 30% can be separated. Plasma membrane sheets present in a density gradient of 10% to 30% are essentially free of nanovesicles.

[0069] In some aspects, methods for generating gNVs may include applying energy or force sufficient to convert the purified plasma membrane sheet into gNVs. Suitable energy sources include mild acoustic treatment, shear force, acoustic force, freeze-thaw cycles, etc. In some aspects, the purified plasma membrane sheet may be acoustically treated for a duration sufficient to convert the sheet into gNVs. In some aspects, the purified plasma membrane sheet may be acoustically treated by applying energy 100 to 1000 times less than that applied to destroy mammalian cells. In some aspects, mild acoustic treatment may include using an ultrasonic bath to convert the plasma membrane sheet into gNVs.

[0070] In some respects, the gNV of this disclosure can be accessed via Figure 1 The method described in the text is used for preparation. For example... Figure 1As shown, mammalian cells can be disrupted by continuously squeezing through a filter with increasingly smaller pores, forcing the cells to rupture into vesicles. A density gradient ultracentrifugation was used to separate vesicles based on size, employing a density gradient of 0% to 50% iodixanol. Nanovesicles present in a density layer of 10% to 50% were separated; the nanovesicles were exposed to an alkaline solution (e.g., pH 11 to pH 14) to open NVs; the opened NVs were then separated using a density gradient ultracentrifugation employing a density gradient of 10% to 50% iodixanol. Open NVs (i.e., membranes) present in a density layer of 10% to 30% were separated; and the separated membranes were acoustically treated to produce gNVs.

[0071] Individuals requiring inflammation reduction may have or be prone to developing inflammatory conditions. Inflammation-related conditions can include cancer, multiple sclerosis, psoriasis, dry eye, asthma, sepsis, infections, rheumatoid arthritis, ulcerative colitis, Crohn's disease, tuberculosis, hepatitis, sinusitis, autoimmune diseases, inflammatory bowel disease, pelvic inflammatory disease, ulcers, atherosclerosis, erythema, necrosis, vasculitis, ankylosing spondylitis, connective tissue diseases, kidney disease, sarcoidosis, thyroiditis, osteoarthritis, rheumatism, chronic inflammatory diseases, demyelinating polyneuropathy, pancreatitis, psoriatic arthritis, periodontitis, Behcet's disease, sinusitis, polymyalgia rheumatica, nephritis, diverticulitis, and granulomatosis with... Polyangiitis, granulomas, encephalitis, immune-mediated inflammatory diseases, esophagitis, gout, uveitis, myopathy, gallbladder disease, periodic febrile syndrome, interstitial cystitis, peritonitis, appendicitis, neurodegenerative diseases, Parkinson's disease, Alzheimer's disease, cerebellar ataxia, systemic lupus erythematosus, fibromyalgia, diverticulitis, dermatitis, spinobulbar muscular atrophy (SBMA), lysosomal storage diseases, cerebral palsy, glioma, glioblastoma, muscular dystrophy, ataxia telangiectasia (AT), schizophrenia, depression, bipolar disorder, attention deficit disorder, Down syndrome (trisomy 21), amyotrophic lateral sclerosis (ALS), and ankylosing spondylitis. In some respects, inflammatory conditions can be asthma. In some respects, inflammatory conditions can be sepsis. In some respects, inflammatory conditions can be infections. In some cases, inflammatory conditions can be caused by bacterial, viral, or parasitic infections.

[0072] In some aspects, methods for reducing inflammation may include administering an additional therapeutic agent to the subject. In some aspects, the additional therapeutic agent is present in gNV. In some aspects, the method includes administering a composition comprising the additional therapeutic agent and gNV. In some aspects, the method includes administering gNV in combination with the additional therapeutic agent, for example, co-administering (as a single composition or substantially simultaneously) or administering gNV and the additional therapeutic agent sequentially.

[0073] Therapeutic agents can be small molecules, peptides, nucleic acids, or polypeptides. Therapeutic agents can be as provided in the previous sections. Therapeutic agents can have general anti-inflammatory properties or can target different steps in inflammatory pathways within cells, such as downstream TLR receptor activation (Myd88 or NFKB), downstream cytokine receptors, or the Stimulator of Interferon Genomes (STING) pathway.

[0074] GNV application

[0075] This disclosure contemplates the application of the disclosed compositions in any suitable manner for the prevention and / or treatment of conditions as described herein. Suitable routes of administration include parenteral (e.g., intramuscular, intravenous, intra-arterial, subcutaneous (e.g., injection), intraperitoneal, intracisional, intra-articular, intraperitoneal, intracerebral (parenchymal), and intraventricular), oral, nasal, vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal), sublingual, and inhalation, as well as direct injection into diseased tissues, such as tumor tissue.

[0076] In some respects, application includes local application to a target site in the subject. In some respects, the target site contains an inflammatory response or is prone to an inflammatory response. In some respects, the target site has lesions. The target site may be adjacent to a site with lesions. The target site may include sites in the central nervous system. The target site may be the brain. The target site may have an arterial occlusion. In some respects, application may be intra-arterial at the site of arterial occlusion; for example, a catheter for clot removal may be used to apply gNV after clot removal.

[0077] In some respects, the gNV composition can be injected into or near a tumor. In some respects, the anticancer composition and the gNV composition can be administered simultaneously to the subject.

[0078] This disclosure contemplates methods in which the composition of this disclosure is applied to a subject at least twice a day, at least once a day, at least once every 48 hours, at least once every 72 hours, at least once a week, at least once every two weeks, or once a month.

[0079] Combination therapy

[0080] This disclosure contemplates the use of the compositions provided herein in combination with one or more active therapeutic agents or other preventative or therapeutic modalities. In such combination therapies, the multiple active agents typically have different mechanisms of action. Such combination therapies can be particularly advantageous by allowing for a reduction in the dosage of one or more agents, thereby reducing or eliminating side effects associated with one or more agents; furthermore, such combination therapies may have a synergistic therapeutic or preventative effect against underlying diseases, disorders, or conditions.

[0081] As used herein, the term “combination” is intended to include treatments that can be administered separately (e.g., separately formulated for separate administration (e.g., as may be provided in a kit)) as well as treatments that can be administered together in a single formulation (i.e., “co-formulation”).

[0082] In some embodiments, the compositions of this disclosure are applied sequentially or in combination, for example, one agent is applied before one or more other agents. In other embodiments, the compositions are applied simultaneously, for example, two or more compositions are applied simultaneously or approximately simultaneously; the two or more compositions may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Whether the two or more compositions are applied sequentially or simultaneously, they are considered to be applied in combination for the purposes of this disclosure.

[0083] The compositions disclosed herein may be used in combination with other pharmaceutical agents (including those commonly administered to subjects suffering from inflammation) that can be used to treat, prevent, suppress or improve the diseases, disorders or conditions described herein.

[0084] Examples of some non-limiting aspects of this disclosure

[0085] Some aspects of the subject matter of the invention described above, including embodiments, may be advantageous, either alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of this disclosure are provided below. As will be apparent to those skilled in the art upon reading this disclosure, each individually numbered aspect, or any combination thereof before or after an individually numbered aspect, may be used. This is intended to support all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below:

[0086] 1. A method for reducing inflammation in a subject with this need, the method comprising:

[0087] An effective amount of blood shadow nanovesicles (gNVs) derived from mammalian cells was applied to the object.

[0088] The gNV described therein lacks cytoplasmic proteins and nucleic acids.

[0089] The gNV reduces the level of at least one pro-inflammatory cytokine in the object.

[0090] 2. The method of aspect 1, wherein the mammalian cell is an autologous mammalian cell.

[0091] 3. The method of aspect 1, wherein the mammalian cell is a heterologous mammalian cell.

[0092] 4. The method of any one of aspects 1 to 3, wherein the mammalian cell is a stem cell.

[0093] 5. The method of any one of aspects 1 to 4, wherein the mammalian cell is genetically modified.

[0094] 6. The method of any one of aspects 1 to 5, wherein the mammalian cells comprise a therapeutic agent, wherein optionally, the therapeutic agent does not comprise an anti-inflammatory agent.

[0095] 7. The method of any one of aspects 1 to 6, wherein the gNV is a large gNV with a diameter of 100 nm to 200 nm.

[0096] 8. The method of any one of aspects 1 to 6, wherein the gNV is a small gNV with a diameter of 40 nm to 100 nm.

[0097] 9. The method of any one of aspects 1 to 6, wherein the gNV comprises a large gNV with a diameter of 100 nm to 200 nm and a small gNV with a diameter of 40 nm to 100 nm.

[0098] 10. The method of any one of aspects 1 to 9, wherein the gNV is prepared by a method comprising:

[0099] Disruption of the mammalian cells to produce vesicles;

[0100] The vesicles were separated based on density, and nanovesicles were also separated.

[0101] The isolated nanovesicles were exposed to an alkaline pH to open them, thereby producing a plasma membrane sheet;

[0102] Purify the plasma membrane sheet; and

[0103] Apply energy sufficient to convert the purified plasma membrane sheet into gNV.

[0104] 11. The method of aspect 10, further comprising adding a therapeutic agent to a composition comprising the purified membrane sheet, and applying energy to the composition sufficient to convert the membrane sheet into gNV comprising the therapeutic agent, wherein optionally, the therapeutic agent does not contain an anti-inflammatory agent.

[0105] 12. The method of any one of aspects 1 to 11, wherein the at least one pro-inflammatory cytokine comprises IL-2, IL-4, IL-6, IL-12, IL-12p70, IL-17, tumor necrosis factor α (TNF-α), or interferon γ (IFN-γ).

[0106] 13. The method of any one of aspects 1 to 12, wherein the subject suffers from or is susceptible to developing an inflammatory condition selected from the following: cancer, multiple sclerosis, psoriasis, dry eye disease, asthma, sepsis, infection, rheumatoid arthritis, ulcerative colitis, Crohn's disease, tuberculosis, hepatitis, sinusitis, autoimmune diseases, inflammatory bowel disease, pelvic inflammatory disease, ulcer, atherosclerosis, erythema, necrosis, vasculitis, ankylosing spondylitis, connective tissue disease, nephropathy, sarcoidosis, thyroiditis, osteoarthritis. Rheumatism, chronic inflammatory diseases, demyelinating polyneuropathy, pancreatitis, psoriatic arthritis, periodontitis, Behcet's disease, sinusitis, polymyalgia rheumatica, nephritis, diverticulitis, granulomatous polyangiitis, granuloma, encephalitis, immune-mediated inflammatory diseases, esophagitis, gout, uveitis, myopathy, gallbladder disease, periodic fever syndrome, interstitial cystitis, peritonitis, appendicitis, Parkinson's disease, Alzheimer's disease, systemic lupus erythematosus, fibromyalgia, diverticulitis, dermatitis, and ankylosing spondylitis.

[0107] 14. The method of aspect 13, wherein the inflammatory-related condition is asthma.

[0108] 15. The method of aspect 13, wherein the inflammatory-related condition is sepsis.

[0109] 16. The method of aspect 13, wherein the inflammatory-related condition is an infection.

[0110] 17. The method of aspect 13, wherein the infection is a bacterial, viral, or parasitic infection.

[0111] 18. The method of any one of aspects 1 to 17, wherein the method further comprises administering a therapeutic agent to the object.

[0112] 19. The method of aspect 18, wherein the method comprises administering to the subject a composition comprising the gNV and the therapeutic agent.

[0113] 20. The method of aspect 18 or 19, wherein the therapeutic agent comprises a small molecule, peptide, nucleic acid, or polypeptide.

[0114] 21. The method of aspect 18 or 19, wherein the therapeutic agent comprises an antibody.

[0115] 22. The method of any one of aspects 1 to 21, wherein the administration includes intravenous administration.

[0116] 23. The method of any one of aspects 1 to 21, wherein the application includes subcutaneous application.

[0117] 24. The method of any one of aspects 1 to 21, wherein the administration includes intramuscular, intraperitoneal, intra-articular, intra-articular, intracerebral (internal) or intraventricular administration.

[0118] 25. The method of any one of aspects 1 to 21, wherein the application comprises local application to a target site in the object.

[0119] 26. The method of aspect 25, wherein the target site comprises an inflammatory response or is susceptible to an inflammatory response.

[0120] 27. The method of aspect 25 or 26, wherein the target site has damage.

[0121] 28. The method of aspect 25 or 26, wherein the target site is adjacent to the site of damage.

[0122] 29. The method of aspects 27 to 28, wherein the target site includes a site in the central nervous system.

[0123] 30. The method of aspect 29, wherein the target site comprises the brain.

[0124] 31. The method of aspect 30, wherein the target site includes an arterial occlusion site.

[0125] 32. The method of aspect 31, wherein the administration is intra-arterial administration.

[0126] 33. The method of any one of aspects 1 to 31, wherein the gNV is not derived from cancer cells.

[0127] 34. The method of any one of aspects 1 to 31, wherein the gNV is not derived from a tumor.

[0128] 35. The method of any one of aspects 1 to 31, wherein the gNV is derived from isolated mammalian cells present in a composition lacking vesicles released from the cells.

[0129] 36. A composition comprising:

[0130] Blood-shadow nanovesicles (gNVs) derived from mammalian cells

[0131] The gNV described therein lacks cytoplasmic proteins and nucleic acids.

[0132] The gNV reduces the level of at least one pro-inflammatory cytokine when applied to subjects in need of it.

[0133] 37. The composition of aspect 36, wherein the mammalian cell is an autologous mammalian cell.

[0134] 38. The composition of aspect 36, wherein the mammalian cell is a heterologous mammalian cell.

[0135] 39. The composition of any one of aspects 36 to 38, wherein the mammalian cell is a stem cell.

[0136] 40. The composition of any one of aspects 36 to 39, wherein the mammalian cell is genetically modified.

[0137] 41. The composition of any one of aspects 36 to 40, wherein the mammalian cells comprise a therapeutic agent, wherein optionally, the therapeutic agent does not comprise an anti-inflammatory agent.

[0138] 42. The composition of any one of aspects 36 to 41, wherein the gNV is a large gNV with a diameter of 100 nm to 200 nm.

[0139] 43. The composition of any one of aspects 36 to 41, wherein the gNV is a small gNV with a diameter of 40 nm to 100 nm.

[0140] 44. The composition of any one of aspects 36 to 41, wherein the gNV comprises a large gNV with a diameter of 100 nm to 200 nm and a small gNV with a diameter of 40 nm to 100 nm.

[0141] 45. The composition of any one of aspects 36 to 44, wherein the gNV is prepared by a method comprising:

[0142] Disruption of the mammalian cells to produce vesicles;

[0143] The vesicles were separated based on density, and nanovesicles were also separated.

[0144] The isolated nanovesicles were exposed to an alkaline pH to open them, thereby producing a plasma membrane sheet;

[0145] Purify the plasma membrane sheet; and

[0146] Apply energy sufficient to convert the purified plasma membrane sheet into gNV.

[0147] 46. ​​The composition of aspect 45, wherein the gNV is prepared by adding a therapeutic agent to a composition comprising the purified membrane and applying energy to the composition sufficient to convert the membrane into a gNV comprising the therapeutic agent, wherein optionally, the therapeutic agent does not contain an anti-inflammatory agent.

[0148] 47. The composition of any one of aspects 36 to 46, wherein the at least one pro-inflammatory cytokine comprises IL-2, IL-4, IL-6, IL-12, IL-12p70, IL-17, tumor necrosis factor α (TNF-α), or interferon γ (IFN-γ).

[0149] 48. The composition of any one of aspects 36 to 46, in a method of reducing at least one pro-inflammatory cytokine in a subject in which such a need exists, the method comprising administering the composition to the subject.

[0150] 49. The composition of aspect 48, wherein the subject suffers from or is susceptible to developing an inflammatory condition selected from the following: cancer, multiple sclerosis, psoriasis, dry eye disease, asthma, sepsis, infection, rheumatoid arthritis, ulcerative colitis, Crohn's disease, tuberculosis, hepatitis, sinusitis, autoimmune diseases, inflammatory bowel disease, pelvic inflammatory disease, ulcer, atherosclerosis, erythema, necrosis, vasculitis, ankylosing spondylitis, connective tissue disease, nephropathy, sarcoidosis, thyroiditis, osteoarthritis, rheumatoid arthritis, rheumatoid arthritis, etc. Eczema, chronic inflammatory diseases, demyelinating polyneuropathy, pancreatitis, psoriatic arthritis, periodontitis, Behcet's disease, sinusitis, polymyalgia rheumatica, nephritis, diverticulitis, granulomatous polyangiitis, granuloma, encephalitis, immune-mediated inflammatory diseases, esophagitis, gout, uveitis, myopathy, gallbladder disease, periodic fever syndrome, interstitial cystitis, peritonitis, appendicitis, Parkinson's disease, Alzheimer's disease, systemic lupus erythematosus, fibromyalgia, diverticulitis, dermatitis, and ankylosing spondylitis.

[0151] 50. The composition of aspect 49, wherein the inflammatory-related condition is asthma, sepsis, or infection, wherein optionally, the infection is a bacterial, viral, or parasitic infection.

[0152] 51. The composition of aspect 41, wherein the therapeutic agent comprises a small molecule, peptide, nucleic acid, or polypeptide.

[0153] 52. The composition of aspect 41, wherein the therapeutic agent comprises an antibody.

[0154] 53. The composition of any one of aspects 36 to 52, wherein the gNV is not derived from cancer cells.

[0155] 54. The composition of any one of aspects 36 to 52, wherein the gNV is not derived from a tumor.

[0156] 55. The composition of any one of aspects 36 to 52, wherein the gNV is derived from isolated mammalian cells present in a composition lacking vesicles released from the cells.

[0157] Example

[0158] The following embodiments are provided to provide a complete disclosure and description of how to manufacture and use the invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent all or only the experiments conducted. Efforts have been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and biases should be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc.

[0159] Example 1: Generation of gNV from mesenchymal stem cells

[0160] method

[0161] Preparation of gNV

[0162] MSC at 5×10 6 The cells were resuspended at a density of 10 cells / mL in a total of 10 mL of phosphate-buffered saline. The cell suspension was passed sequentially through each membrane filter with pore sizes of 10 μm, 5 μm, and 1 μm five times. 1 and 2 mL of 50% and 10% iodixanol solution (Axis-Shield PoC AS), followed by 7 mL of cell suspension effluent from the membrane filters, were added sequentially to each 10 mL ultracentrifuge tube. The layer formed between 50% and 10% iodixanol after ultracentrifugation at 100,000 × g for 2 hours was collected and considered as the NV. The NV was incubated with a high pH solution (200 mM Na₂CO₃, pH 14.0) at 25°C for 1 hour. The solution was added to 4 mL of 50% iodixanol, followed by 4 mL of 30% iodixanol and 2 mL of 10% iodixanol to the ultracentrifuge tube. The layer formed between 10% and 30% iodixanol after ultracentrifugation at 100,000 × g for 2 hours was collected. Finally, the sample was acoustically treated for 30 minutes and considered as gNV ( Figure 1 ).

[0163] Preparation of OMV

[0164] E. coli culture was precipitated twice at 6,000×g, 4°C for 20 minutes. The supernatant was then filtered through a 0.45-μm vacuum filter and concentrated using a Vivaflow 200 ultrafiltration module (Sartorius) with a 100 kDa cut-off membrane. The residue was filtered again through a 0.22-μm vacuum filter to remove any remaining cells. The resulting filtrate was ultracentrifuged at 150,000×g, 4°C for 3 hours and resuspended in PBS.

[0165] RNA and DNA analysis

[0166] Using miRCURY for biological fluids TM RNA was isolated from NV and gNV using the RNA Isolation Kit (Exiqon) according to the manufacturer's protocol. DNA was isolated using the Qiamp DNA Blood Mini Kit (Qiagen) according to the manufacturer's protocol. The DNA was isolated using an Agilent 2100 Bioanalyzer. (Agilent Technologies) used the Agilent RNA6000 nanochip and the Agilent High Sensitivity DNA chip, respectively, to analyze the quality, yield, and nucleotide length of one microliter of separated RNA or DNA by capillary electrophoresis.

[0167] Example 2: Proteomics analysis of gNV

[0168] LC-MS / MS analysis

[0169] NV and gNV were digested with trypsin, which was then desalted using a filter-aided sample preparation (FASP) method and a C18 centrifuge column according to the manufacturer's instructions. All fractions were dried on a Speedvac and reconstituted in 3% acetonitrile and 0.2% formic acid, and analyzed on an Orbitrap Fusion Tribrid mass spectrometer interfaced with an Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA). Peptides were captured on an Acclaim Pepmap 100 C18 capture column (100 μm × 2 cm, 5 μm particle size; Thermo Fischer Scientific) and separated on an in-house packed C18 analytical column (75 μm × 30 cm, 3 μm particle size) using gradients from 5% to 33% B over 160 min and from 33% to 100% B over 5 min. Solvent A was 0.2% formic acid, and solvent B was 80% acetonitrile and 0.2% formic acid. Precursor ion mass spectrometry was recorded at 120,000 resolution, selecting the strongest precursor ion, and fragmentation was performed using HCD at a collision energy of 30. MS / MS spectra were recorded at 30,000 resolution, with a maximum injection time of 125 ms and a separation window of 1.0 Da. Charge states from 2 to 7 were selected for fragmentation, with dynamic exclusion set to 45 seconds and a tolerance of 10 ppm.

[0170] result

[0171] Principal component analysis showed that the first component, based on vesicle type (gNV and NV), separated 59% of the data, and the second component, based on replication, separated 18% of the data. Figure 4A This indicates that the gNV protein is different from the NV protein. Hierarchical clustering analysis produced similar results, where samples were first clustered into vesicle types (gNV and NV), and then further clustered by replication ( Figure 4B The Venn diagram shows the closest unique quantification spectra for each group. We then identified 3536 and 3484 proteins from NV and high-pH-treated gNV, respectively. (See Venn diagram). Figure 5A As shown in the figure, 3483 proteins were identified in the two vesicle formulations, while 53 and 1 proteins were identified in NV and gNV, respectively. The relative abundance of different proteins was obtained using MaxQuant software and plotted as shown in the figure. Figure 5BAs shown. Based on relative protein abundance, 2231 out of 3483 proteins showed no significant change in abundance. However, in gNV, 515 and 737 proteins showed relative increases (1.5-fold) and decreases (1.5-fold), respectively. Volcano plots of the proteins identified in NV and gNV also showed similar patterns. Figure 6 This indicates that protein expression changes differently due to high pH treatment.

[0172] In GO terminology subcellular localization analysis, the gNV-rich proteome showed distinct characteristics from the NV-rich proteome. Figure 7 The gNV proteome is rich in cell membrane proteins, while the NV proteome is rich in cytoplasmic and nuclear proteins. In GO terminology biological process analysis, the gNV proteome is rich in biological processes including transport. Figure 8 In contrast, the NV proteome is rich in biological processes, including nucleic acid metabolism.

[0173] Example 3: gNV reduces OMV-induced inflammation

[0174] method

[0175] RAW 264.7 cytokines

[0176] The mouse macrophage cell line RAW 264.7 (1×10⁻⁶) was used. 5 Cells were seeded into 24-well plates. OMV was applied to the cells to induce pro-inflammatory cytokines (TNF-α and IL-6) for 3 hours. NV or gNV was added for an additional 15 hours. Cytokine concentrations in the supernatant were measured using an ELISA kit (R&D systems).

[0177] Mouse experiment

[0178] Mice (wild-type mice with a C57BL / 6 genetic background, 6 weeks old) were injected intraperitoneally (ip) with 15 μg of OMV. Mice were then treated intraperitoneally with NV or gNV and sacrificed 6 hours after OMV injection. Peritoneal fluid (PF) and blood were collected from the mice, and cytokines in the supernatant were analyzed using a DuoSet ELISA development kit (R&D Systems).

[0179] Bacterial OMV has been identified as an infectious agent that induces inflammation. rgNV significantly reduced OMV-induced TNF-α and IL-6 release from RAW 264.7 cells, comparable to NV, revealing the anti-inflammatory effect of gNV. Figure 9Furthermore, the pattern of decreased weight and body temperature loss typically observed after OMV exposure was restored by gNV at 6 hours. Figure 10 Furthermore, the administration of gNV significantly reduced peritoneal fluid induced by OMV ( Figure 11 ) and serum cytokines ( Figure 12 The increase in gNV indicates that the therapeutic efficacy was retained after high pH treatment.

Claims

1. A composition configured to reduce the levels of pro-inflammatory cytokines in subjects suffering from inflammation-related conditions, comprising: Blood-shadow nanovesicles (gNVs) derived from mammalian cells, wherein the mammalian cells are mesenchymal stem cells (MSCs). The gNV described therein lacks cytoplasmic proteins and nucleic acids. The gNV is prepared by a method comprising the following steps: Disruption of the MSCs to generate vesicles; The vesicles are separated based on density, and nanovesicles are isolated. The isolated nanovesicles were exposed to an alkaline pH to open them, thereby producing a plasma membrane sheet; Purify the plasma membrane sheet; as well as Apply energy sufficient to convert the purified plasma membrane sheet into gNV.

2. The composition of claim 1, wherein the mammalian cell is an autologous mammalian cell.

3. The composition of claim 1, wherein the mammalian cell is a heterologous mammalian cell.

4. The composition of any one of claims 1 to 3, wherein the MSC comprises a therapeutic agent.

5. The composition of claim 4, wherein the therapeutic agent does not contain an anti-inflammatory agent.

6. The composition of any one of claims 1 to 5, wherein the gNV is a large gNV with a diameter of 100 nm to 200 nm.

7. The composition of any one of claims 1 to 5, wherein the gNV is a small gNV with a diameter of 40 nm to 100 nm.

8. The composition of any one of claims 1 to 5, wherein the gNV comprises large gNVs with a diameter of 100 nm to 200 nm and small gNVs with a diameter of 40 nm to 100 nm.

9. The composition of any one of claims 1 to 8, wherein the gNV is prepared by adding a therapeutic agent to a composition comprising the purified plasma membrane sheet and applying energy to the composition sufficient to convert the plasma membrane sheet into a gNV comprising the therapeutic agent.

10. The composition of claim 9, wherein the therapeutic agent does not contain an anti-inflammatory agent.

11. The composition of any one of claims 1 to 10, wherein the pro-inflammatory cytokines comprise IL-2, IL-4, IL-6, IL-12, IL-17, tumor necrosis factor α, or interferon γ.

12. The composition of any one of claims 1 to 11, in a method for reducing at least one pro-inflammatory cytokine in a subject in which such a need exists, the method comprising administering the composition to the subject.

13. The composition of claim 9 or 10, wherein the therapeutic agent comprises a small molecule.

14. The composition of claim 9 or 10, wherein the therapeutic agent comprises a peptide.

15. The composition of claim 9 or 10, wherein the therapeutic agent comprises a nucleic acid.

16. The composition of claim 9 or 10, wherein the therapeutic agent comprises a polypeptide.

17. The composition of claim 9 or 10, wherein the therapeutic agent comprises an antibody.

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