Biomolecule-loaded extracellular vesicles

Abstract

The compositions of therapeutic extracellular vesicles, as well as methods and systems for producing therapeutic extracellular vesicles are described herein.Methods for treating disease or symptoms using therapeutic extracellular vesicles are also described herein.In some cases, the multiple extracellular vesicles are produced by transfecting cells with a plasmid encoding an extracellular matrix protein.In some cases, transfecting cells is performed by cell nanoporation.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 179,058, filed April 23, 2021, which is incorporated herein by reference.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Background technology]

[0003] background Extracellular vesicles are secreted by a wide variety of cell types. Generally, extracellular vesicles, such as exosomes, microvesicles, and apoptotic bodies, are membrane-bound and can be loaded with therapeutic cargo. Exosomes are a type of membrane-bound extracellular vesicle secreted by most eukaryotic cells. Exosome biogenesis can begin with the constriction of endosomal invaginations into multivesicular bodies to form intraluminal vesicles. When multivesicular bodies fuse with the cell's plasma membrane, the intraluminal vesicles can be released as exosomes. Microvesicles are another type of extracellular vesicles that bud outward from the cell surface membrane. Meanwhile, apoptotic bodies are extracellular vesicles formed from dead cell debris.

[0004] The extracellular matrix (ECM) provides structural support for many different types of tissues and also influences certain cellular processes in the body. Components of the ECM in skin include collagen, elastic fibers, proteoglycans, and glycosaminoglycans. Age-related changes in ECM proteins can have several different consequences. Wrinkling and loss of elasticity are typical phenomena of skin aging and are likely the result of a gradual decrease in the amount of extracellular matrix (ECM) (e.g., collagen) in the dermis. The decrease in collagen in aging skin may be due to decreased collagen production and / or increased collagen degradation. Structural changes in collagen and other ECM proteins may also be involved in dermal aging.

[0005] Vascular endothelial growth factor (VEGF) is a signaling protein that promotes angiogenesis, the growth of new blood vessels. VEGF forms part of the mechanism that restores blood supply to cells and tissues when impaired circulation deprives them of oxygenated blood. Summary of the Invention [Means for solving the problem]

[0006] Abstract In some embodiments, a plurality of extracellular vesicles comprising exogenous extracellular matrix messenger RNA (mRNA) are described herein. In some cases, the exogenous extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is type I collagen alpha 1 chain (Col1A1). In some cases, the exogenous extracellular matrix mRNA is mRNA encoding the pro-alpha 1 (I) chain. In some cases, the collagen is Col1A1 and the extracellular vesicles do not contain the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2.

[0007] In some cases, the plurality of extracellular vesicles comprises an average of at least one copy of exogenous extracellular matrix mRNA per 450 EVs. In some cases, the plurality of extracellular vesicles comprises an average of at least one copy of exogenous extracellular matrix mRNA per 200 EVs. In some cases, the plurality of extracellular vesicles comprises an average of at least one copy of exogenous extracellular matrix mRNA per about 0.1-100 EVs. In some cases, the plurality of extracellular vesicles comprises an average of at least 1 copy of exogenous extracellular matrix mRNA per 2000 EVs, per 1000 EVs, per 500 EVs, per 400 EVs, per 300 EVs, per 200 EVs, per 150 EVs, per 100 EVs, per 90 EVs, per 80 EVs, per 70 EVs, per 60 EVs, 50 EVs, per 40 EVs, per 30 EVs, per 20 EVs, per 15 EVs, per 10 EVs, per 5 EVs, per 2 EVs, or per 1 EV. In some cases, the plurality of extracellular vesicles comprises an average of at least 1, 2, 5, 10, 50, 100, 200, 300, 500, or 1000 copies of exogenous extracellular matrix mRNA per EV. In some cases, the plurality of extracellular vesicles comprises an average of about 1-10 copies of exogenous extracellular matrix mRNA per EV. In some cases, the plurality of extracellular vesicles comprises an average of about 1 copy of exogenous extracellular matrix mRNA per EV. In some cases, the plurality of extracellular vesicles comprises an average of at least 1 copy of exogenous extracellular matrix mRNA per 30 EVs.

[0008] In some cases, the plurality of extracellular vesicles are produced by transfecting cells with a plasmid encoding an extracellular matrix protein. In some cases, transfecting the cells is performed by cell nanoporation. In some cases, transfecting the cells is performed by electroporation. In some cases, the cells are human cells. In some cases, the cells are fibroblasts selected from the group consisting of dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, and neonatal human dermal fibroblasts. In some cases, the cells are adherent cells. In some cases, the cells are human dermal fibroblasts (e.g., neonatal human dermal fibroblasts).

[0009] In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the plurality of extracellular vesicles comprises at least one exosome. In some cases, the plurality of extracellular vesicles comprises at least one apoptotic body. In some cases, the plurality of extracellular vesicles comprises at least one microvesicle. In some cases, the plurality of extracellular vesicles comprises a mixture of at least two of exosomes, microvesicles, or apoptotic bodies.

[0010] In some cases, the plurality of extracellular vesicles are formulated for injection via intravenous route, intramuscular route, subcutaneous route, or any combination thereof.In some cases, the formulation comprises one or more stabilizers and / or one or more preservatives.In some cases, the formulation comprises one or more DNA and / or RNase inhibitors.In some cases, the formulation comprises one or more DNAse, DNAse inhibitor, RNAse and / or RNAse inhibitor.

[0011] In some cases, multiple extracellular vesicles are present, at least 1 x 10 6 , 5×10 6 , 1×107 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 13 In some cases, the number of extracellular vesicles is 1 x 10 14 In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 9 extracellular vesicles and up to 1 × 10 20 In some cases, the number of extracellular vesicles may be up to 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 1 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 6 of extracellular vesicles and up to 1 × 10 30In some cases, the plurality of extracellular vesicles comprises at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of extracellular matrix mRNA. In some cases, the plurality of extracellular vesicles comprises at most 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of extracellular matrix mRNA. In some cases, the plurality of extracellular vesicles comprises at least 1 ng and at most 20 μg of extracellular matrix mRNA. In some cases, the plurality of extracellular vesicles comprises at least 5 ng and at most 30 μg of extracellular matrix mRNA.

[0012] In some cases, the plurality of extracellular vesicles includes extracellular vesicles comprising a targeting polypeptide. In some cases, the targeting polypeptide targets dermal cells. In some cases, the targeting polypeptide targets aminopeptidase N, CD26 / DPP4, fibroblast activation protein alpha, or any combination thereof. In some cases, the plurality of extracellular vesicles does not contain one or more of the following miRNAs: hsa-miR-29c-3p, hsa-miR-29a-3p, hsa-miR-378a-3p, hsa-miR-125b-5p, hsa-miR-23a-3p, hsa-miR-449a, hsa-miR-196a-5p, hsa-miR-744-5p, hsa-miR-223-3p, hsa-miR-23a-3p, hsa-miR-133a-3p, hsa-miR-223-3p, hsa-miR-5011-5p, hsa-miR-325, or hsa-miR-199b-5p.

[0013] In some cases, the plurality of extracellular vesicles comprises a size distribution having a peak at about 50 to 200 nm in diameter. In some cases, the plurality of extracellular vesicles comprises a size distribution having a peak at about 100 nm in diameter. In some cases, the plurality of extracellular vesicles comprises a size distribution having a peak at about 75 to 130 nm in diameter. In some cases, the extracellular vesicles have a diameter of more than 20 nm, 30 nm, 50 nm, 75 nm, or 100 nm. In some cases, at least 90% of the extracellular vesicles have a diameter of more than 20 nm, 30 nm, 50 nm, 75 nm, or 100 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 50 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 30 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 20 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 10 nm. In some cases, the extracellular vesicles do not include extracellular vesicles less than 5 nm in diameter.

[0014] In some cases, the exogenous extracellular matrix mRNA is present at a level at least two times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level at least three times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 75 times, at least 100 times, at least 500 times, at least 1000 times, at least 1500 times, or at least 2000 times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level about 3000 times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level about 2000 times higher than the level of exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the plurality of extracellular vesicles expresses a higher level of at least one of the following markers: CD9, CD63, TSG101, or ARF6, compared to a comparable number of naturally occurring extracellular vesicles.

[0015] In some embodiments, extracellular vesicles containing exogenous extracellular matrix messenger RNA (mRNA) are described herein. In some cases, the exogenous extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is type I collagen alpha 1 chain (Col1A1). In some cases, the exogenous extracellular matrix mRNA is mRNA encoding a pro-alpha 1 (I) chain. In some cases, the collagen is Col1A1, and the extracellular vesicles do not contain the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2. In some cases, the extracellular vesicles comprise extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the extracellular vesicles are exosomes. In some cases, the extracellular vesicles are apoptotic bodies. In some cases, the extracellular vesicles are microvesicles.

[0016] In some embodiments, the present disclosure provides a container comprising a plurality of extracellular vesicles according to any one of the preceding claims, wherein the container further comprises cells. In some cases, the cells comprise human cells, human fibrocytes, fibroblasts, dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, neonatal human dermal fibroblasts, or any combination thereof. In some cases, the plurality of extracellular vesicles are present in the container at a ratio of at least 1,000, 2,000, 5,000, 10,000, or 12,000 extracellular vesicles per cell. In some cases, the plurality of extracellular vesicles are present in the container at a ratio of no more than 1,000, 2,000, 5,000, 10,000, 12,000, 15,000, 20,000, 25,000, 50,000, 75,000, 90,000, or 100,000 extracellular vesicles per cell.

[0017] In some embodiments, needles comprising a plurality of extracellular vesicles disclosed herein are described herein. In some cases, the needles are microneedles. In some cases, the needles are solid. In some cases, the needles are hydrogel needles. In some cases, at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the needles comprise hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid. In some cases, the hydrogel comprises at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel contains up to 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid. In some cases, the hydrogel contains about 15% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0018] In some aspects, described herein is a syringe containing a plurality of extracellular vesicles disclosed herein. In some cases, the plurality of extracellular vesicles are suspended in hyaluronic acid within the syringe.

[0019] In some embodiments, hydrogel microneedles containing a plurality of extracellular vesicles are described herein. In some cases, the hydrogel comprises at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel comprises at least 5%, 7%, 10%, 15%, or 20% hyaluronic acid. In some cases, the hydrogel comprises 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or less hyaluronic acid. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid. In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the plurality of extracellular vesicles comprises at least one exosome. In some cases, the plurality of extracellular vesicles comprises at least one apoptotic body. In some cases, the plurality of extracellular vesicles comprises at least one microvesicle. In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30In some cases, the number of extracellular vesicles is 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , 1×10 30 , 1×10 30 , 1×10 40 , 1×10 50 , 1×10 60 , 1×10 70 , 1×10 80 , 1×10 90 , or 1 × 10 100 In some cases, the number of extracellular vesicles is about 1 x 10 6 ~1×10 30 In some cases, the plurality of extracellular vesicles comprises about 1 x 10 10 ~1×10 14 In some cases, the plurality of extracellular vesicles comprises about 1 x 10 13 ~1×10 14 Contains extracellular vesicles.

[0020] In some cases, the hydrogel microneedles have a length of less than 2 mm. In some cases, the hydrogel microneedles have a length of less than 1 mm. In some cases, the hydrogel microneedles have a length of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 μm. In some cases, the hydrogel microneedles have a length of 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, 500 μm or less, 600 μm or less, 700 μm or less, 800 μm or less, 900 μm or less, 1000 μm or less, 1100 μm or less, 1200 μm or less, 1300 μm or less, 1400 μm or less, 1500 μm or less, 1600 μm or less, 1700 μm or less, 1800 μm or less, 1900 μm or less, or 2000 μm or less. In other cases, the hydrogel microneedles have a length of 100 μm to 3000 μm, 500 μm to 2500 μm, 1000 μm to 2500 μm, or 1000 μm to 2000 μm. In certain embodiments, the hydrogel microneedles have a length of about 1000 μm. In certain embodiments, the hydrogel microneedles have a length of about 2000 μm.

[0021] In some cases, the hydrogel microneedles have a base with a diameter of 10,000 μm, 9,000 μm, 8,000 μm, 7,000 μm, 6,000 μm, 5,000 μm, 4,000 μm, 3,000 μm, 2,000 μm, 1,800 μm, 1,500 μm, 1,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, or 100 μm or less. In some cases, the hydrogel microneedles have a base with a diameter greater than 10,000 μm, 9,000 μm, 8,000 μm, 7,000 μm, 6,000 μm, 5,000 μm, 4,000 μm, 3,000 μm, 2,000 μm, 1,800 μm, 1,500 μm, 1,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, or 100 μm. In some cases, the hydrogel microneedles contain extracellular vesicles loaded with one or more mRNA cargos.

[0022] In some embodiments, needles comprising a plurality of extracellular vesicles are described herein, wherein the extracellular vesicles comprise at least one extracellular matrix messenger RNA (mRNA). In some cases, the extracellular matrix mRNA encodes a collagen. In some cases, the extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is the alpha 1 chain of type I collagen (Col1A1). In some cases, the collagen is Col1A1 and the extracellular vesicles do not contain the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2. In some cases, the extracellular matrix mRNA is mRNA encoding the pro-alpha 1 (I) chain.

[0023] In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the plurality of extracellular vesicles comprises at least one exosome. In some cases, the plurality of extracellular vesicles comprises at least one apoptotic body. In some cases, the plurality of extracellular vesicles comprises at least one microvesicle. In some cases, the plurality of extracellular vesicles comprises a mixture of any two of exosomes, microvesicles, or apoptotic bodies.

[0024] In some cases, the needle is a microneedle. In some cases, the needle is solid. In some cases, the needle is a hydrogel needle, and optionally, at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the needle comprises a hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid. In some cases, the hydrogel comprises at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel comprises at least 5% hyaluronic acid. In some cases, the hydrogel contains 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or less hyaluronic acid. In some cases, the hydrogel contains about 15% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0025] In some cases, the needle has a length of less than 2 mm. In some cases, the needle has a length of less than 1 mm. In some cases, the needle has a length of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 μm. In some cases, the needle has a length of 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, 500 μm or less, 600 μm or less, 700 μm or less, 800 μm or less, 900 μm or less, 1000 μm or less, 1100 μm or less, 1200 μm or less, 1300 μm or less, 1400 μm or less, 1500 μm or less, 1600 μm or less, 1700 μm or less, 1800 μm or less, 1900 μm or less, or 2000 μm or less. In other cases, the needle has a length of 100 μm to 3000 μm, 500 μm to 2500 μm, 1000 μm to 2500 μm, or 1000 μm to 2000 μm. In certain embodiments, the needle has a length of about 1000 μm. In certain embodiments, the needle has a length of about 2000 μm.

[0026] In some cases, the needle has a base with a diameter of 10,000 μm, 9,000 μm, 8,000 μm, 7,000 μm, 6,000 μm, 5,000 μm, 4,000 μm, 3,000 μm, 2,000 μm, 1,800 μm, 1,500 μm, 1,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, or 100 μm or less. In some cases, the needle has a base with a diameter greater than 10,000 μm, 9,000 μm, 8,000 μm, 7,000 μm, 6,000 μm, 5,000 μm, 4,000 μm, 3,000 μm, 2,000 μm, 1,800 μm, 1,500 μm, 1,000 μm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, 400 μm, 300 μm, 200 μm, or 100 μm.

[0027] In some embodiments, the present disclosure provides a method for treating a skin condition in a subject, comprising administering at least one extracellular matrix mRNA to a subject in need of such treatment, thereby treating the skin condition. In some cases, the exogenous extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is the alpha 1 chain of type I collagen (Col1A1). In some cases, the collagen is Col1A1 but not the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2. In some cases, the exogenous extracellular matrix mRNA is mRNA encoding the pro-alpha 1 (I) chain.

[0028] In some cases, the skin condition is a skin lesion. In some cases, the skin lesion is caused by aging or sun damage. In some cases, the skin condition is a wound.

[0029] In some cases, administering comprises administering to the subject at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of extracellular matrix mRNA. In some cases, administering comprises administering to the subject no more than 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, or 500 μg of extracellular matrix mRNA. In some cases, administering comprises administering to the subject between 1 ng and 20 μg of extracellular matrix mRNA. In some cases, administering comprises administering 1 ng to 10 μg of extracellular matrix mRNA to the subject. In some cases, administering comprises administering 10 ng to 10 μg, 10 ng to 1 μg, 50 ng to 1 μg, 100 ng to 1 μg, or 500 ng to 1 μg of extracellular matrix mRNA to the subject. In some cases, administering comprises administering about 50 ng of extracellular matrix mRNA to the subject.

[0030] In some cases, the extracellular matrix mRNA is encapsulated in at least one extracellular vesicle (EV). In some cases, the EV does not contain one or more of the following miRNAs: hsa-miR-29c-3p, hsa-miR-29a-3p, hsa-miR-378a-3p, hsa-miR-125b-5p, hsa-miR-23a-3p, hsa-miR-449a, hsa-miR-196a-5p, hsa-miR-744-5p, hsa-miR-223-3p, hsa-miR-23a-3p, hsa-miR-133a-3p, hsa-miR-223-3p, hsa-miR-5011-5p, hsa-miR-325, or hsa-miR-199b-5p.

[0031] In some cases, at least one EV is an exosome. In some cases, at least one EV is an apoptotic body or a microvesicle. In some cases, at least one EV comprises a mixture of at least two of exosomes, microvesicles and / or apoptotic bodies.

[0032] In some cases, treating skin damage results in at least a 10% reduction in the appearance of wrinkles. In some cases, at least a 10% reduction in the appearance of wrinkles occurs within 7 to 14 days after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject. In some cases, treating skin damage results in at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the appearance of wrinkles. In some cases, at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the appearance of wrinkles occurs within 7 to 14 days after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject. In some cases, treating skin damage results in at least a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the total number of wrinkles. In some cases, treating skin damage results in a reduction of about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in the total number of wrinkles. In some cases, treating skin damage results in a reduction of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in the total wrinkle area. In some cases, treating skin damage results in a reduction of not more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in the total wrinkle area. In some cases, treating skin damage results in a reduction of about 70%, 80%, or 90% in the total wrinkle area. In some cases, the wrinkle area is measured by photomicrography of the treated area of ​​skin and quantified by photoanalysis software.

[0033] In some cases, treating the skin condition includes treating skin damage, and treating the skin damage results in a reduction in skin damage that persists for at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days after treating the skin condition. In some cases, the reduction in skin damage is one or more of a reduction in total wrinkle number, a reduction in total wrinkle area, a reduction in the appearance of wrinkles, or any combination thereof.

[0034] In some cases, the administration is by subcutaneous injection. In some cases, the subcutaneous injection is performed at or near the site of a skin injury or wound. In some cases, the subcutaneous injection is performed using a needle with a gauge of at least 23s, 23, 22s, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10. In some cases, the subcutaneous injection is performed using a needle with a gauge of 34, 33, 32, 31, 30, 29, 28, 27, 26s, 26, 25s, 25, 24, 23s, 23, 22s, 22, 21, or 20 or less. In some cases, the subcutaneous injection is performed using a needle having a gauge of 34, 33, 32, 31, 30, 29, 28, 27, 26s, 26, 25s, 25, 24, 23s, 23, 22s, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10. In some cases, the subcutaneous injection is performed using a 28 gauge needle.

[0035] In some cases, administering comprises administering at least 1 x 10 extracellular vesicles to a subject in need thereof. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×1015 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, administering comprises administering 1 x 10 extracellular vesicles to a subject in need thereof, at least some of the extracellular vesicles comprising at least one extracellular matrix mRNA. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, administering comprises administering no more than about 1 x 10 extracellular vesicles to a subject in need thereof, at least some of the extracellular vesicles comprising at least one extracellular matrix mRNA. 7 ~1×10 17 , 1×10 8 ~1×10 16 , 1×10 9 ~1×10 15 In some cases, administering comprises administering about 1 x 10 extracellular vesicles to a subject in need thereof, at least some of the extracellular vesicles comprising at least one extracellular matrix mRNA. 10 ~1×10 14 The method includes administering extracellular vesicles, at least some of which contain at least one extracellular matrix mRNA.

[0036] In some cases, the extracellular vesicles are administered in multiple doses or as a single dose. In some cases, the extracellular vesicles are administered to the subject at intervals of at least once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks. In some cases, the extracellular vesicles are administered to the subject at most once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks. In some cases, the administration is performed once, and at most 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 extracellular vesicles are administered to a subject. In some cases, the administration is a single dose, and the dose is at least 10, 1 x 10, or 1 x 10. 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×1011 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 or 1×10 30 extracellular vesicles are administered to a subject. In some cases, multiple administrations are performed, each administration containing up to 1 x 10 extracellular vesicles. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 extracellular vesicles are administered to a subject. In some cases, the administration is multiple times, each time at least 10, 1 x 10 2 , 5×10 2 , 1×10 3 , 5×10 3 , 1×10 4 , 5×10 4 , 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×1015 , 1×10 16 , 1×10 20 , 1×10 25 or 1×10 30 Extracellular vesicles are administered to a subject. In some cases, the extracellular vesicles are administered in at least one dose containing at least 1,000 extracellular vesicles, and at least a portion of the extracellular vesicles contain at least one extracellular matrix mRNA. In some cases, administering comprises administering at least one extracellular matrix mRNA to a tissue of a subject.

[0037] In some cases, the tissue is subcutaneous tissue. In some cases, the tissue is dermis. In some cases, the tissue is epidermis. In some cases, the tissue is gum. In some cases, the tissue is lip.

[0038] In some cases, within 72 hours of administering at least one extracellular vesicle containing extracellular matrix mRNA to a subject, the tissue has an extracellular matrix protein concentration of at least 6000 pg / ml. In some cases, after administering the extracellular matrix mRNA to the subject's tissue, the subject's tissue has an extracellular matrix protein concentration of at least 200, 300, 500, 750, 1000, 2000, 3000, 4000, or 5000 pg / ml. In some cases, after administration of extracellular matrix mRNA to a tissue of a subject, the tissue of the subject has an extracellular matrix protein concentration of 200, 300, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, or 18000 pg / ml or less. In some cases, after administration of extracellular vesicles containing extracellular matrix mRNA to a tissue of a subject, the tissue of the subject has an extracellular matrix protein concentration of at least 200, 300, 500, 750, 1000, 2000, 3000, 4000, or 5000 pg / ml. In some cases, within 72 hours after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject, the tissue has an extracellular matrix protein concentration of at least 200, 300, 500, 750, 1000, 2000, 3000, 4000, or 5000 pg / ml. In some cases, within 72 hours after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject, the tissue has an extracellular matrix protein concentration of 200, 300, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, or 18000 pg / ml or less. In some cases, the tissue has a peak concentration of the extracellular matrix protein within about four days after administering to the subject at least one extracellular vesicle comprising extracellular matrix mRNA.

[0039] In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated at least every day, every 3 days, every 5 days, every 7 days, every 10 days, every 20 days, every 30 days, every 40 days, or every 50 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 10 days, every 20 days, every 30 days, every 40 days, or every 50 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 30 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 60 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 90 days.

[0040] In some cases, the method further includes producing extracellular vesicles containing at least one extracellular matrix mRNA by (a) introducing a vector or plasmid corresponding to the extracellular matrix mRNA into donor cells via transfection, (b) culturing the donor cells in culture medium for a sufficient time to produce extracellular vesicles encapsulating at least one extracellular matrix mRNA transcribed from the vector or plasmid, and (c) recovering the extracellular vesicles from the culture medium. In some cases, in step (c), the recovery of the extracellular vesicles is performed about 8 to 24 hours after transfection. In some cases, the extracellular vesicles are present at a ratio of at least 1,000, 2,000, 5,000, 10,000, or 12,000 extracellular vesicles per donor cell. In some cases, the extracellular vesicles are present at a ratio of no more than 1,000, 2,000, 5,000, 10,000, 12,000, 15,000, 17,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 extracellular vesicles per donor cell. In some cases, the donor cells comprise human cells, human fibrocytes, fibroblasts, dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, neonatal human dermal fibroblasts, or any combination thereof.

[0041] In some embodiments, methods for producing extracellular vesicles containing extracellular matrix mRNA are described herein, including (a) introducing a vector or plasmid corresponding to an extracellular matrix mRNA into donor cells via transfection; (b) culturing the donor cells in a culture medium for a sufficient period of time to produce extracellular vesicles encapsulating at least one extracellular matrix mRNA transcribed from the vector or plasmid; and (c) recovering the extracellular vesicles from the culture medium. In some cases, recovery of the extracellular vesicles in step (c) is performed approximately 8 to 24 hours after transfection. In some cases, the extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid are present at a level of at least 1,000, 2,000, 5,000, 10,000, or 12,000 extracellular vesicles per donor cell. In some cases, the extracellular vesicles are present at a ratio of no more than 1,000, 2,000, 5,000, 10,000, 12,000, 15,000, 17,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, or 50,000 extracellular vesicles per donor cell. In some cases, the donor cells comprise human cells, human fibrocytes, fibroblasts, dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, neonatal human dermal fibroblasts, or any combination thereof.

[0042] In some aspects, described herein are microneedle devices, the microneedle device comprising a substrate and a plurality of microneedles, the plurality of microneedles protruding from the substrate, and at least one microneedle of the plurality of microneedles comprising at least one extracellular vesicle (EV).

[0043] In some cases, the density of the microneedles on the substrate is such that 2 In some cases, the density of the microneedles on the substrate can range from 0.1 microneedles to 100 microneedles per mm of substrate. 2In some cases, the density of the microneedles on the substrate is at least 0.3 microneedles per mm of substrate. 2 In some cases, the plurality of microneedles includes about 10 to 1000 microneedles, and the area of ​​the substrate is about 20 to 1000 mm. 2 In some cases, the plurality of microneedles includes about 100 microneedles and the area of ​​the substrate is about 169 mm 2 is.

[0044] In some cases, the plurality of microneedles is arranged in at least two rows, with at least two microneedles in each row, and in some cases, the plurality of microneedles is arranged in ten rows, with ten microneedles in each row.

[0045] In some cases, at least one microneedle of the plurality of microneedles comprises a hydrogel, in some cases the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof.

[0046] In some cases, at least one microneedle of the plurality of microneedles is conical. In some cases, at least one microneedle of the plurality of microneedles has a tapered shape.

[0047] In some cases, at least one microneedle of the plurality of microneedles includes a base having a diameter of less than 1000 μm. In some cases, at least one microneedle of the plurality of microneedles includes a base having a diameter of about 100 μm to about 800 μm. In some cases, at least one microneedle of the plurality of microneedles includes a base having a diameter of about 400 μm.

[0048] In some cases, at least one microneedle of the plurality of microneedles has a length of at least 100 μm. In some cases, at least one microneedle of the plurality of microneedles has a length of between 100 μm and 3000 μm. In some cases, at least one microneedle of the plurality of microneedles has a length of about 1000 μm. In some cases, at least one microneedle of the plurality of microneedles has a length of about 2000 μm.

[0049] In some cases, the center-to-center distance between the microneedles of the plurality of microneedles is at least 100 μm. In some cases, the center-to-center distance between the microneedles of the plurality of microneedles is about 100-2000 μm. In some cases, the center-to-center distance between the microneedles of the plurality of microneedles is about 1000 μm.

[0050] In some cases, at least one microneedle of the plurality of microneedles has at least 3×10 5 In some cases, at least one microneedle of the plurality of microneedles contains about 3 x 10 EVs. 8 ~3×10 10 Includes EVs.

[0051] In some cases, EVs are suspended in a hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid.

[0052] In some cases, the EV comprises exogenous extracellular matrix mRNA. In some cases, the exogenous extracellular matrix mRNA comprises type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the exogenous extracellular matrix mRNA is mRNA encoding a proalpha 1(I) chain.

[0053] In some cases, the EV comprises exogenous VEGF mRNA. In some cases, the exogenous VEGF mRNA comprises VEGFA, VEGFB, VEGFC, VEGFD, PIGF, or any combination thereof.

[0054] In some embodiments, methods are described herein for administering extracellular vesicles (EVs) to a subject's tissue, the method comprising administering a needle, syringe, hydrogel needle, microneedle, or microneedle device disclosed herein to the subject's tissue. In some cases, the method comprises administering a microneedle device disclosed herein to the subject's tissue. In some cases, the microneedle device is removed after at least 5, 10, 15, 20, 25, or 30 minutes. In some cases, the microneedle device is removed after about 10, 15, 20, or 30 minutes.

[0055] In some cases, the EVs comprise at least one mRNA. In some cases, administering comprises administering at least 1 ng, 10 ng, 50 ng, 100 ng, 1 μg, 10 μg, or 20 μg of at least one mRNA to the tissue of the subject. In some cases, administering comprises administering 1 ng to 10 μg of extracellular matrix mRNA to the tissue of the subject. In some cases, administering comprises administering 1 ng to 20 μg of extracellular matrix mRNA to the tissue of the subject.

[0056] In some cases, the tissue is subcutaneous tissue. In some cases, the tissue is dermis. In some cases, the tissue is epidermis. In some cases, the tissue is gum. In some cases, the tissue is lip. In some cases, the subject is a mammal. In some cases, the subject is a human. In some cases, the subject is a rodent. In some cases, the subject is a monkey. In some cases, the subject is a rabbit.

[0057] In some cases, the administration is a single dose and is at least 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 In some cases, administration is a single dose, with a maximum of 1 x 10 extracellular vesicles. 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 17 , 1×10 18 , 1×1019 , 1×10 20 , or 1 × 10 30 In some cases, administration is a single dose, with a maximum of 1 x 10 extracellular vesicles. 13~14 In some cases, multiple administrations are performed, each time administering at least 1 x 10 extracellular vesicles. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 extracellular vesicles are administered. In some cases, multiple administrations are performed, each time up to 1 × 10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , or 1 × 10 30 extracellular vesicles are administered. In some cases, multiple administrations are performed, each time up to 1 × 10 13~14 In some cases, multiple doses are administered, up to a maximum of 1 x 10 extracellular vesicles within 6-8 weeks. 13~14 In some cases, multiple doses are administered over a period of time, up to 1 x 10 extracellular vesicles. 7 pieces, 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×1014 , 1×10 15 , 1×10 17 , 1×10 18 , 1×10 19 , or 1 × 10 20 pieces, 1×10 30 extracellular vesicles are administered.

[0058] In some cases, the method results in less EV breakage than administering EVs with a conventional microneedle or syringe. In some cases, the method results in less irritation at the administration site than administering EVs with a conventional microneedle or syringe. In some cases, the method results in more uniform distribution of EVs within tissue than administering EVs with a conventional microneedle or syringe. In some cases, the conventional microneedle is a solid microneedle or a hollow microneedle.

[0059] In some aspects, methods are described herein for treating a skin condition in a subject in need thereof, the method comprising applying to the subject a needle, syringe, hydrogel needle, microneedle, or microneedle device as disclosed herein. In some cases, the method comprises administering to the subject a microneedle as disclosed herein.

[0060] In some cases, the skin condition is a skin lesion. In some cases, the skin lesion is caused by aging or sun damage. In some cases, the skin condition is a wound.

[0061] In some cases, administering includes administering at least 0.1 ng, 1 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, or 10 μg of extracellular matrix mRNA. In some cases, administering includes administering 0.1 ng, 1 ng, 5 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, or 900 μg or less of the extracellular matrix mRNA. In some cases, administering comprises administering about 0.1 ng to 50 μg, 1 ng to 50 μg, 1 ng to 30 μg, or 1 ng to 25 μg of extracellular matrix mRNA. In some cases, administering comprises administering about 1 ng to 20 μg of extracellular matrix mRNA.

[0062] In some cases, the methods result in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the appearance of wrinkles. In some cases, the methods result in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more collagen fibers. In some cases, the methods result in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater dermal thickness. In some cases, the methods result in long-term effects of at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, or at least 90 days.

[0063] In some cases, at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the needles, microneedles, or microneedles of the microneedle device comprise a hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid. In some cases, the hydrogel comprises at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel comprises at least 5% hyaluronic acid. In some cases, the hydrogel comprises about 15% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0064] In some cases, the administration is a single dose and is at least 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 In some cases, administration is a single dose, with a maximum of 1 x 10 extracellular vesicles. 7 , 1×10 8 , 1×10 9 , 1×1010 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , or 1 × 10 30 In some cases, administration is a single dose, with a maximum of 1 x 10 extracellular vesicles. 13~14 In some cases, multiple administrations are performed, each time administering at least 1 x 10 extracellular vesicles. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , or 1 × 10 14 extracellular vesicles are administered. In some cases, multiple administrations are performed, each time up to 1 × 10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , or 1 × 10 30 extracellular vesicles are administered. In some cases, multiple administrations are performed, each time up to 1 × 10 13~14 In some cases, multiple doses are administered, up to a maximum of 1 x 10 extracellular vesicles within 6-8 weeks. 13~14In some cases, multiple doses are administered over a period of time, up to 1 x 10 extracellular vesicles. 7 pieces, 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 17 , 1×10 18 , 1×10 19 , or 1 × 10 20 pieces, 1×10 30 extracellular vesicles are administered.

[0065] In some embodiments, methods of fabricating a microneedle device are described herein, the methods comprising: (a) mixing extracellular vesicles (EVs) with a first batch of a polymerizable solution; and (b) casting the mixture from (a) into a polydimethylsiloxane (PDMS) mold having at least one needle-like shape. In some cases, the mixing in (a) is performed under vacuum.

[0066] In some cases, the method further includes further concentrating the EVs at the tip of at least one needle-like shape of the PDMS mold. In some cases, the concentrating is by maintaining the PDMS mold at a temperature of at most 10° C. In some cases, the concentrating is by maintaining the PDMS mold at about 4° C. In some cases, the concentrating lasts for about 2 to about 6 hours.

[0067] In some cases, the method further includes adding a second batch of polymerizable solution onto the PDMS mold. In some cases, the polymerizable solution includes a hydrogel. In some cases, the hydrogel includes hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel includes hyaluronic acid. In some cases, the hydrogel includes at least 5% hyaluronic acid. In some cases, the hydrogel includes at least 5% hyaluronic acid. In some cases, the hydrogel includes about 15% hyaluronic acid. In some cases, the hydrogel includes at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel includes more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel includes more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel comprises more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0068] In some cases, the final ratio of EVs to polymerizable solution is at least 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:80, 1:90, or 1:100. In some cases, the final ratio of EVs to polymerizable solution is no greater than 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some cases, the final ratio of EVs to polymerizable solution is about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, or 1:40.

[0069] In some cases, the EV comprises exogenous extracellular matrix mRNA. In some cases, the exogenous extracellular matrix mRNA comprises type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the EV comprises exogenous VEGF mRNA. In some cases, the exogenous VEGF mRNA comprises VEGFA, VEGFB, VEGFC, VEGFD, PIGF, or any combination thereof.

[0070] In some embodiments, the present invention provides a method for producing a type I collagen heterodimer or heterotrimer in a tissue, the method comprising administering a plurality of extracellular vesicles disclosed herein to the tissue. In some cases, the heterodimer or heterotrimer comprises at least one type I collagen alpha chain (Col1A1) and at least one type I collagen alpha chain (Col1A2), wherein Col1A1 is exogenously delivered by a plurality of extracellular vesicles disclosed herein. In some cases, Col1A2 is endogenous to the tissue.

[0071] In some embodiments, the present invention provides a plurality of extracellular vesicles, each containing at least one copy of exogenous VEGF mRNA per 400 extracellular vesicles on average.In some cases, the exogenous VEGF mRNA is selected from the group consisting of VEGFA, VEGFB, VEGFC, VEGFD, PIGF, and any combination thereof.In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, or any combination thereof.In some cases, the plurality of extracellular vesicles comprises extracellular vesicles containing targeting polypeptides.

[0072] In some cases, the plurality of extracellular vesicles comprises at least one copy of exogenous VEGF mRNA per up to 1, 5, 10, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or 350 extracellular vesicles. In some cases, the plurality of extracellular vesicles comprises at least 1, 2, 5, 10, 20, 25, 30, 35, 30, 50, 60, 70, 90, or 100 copies of VEGF mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises at least 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 copies of VEGF mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises up to 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 copies of VEGF mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises at least one copy of exogenous VEGF mRNA per about 0.001-100, 0.01-100, or 0.01-50 extracellular vesicles. In some cases, the plurality of extracellular vesicles comprises at least one copy of exogenous VEGF mRNA per about 0.02-50 extracellular vesicles.

[0073] In some cases, multiple extracellular vesicles are present, at least 1 x 10 6 , 5×10 6 , 1×107 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the number of extracellular vesicles may be up to 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 Contains extracellular vesicles.

[0074] In some cases, the extracellular vesicles are formulated for intravenous injection, intramuscular injection, subcutaneous injection, or injection via a coronary catheter.

[0075] In some cases, the mixture includes at least two extracellular vesicles containing 1 to 6 copies of exogenous VEGF mRNA, and the mixture includes exosomes, microvesicles, and apoptotic bodies. In some cases, the mixture is formulated for injection via intravenous, intramuscular, or subcutaneous routes. In some cases, the mixture is formulated for injection via a coronary artery catheter. In some cases, the VEGF mRNA is present at a level at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 1500-fold, or at least 2000-fold higher than the level of VEGF mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the plurality of extracellular vesicles comprises at least 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, or 200 μg of VEGF mRNA. In some cases, the plurality of extracellular vesicles contains VEGF mRNA at or below 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg. In some cases, the plurality of extracellular vesicles contains between about 1 ng and 200 μg of VEGF mRNA.

[0076] In some embodiments, the present invention provides a method for treating blood flow disorder in an object, comprising administering at least one extracellular vesicle that comprises VEGF mRNA to the object, thereby treating blood flow disorder.In some cases, treating blood flow disorder causes at least 5% increase in revascularization.In some cases, at least 5% increase in revascularization occurs within 14 days after administering at least one extracellular vesicle that comprises VEGF mRNA.

[0077] In some cases, administering at least one extracellular vesicle comprising VEGF mRNA comprises administering at least one extracellular vesicle comprising VEGF mRNA to a subject via intravenous, intramuscular or subcutaneous injection, or via a coronary artery catheter.In some cases, intravenous, intramuscular or subcutaneous injection is performed using a needle with a gauge of at least 14 gauge.

[0078] In some cases, the blood flow disorder is ischemia. In some cases, at least one extracellular vesicle containing VEGF mRNA is administered in at least one dose. In some cases, at least one extracellular vesicle containing VEGF mRNA comprises at least 1,000 extracellular vesicles containing VEGF mRNA. In some cases, at least one extracellular vesicle containing VEGF mRNA is administered in at least two doses.

[0079] In some cases, the administration is at least 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 or 1×10 16 In some cases, the administration includes a single administration of 1 x 10 EVs. 5 , 1×10 6 , 1×107 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , 1×10 21 , 1×10 22 , 1×10 23 or 1×10 24 In some cases, the administration includes administering a single dose of about 1 x 10 EVs or less. 5 ~1×10 20 , 1×10 6 ~1×10 19 , 1×10 7 ~1×10 18 , 1×10 8 ~1×10 17 or 1×10 9 ~1×10 16 In some cases, the administration includes administering about 1 x 10 EVs. 10 ~1×10 16 In some cases, the administering comprises a single administration of at least 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, or 200 μg of VEGF mRNA. In some cases, administering comprises a single administration of VEGF mRNA of 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg or less.

[0080] In some cases, the administration is at least 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 or 1×10 16 In some cases, the administration may include administering 1 x 10 EVs multiple times and each time. 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , 1×10 21 , 1×10 22 , 1×10 23 or 1×10 24 In some cases, the administration may include administering up to about 1 x 10 EVs multiple times and each time. 5 ~1×10 20 , 1×10 6 ~1×10 19 , 1×10 7 ~1×10 18 , 1×10 8 ~1×10 17 or 1×10 9 ~1×10 16 In some cases, the administration includes administering about 1 x 10 EVs multiple times and each time. 10 ~1×10 16In some cases, the administering comprises administering at least 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, or 200 μg of VEGF mRNA multiple times and each time. In some cases, administering comprises administering no more than 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg of VEGF mRNA multiple times and each time.

[0081] In some cases, administering at least one extracellular vesicle comprising VEGF mRNA to a subject comprises administering at least one extracellular vesicle comprising VEGF mRNA to a tissue of the subject. In some cases, the administering is carried out using a microneedle loaded with extracellular vesicles comprising VEGF mRNA.

[0082] In some cases, the microneedles comprise a hydrogel, which in some cases comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof.

[0083] In some cases, the extracellular vesicles are administered to the subject at least once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks. In some cases, the extracellular vesicles are administered to the subject at most once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks.

[0084] In some embodiments, described herein are methods for producing extracellular vesicles containing VEGF mRNA, the methods comprising: (a) introducing a vector or plasmid encoding VEGF into donor cells via transfection; (b) incubating the donor cells for a period of time sufficient to produce extracellular vesicles encapsulating the VEGF mRNA transcribed from the vector or plasmid; and (c) collecting the extracellular vesicles encapsulating the VEGF mRNA transcribed from the vector or plasmid.

[0085] In some aspects, methods are described herein for treating a blood flow disorder in a subject in need thereof, the method comprising administering an extracellular vesicle disclosed herein. In some cases, the blood flow disorder is ischemia.

[0086] In some embodiments, the present disclosure provides a plurality of extracellular vesicles comprising an average of at least one copy of exogenous VEGF mRNA. In some embodiments, the exogenous VEGF mRNA is selected from the group consisting of VEGFA, VEGFB, VEGFC, VEGFD, PIGF, and any combination thereof. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles comprising a targeting polypeptide.

[0087] In some embodiments, a mixture of extracellular vesicles is described, the mixture comprising at least two extracellular vesicles containing 1 to 6 copies of exogenous VEGF mRNA, the mixture comprising exosomes, microvesicles, and apoptotic bodies. In some embodiments, the mixture is formulated for injection via intravenous, intramuscular, or subcutaneous routes. In some embodiments, the mixture is formulated for injection via a coronary artery catheter.

[0088] In some aspects, methods for treating a blood flow disorder in a subject are described herein, comprising administering at least one extracellular vesicle comprising VEGF mRNA to the subject, thereby treating the blood flow disorder. In some embodiments, treating the blood flow disorder results in at least a 5% increase in revascularization. In some embodiments, the at least 5% increase in revascularization occurs within 14 days of administering the at least one extracellular vesicle comprising VEGF mRNA. In some embodiments, administering the at least one extracellular vesicle comprising VEGF mRNA to the subject via intravenous, intramuscular, or subcutaneous injection, or via a coronary artery catheter. In some embodiments, the intravenous, intramuscular, or subcutaneous injection is performed using a needle having a gauge of at least 14. In some embodiments, the blood flow disorder is ischemia. In some embodiments, the at least one extracellular vesicle comprising VEGF mRNA is administered in at least one dose. In some embodiments, the dose comprises at least 1,000 extracellular vesicles comprising VEGF mRNA. In some embodiments, at least one extracellular vesicle comprising VEGF mRNA is administered in at least two doses. In some embodiments, administering at least one extracellular vesicle comprising VEGF mRNA to a subject comprises administering at least one extracellular vesicle comprising VEGF mRNA to a tissue of the subject.

[0089] In some aspects, a plurality of extracellular vesicles are described herein, each containing an average of at least one copy of exogenous extracellular matrix mRNA. In some embodiments, the exogenous extracellular matrix mRNA is selected from the group consisting of collagen, COL1, type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, type IX collagen, type XII collagen, type XIV collagen, type VIII collagen, type X collagen, type IV collagen, type VI collagen, type VII collagen, type XIII collagen, type XV collagen, type XVII collagen, and type XVIIII collagen. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles comprising a targeting polypeptide.

[0090] In some aspects, a mixture of extracellular vesicles is described, the mixture comprising at least two extracellular vesicles containing one to two copies of exogenous extracellular matrix mRNA, the mixture comprising exosomes, microvesicles, and apoptotic bodies. In some embodiments, the mixture is formulated for injection via intravenous, intramuscular, or subcutaneous routes.

[0091] In some aspects, methods for treating skin damage in a subject are described herein, the methods comprising administering at least one extracellular vesicle comprising extracellular matrix mRNA to the subject, thereby treating the skin damage in the subject. In some embodiments, treating the skin damage results in at least a 10% reduction in the appearance of wrinkles. In some embodiments, the at least 10% reduction in the appearance of wrinkles occurs within 14 days of administering the at least one extracellular vesicle comprising extracellular matrix mRNA. In some embodiments, administering the at least one extracellular vesicle comprising extracellular matrix mRNA comprises administering the at least one extracellular vesicle comprising extracellular matrix mRNA to the subject via subcutaneous injection. In some embodiments, the subcutaneous injection is performed using a needle having at least 14 gauge. In some embodiments, the skin damage is caused by aging or sun damage. In some embodiments, the at least one extracellular vesicle comprising extracellular matrix mRNA is administered in at least one dose. In some embodiments, the dose comprises at least 1,000 extracellular vesicles comprising extracellular matrix mRNA. In some embodiments, at least one extracellular vesicle comprising extracellular matrix mRNA is administered in at least two doses. In some embodiments, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject comprises administering at least one extracellular vesicle comprising VEGF mRNA to the subject's tissue. In some embodiments, within 72 hours after administering at least one extracellular vesicle comprising extracellular matrix mRNA to the subject, the tissue has an extracellular matrix protein concentration of at least 6000 pg / ml.

[0092] In some embodiments, described herein are methods for producing extracellular vesicles containing VEGF mRNA, the methods comprising introducing a vector or plasmid encoding VEGF into donor cells via transfection, incubating the donor cells for a sufficient time to produce extracellular vesicles encapsulating the VEGF mRNA transcribed from the vector or plasmid, and recovering the extracellular vesicles encapsulating the VEGF mRNA transcribed from the vector or plasmid.

[0093] In some embodiments, described herein are methods for producing extracellular vesicles containing extracellular matrix mRNA, the methods comprising introducing a vector or plasmid encoding an extracellular matrix protein into donor cells via transfection, incubating the donor cells for a sufficient time to produce extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid, and recovering the extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid.

[0094] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0095] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments. [Brief explanation of the drawings]

[0096] [Figure 1]Figure 1 illustrates cell nanoporation ("CNP," also referred to herein as cell nanoelectroporation, or "NEP"), which generates large quantities of extracellular vesicles (EVs) loaded with transcribed mRNA. Schematic of CNP-generated EVs for targeted nucleic acid delivery. An exemplary CNP system consists of a nanochannel array, with each channel measuring approximately 500 nm in diameter (top inset).

[0097] [Figure 2] FIG. 2 shows quantification of VEGF mRNA loading levels produced by cell nanoporation (“NEP”) or bulk electroporation (“BEP”).

[0098] [Figure 3] Figure 3 shows an in vitro study of cell nanoporation-generated exosomes for VEGF mRNA delivery. Figure 3A shows representative confocal microscopy images of VEGF staining in human fibroblasts 48 hours after in vitro delivery of VEGF mRNA-loaded EVs ("NEP"). Figure 3B shows qPCR of human fibroblasts treated with VEGF mRNA-loaded EVs for 24 or 48 hours.

[0099] [Figure 4-1] Figure 4 shows the in vivo therapeutic efficacy of VEGF mRNA-loaded CNP-generated exosomes in a hindlimb ischemia (HLI) model. Figure 4A shows in vivo imaging demonstrating increased hindlimb perfusion / revascularization after VEGF mRNA-loaded EV delivery to an HLI model. Figure 4B shows quantification of the mean perfusion value of the ischemic footpad relative to the mean perfusion value of the control footpad at various time points after VEGF mRNA-loaded EV injection. Figure 4C shows ELISA quantification of VEGF concentration in 0.20 g of ischemic hindlimb tissue 48 hours after VEGF mRNA-loaded EV injection. Figure 4D shows representative confocal microscopy images of VEGF staining in excised gastrocnemius muscle tissue 48 hours after VEGF mRNA-loaded EV injection. [Figure 4-2] Same as above.

[0100] [Figure 5] Figure 5 shows an in vitro study of CNP-generated exosomes for type I collagen alpha I (Col1) mRNA delivery. Figure 5A shows representative confocal microscopy images of Col1 staining in human fibroblasts 48 hours after Col1 mRNA-loaded EV in vitro delivery ("NEP"). Figure 5B shows qPCR of human fibroblasts treated for 24 or 48 hours with Col1 mRNA-loaded EVs produced by CNP ("NEP") and bulk electroporation ("BEP").

[0101] [Figure 6-1] Figure 6 shows the in vivo therapeutic efficacy of Col1 mRNA-loaded CNP-generated exosomes in a photoaged skin model. Figure 6A shows in vivo imaging demonstrating improved skin symptoms (e.g., increased anti-aging) after Col1 mRNA-loaded EV delivery to a skin photoaging model. Figure 6B shows in vivo imaging demonstrating improved skin symptoms (e.g., increased anti-aging) after Col1 mRNA-loaded EV delivery to a skin photoaging model. Figure 6C shows representative immunohistochemistry (IHC) staining of skin tissue after Col1 mRNA-loaded EV delivery to a skin photoaging model. Figure 6D shows representative confocal microscopy images of Col1 staining in skin tissue after Col1 mRNA-loaded EV delivery to a skin photoaging model. Figure 6E shows confocal microscopy images of IHC staining (left) and Col1 staining (right) of skin tissue after Col1 mRNA EV delivery to a skin photoaging model. Figure 6F shows representative Masson's Trichrome staining of skin tissue after Col1 mRNA-loaded EV delivery to a skin photoaging model. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0102] [Figure 7-1]Figure 7 shows the successful in vitro delivery of CNP-generated EVs loaded with COL1A1 mRNA and COL1A1 mRNA-containing EVs. Figure 7A shows the display of CNP-generated EVs for targeted nucleic acid delivery. Figure 7B shows the plasmid map of pCMV-COL1A1-GFP. Figure 7C shows the number of EVs per cell produced by untreated neonatal human dermal fibroblasts (nHDFs) using PBS buffer as a control; bulk electroporation (BEP); or cell nanoporation (CNP) using PBS buffer alone (n = 3 for each group; *P = 0.048 control vs. BEP; **P = 0.005 control vs. CNP; ##P = 0.002 BEP vs. CNP). Figure 7D shows the characteristics of exosomes by nanoparticle tracking analysis. Figure 7E shows Western blot results demonstrating the difference in the expression levels of EV membrane markers in the control and CNP-treated groups. Figure 7F shows the number of EVs per cell under different voltage conditions (0, 50, 100, or 150 V). Figure 7G shows the kinetic analysis of CNP-induced EV release at different time points (0, 4, 8, 12, 16, 20, and 24 h) (n = 3, **P = 0.0071 0 V vs. 100 V; **P = 0.0032 50 V vs. 100 V; NS, not significant). Figure 7H shows RT-qPCR of COL1A1 mRNA, revealing that exosomes produced by CNP contained significantly more transcribed mRNA than exosomes produced by BEP and the control (n = 3 for all groups, **P = 0.002 control vs. BEP; ***P < 0.001 control vs. CNP; ###P < 0.001 BEP vs. CNP). Figure 7I shows an electron gel image of total RNA extracted from the supernatant of 2 x 10 untreated neonatal human dermal fibroblasts (nHDFs) without fetal bovine serum, total RNA recovered from 2 x 10 nHDF supernatants after the CNP platform with COL1A1 plasmid, and 0.4 ng of synthetic COL1A1 mRNA. Figure 7J shows a schematic diagram of in vitro EV-loaded mRNA delivery and expression. Figure 7K shows the proliferation of nHDFs after treatment with different concentrations of CNP EVs for 0, 24, and 48 hours.Figure 7L shows a fluorescence image of serum-starved nHDFs treated with CNP-generated EVs containing COL1A1-GFP mRNA and protein translated from the delivered COL1A1-GFP mRNA. Scale bar, 100 μm. Figure 7M shows the fluorescence intensity of cells treated with COL1A1-EVs (n = 3 for all groups, ***P < 0.001 control vs. COL1A1 EVs). Figure 7N shows RT-qPCR demonstrating higher collagen mRNA transcription levels after in vitro delivery of COL1A1 mRNA from EVs (n = 3 for all groups, ***P < 0.001 control vs. COL1A1 EVs). Figure 7O shows Western blots demonstrating elevated COL1A1 protein in treated fibroblasts (n = 3 for all groups, **P = 0.001 control vs. COL1A1 EVs). Figure 7P shows the fold change in COL1A1 mRNA in the control and CNP-treated groups at 24 and 48 hours (n = 3, **P = 0.0071 control vs. CNP at 24 hours; ***P < 0.001 control vs. CNP at 48 hours). Figure 7Q shows procollagen I recovered from the supernatant and detected by ELISA (n = 3 for all groups, ***P < 0.001 control vs. COL1A1 EV). All data are from three independent experiments and are shown as mean ± sem. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 7-5] Same as above. [Figure 7-6] Same as above. [Figure 7-7] Same as above.

[0103] [Figure 8-1]Figure 8 shows the in vivo kinetics of COL1A1-EV mRNA delivery and protein formation in mouse skin. Figure 8A shows in situ hybridization of human COL1A1 mRNA by RNAscope after subcutaneous injection of COL1A1 EVs, measured at 0 h, 12 h, 24 h, 48 h, 96 h, 7 days, 10 days, and 14 days. Scale bar, 100 μm. Figure 8B shows quantification of RNAscope results by the average number of brown dots per cell. Figure 8C shows immunofluorescence of COL1A1-EV-derived protein over time by visualization of colocalized GFP and COL1A1 protein (RFP). Scale bar, 100 μm. Figure 8D shows fluorescence intensity quantification of COL1A1-GFP protein expression. Figure 8E shows quantification of GFP+ cells confirms a time-dependent decrease in COL1A1-EV-derived protein grafting over 30 days. All data are from three independent experiments and are shown as mean ± SEM. [Figure 8-2] Same as above. [Figure 8-3] Same as above.

[0104] [Figure 9-1]Figure 9 shows the reduction of dermal wrinkles by COL1A1-EV mRNA delivery in a UVB-irradiated mouse model of photoaging. Figure 9A shows a schematic diagram of the UVB-induced skin photoaging mouse model. Figure 9B shows a schematic diagram of five low-dose injections of COL1A1 EVs over time. Skin tissue was harvested on day 28. Figure 9C shows that wrinkle formation was tracked on days 0, 4, 7, 14, 21, and 28 after initial treatment with COL1A1 EVs, unloaded EVs from nHDF cell culture medium, topical 0.05% retinoic acid (RA), or saline (n = 4 for all groups). The sham group contained female nude mice not exposed to UV. Scale bar, 5 cm. Figure 9D shows the total number of dorsal skin wrinkles quantified with custom software (n = 4 for all groups; **P = 0.003 COL1A1-EV vs. saline on day 7; ***P < 0.001 COL1A1-EV vs. saline on days 14, 21, and 28; *P = 0.0189 COL1A1-EV vs. non-loaded EV on day 7; ***P < 0.001 COL1A1-EV vs. non-loaded EV on days 14, 21, and 28; †P = 0.034 RA vs. saline on day 21; ††P = 0.003 RA vs. saline on day 28; #P = 0.014 non-loaded EV vs. saline on day 21; ##P = 0.002 non-loaded EV vs. saline on day 28). Figure 9E shows quantification of wrinkle area of ​​dorsal skin (n = 4 for all groups; *P = 0.023 COL1A1-EV vs. saline on day 7; *P = 0.017 COL1A1-EV vs. saline on day 14; ***P < 0.001 COL1A1-EV vs. saline on days 21 and 28; *P = 0.0205 COL1A1-EV vs. non-loaded EV on day 7; **P = 0.0011 COL1A1-EV vs. non-loaded EV on day 14; ***P < 0.001 COL1A1-EV vs. non-loaded EV on days 21 and 28; †P = 0.039 RA vs. saline on day 28; #P = 0.027 non-loaded EV vs. saline on day 28). Figure 9F shows microscopic observation of dorsal skin and skin replicas. Scale bar, 5 cm.Figure 9G shows the average wrinkle length analyzed in skin replicas (n=4 for all groups, ##P=0.002 unloaded EV vs. saline; ***P<0.001 COL1A1-EV vs. saline; ***P<0.001 COL1A1-EV vs. unloaded EV). Figure 9H shows the average wrinkle depth in skin replicas (n=4 for all groups, ***P<0.001 COL1A1-EV vs. saline; ##P=0.006 unloaded EV vs. saline; †P=0.022 RA vs. saline); ***P<0.001 COL1A1-EV vs. unloaded EV). Figure 9I shows wrinkles tracked on days 0, 4, 7, 14, 21, 28, 35, 42, 49, and 56 after initial treatment (COL1A1-EV, unloaded EV, 0.05% retinoic acid [RA], saline); (n = 4 for all groups). Scale bar, 5 cm. Female nude mice not exposed to UV irradiation comprised the sham group. Figure 9J shows total wrinkle area (n=4 for all groups; *P=0.012 COL1A1-EV vs. saline on day 7; ***P<0.001 COL1A1-EV vs. saline on days 14, 21, 28, and 35; *P=0.015 COL1A1-EV vs. saline on day 42; ††P=0.008 RA vs. saline on day 14; ††P=0.007 RA vs. saline on days 21 and 35; #P=0.012 unloaded EV vs. saline on day 14; #P=0.035 unloaded EV vs. saline on day 21; ##P=0.002 unloaded EV vs. saline on day 28). Figure 9K shows the number of wrinkles on the dorsal skin of mice (n=4 for all groups; **P=0.008 COL1A1-EV vs. saline on day 7; **P=0.004 COL1A1-EV vs. saline on day 21, **P=0.001 COL1A1-EV vs. saline on day 35; ***P<0.001 COL1A1-EV vs. saline on days 14, 28, 42, and 49; †P=0.025 RA vs. saline on day 21; ††P=0.007 RA vs. saline on day 28; #P=0.015 Unloaded EV vs. saline on day 35; ##P=0.004 Unloaded EV vs. saline on day 21, ##P=0.002 Unloaded EV vs. saline on day 28).Figure 9L shows immunofluorescence staining of GFP and COL1A1 (RFP) protein in the sham control group, saline control group, RA-treated group, unloaded EV-treated group, and COL1A1-EV-treated group. COL1A1-EV-treated mice exhibited GFP and COL1A1 protein grafts in the dermis and subcutaneous tissue 30 days after the start of treatment. Scale bar, 200 µm. Figure 9M shows the fluorescence intensity of COL1A1 (RFP) in all treatment groups (n = 3 for all groups; ***P < 0.001 COL1A1-EV vs. saline; †P = 0.022 RA vs. saline; #P = 0.038 unloaded EV vs. saline; ***P < 0.001 COL1A1-EV vs. unloaded EV). Figure 9N shows representative Masson's Trichrome staining of the epidermis, dermis, and subcutaneous tissue in all mouse groups. Scale bar, 300 µm. Figure 9O shows quantification of dermal thickness showing increased collagen fibers in the COL1A1-EV group (n=3 for all groups, ***P<0.001 COL1A1-EV vs. saline; †P=0.022 RA vs. saline; #P=0.018 unloaded EV vs. saline; ***P<0.001 COL1A1-EV vs. unloaded EV). [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 9-7] Same as above. [Figure 9-8] Same as above. [Figure 9-9] Same as above. [Figure 9-10] Same as above.

[0105] [Figure 10-1]Figure 10 shows the construction of a novel microneedle delivery platform to improve in vivo EV distribution and retention. Figure 10A shows a schematic diagram of microneedle fabrication. Figure 10B shows microscopic and scanning electron microscope images of a microneedle (MN) array. Scale bar, 500 μm. Figure 10C shows a schematic diagram of an HA MN patch evaluated with a tensile tester. Figure 10D shows the fracture force of HA+EV MNs containing 10%, 15%, or 20% HA. The dotted line represents the stress intensity of a microneedle inserted into the skin (n = 3 for all groups). Figure 10E shows hematoxylin and eosin (H&E)-stained sections of mouse skin showing the penetration of a single MN. Scale bar, 200 μm. Figure 10F shows the time course of an HA+EV MN tip pressed into the skin; the MN dissolved within 15 minutes of application. Scale bar, 200 μm. Skin recovery after HA+EV MN treatment shows minimal irritation. Scale bar, 5 cm. Figure 10G shows skin histology of Dil-labeled EVs, demonstrating high concentrations of EVs (red) accumulated in the subcutaneous tissue after dermal injection and well-distributed EVs delivered by MN. Scale bar, 100 μm. Figure 10H shows a schematic diagram of EV distribution after needle injection. Figure 10I shows a schematic diagram of EV distribution after MN injection. Figure 10J shows representative EV distribution analyzed by ImageJ software. Figure 10K shows fluorescent tracking of EVs injected by needle injection. EVs were labeled with Dil. Scale bar, 100 μm. Figure 10L shows fluorescent tracking of EVs injected by MN injection. EVs were labeled with Dil. Scale bar, 100 μm. Figure 10M shows a cryo-electron microscopy image. Figure 10N shows quantification of the number of disrupted EVs after injection with a syringe needle or / and MN (n = 3 for all groups; *P = 0.0228 needle injection vs. HA-MN). Scale bar, 100 nm. Figure 10O shows in vivo fluorescence images of nude mice treated with subcutaneous injection of Dil-labeled EVs or HA-MN patch delivery on days 1, 2, 4, 7, 10, and 14 after delivery (n = 3 for all groups). Figure 10P shows quantification of fluorescence intensity over the 14-day treatment period. Data are normalized to the fluorescence intensity on day 1. [Figure 10-2]Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above. [Figure 10-6] Same as above. [Figure 10-7] Same as above. [Figure 10-8] Same as above. [Figure 10-9] Same as above.

[0106] [Figure 11-1]Figure 11 shows that COL1A1-EV mRNA delivery via a custom microneedle patch improved the long-term treatment of dermal wrinkles and resulted in long-term protein replenishment in photoaged mouse skin. Figure 11A shows a schematic diagram of COL1A1 EV released by an HA microneedle and the in vivo transfection pattern. Figure 11B shows long-term (90-day) observations of four treatment groups after a single injection: (1) saline control, (2) COL1A1-EV delivered by a 28G syringe needle, (3) HA MN control, and (4) COL1A1-loaded HA+EV MN (n = 4 for all groups). Scale bar, 5 cm. Figure 11C shows quantification of the total number of wrinkles (n=4 for all groups; *P=0.014 HA+EV MN vs. saline on day 7; *P=0.038 HA+EV MN vs. saline on day 14; *P=0.012 HA+EV MN vs. saline on day 21; *P=0.039 HA+EV MN vs. saline on day 28; *P=0.031 HA+EV MN vs. saline on day 35; *P=0.03 HA+EV MN vs. saline on day 42; *P=0.022 HA+EV MN vs. saline on day 49; *P=0.031 HA+EV MN vs. saline on day 56; **P=0.008 HA+EV MN vs. saline on day 63; †P=0.047 HA MN vs. saline on day 14; #P=0.030 (Needle injection on day 7 vs. saline; #P=0.041 Needle injection on day 14 vs. saline). Figure 11D shows quantification of total dorsal skin wrinkle area (n=4 for all groups, *P=0.016 HA+EV MN on day 7 vs. saline; *P=0.038 HA+EV MN on day 14 vs. saline; *P=0.042 HA+EV MN on day 28 vs. saline; *P=0.026 HA+EV MN on day 42 vs. saline; *P=0.048 HA+EV MN on day 49 vs. saline; #P=0.033 Needle injection on day 7 vs. saline; #P=0.031 Needle injection on day 14 vs. saline; #P=0.042 Needle injection on day 21 vs. saline). Figure 11E. Microscopic observation of dorsal skin and skin replicas on day 30. FIG. 11F shows microscopic observation of the dorsal skin and skin replicas on day 60.Figure 11G shows microscopic observations of dorsal skin and skin replicas on day 90. Figure 11H shows quantification of mean wrinkle length (n=4 for all groups, ***P<0.001 HA+EV MN vs. saline on days 30 and 60; †††P<0.001 HA MN vs. saline on day 30; ###P<0.001 needle injection vs. saline on day 30). Figure 11I shows quantification of mean wrinkle depth from skin replicas (n=4 for all groups, ***P<0.001 HA+EV MN vs. saline on days 30 and 60; #P=0.049 needle injection vs. saline on day 30; not significant HA MN vs. saline on day 30). Figure 11J shows immunofluorescence staining for GFP and COL1A1 (RFP) demonstrating that GFP+COL1A1 protein grafts were present in the skin of mice administered COL1A1-EV via 28G needle injection and HA+EV MN for up to 30 days after delivery. Scale bar, 200 μm. Figure 11K shows that at day 60, only mice treated with HA+EV MN had long-term engraftment of GFP-positive COL1A1. Scale bar, 200 μm. Figure 11L shows that no evidence of GFP-positive collagen protein could be detected in any of the mice by day 90 after delivery. Scale bar, 200 μm. Figure 11M shows quantification of GFP and COL1A1 (RFP) colocalized fluorescent signals, demonstrating long-term engraftment of COL1A1-EV-derived collagen in the skin of mice given HA+EV MN versus mice given COL1A1-EV via 28G needle injection and the control group (n = 3 for all groups; ***P < 0.001 for HA+EV MN versus needle injection at day 60). Figure 11N shows IHC and Masson's trichrome staining at days 30, 60, and 90. Scale bar, 200 µm. Figure 11O shows quantification of dermal thickness by Masson's trichrome staining (n = 3 for all groups; *P = 0.017 for HA+EV MN versus saline at day 30; P = 0.034 for HA+EV MN versus saline at day 60). [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 11-5] Same as above. [Figure 11-6] Same as above. [Figure 11-7] Same as above. [Figure 11-8] Same as above. [Figure 11-9] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0107] Detailed Description While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used in practicing the disclosure. The following claims define the scope of the disclosure, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0108] overview The present disclosure provides extracellular vesicles that carry exogenous RNA cargo (e.g., messenger RNA (mRNA), extracellular matrix mRNA, collagen mRNA, Col1A mRNA, Col1A1 mRNA) and can be used to treat various skin conditions and other disorders, often with long-lasting effects. The present disclosure also provides extracellular vesicles that contain VEGF mRNA and can be used to treat disorders such as impaired blood flow. The present disclosure also provides needles and microneedles that can be used to deliver extracellular vesicles to a subject. The present disclosure also provides hydrogel needles (e.g., hydrogel microneedles) containing extracellular vesicles that carry various cargoes (e.g., DNA, RNA, therapeutic agents) or no exogenous cargo.

[0109] The present disclosure also relates to the design and production of one or more extracellular vesicles (e.g., exosomes, microvesicles, apoptotic bodies, or mixtures thereof) that express and / or carry cargo such as at least one extracellular matrix protein RNA (e.g., extracellular matrix (ECM) mRNA, collagen mRNA, Col1 mRNA, Col1A1 mRNA, ColIV mRNA, elastin mRNA) or vascular endothelial growth factor (VEGF) mRNA (e.g., VEGFA mRNA). In certain embodiments, the present disclosure provides extracellular vesicles (e.g., exosomes) containing exogenous collagen mRNA. In certain embodiments, the present disclosure provides extracellular vesicles (e.g., exosomes) containing exogenous VEGF mRNA. Extracellular vesicles can be designed to carry a payload, such as a therapeutic agent, to be delivered to targeted cells. In some cases, the therapeutic agent delivered by the extracellular vesicles may include a therapeutic compound (e.g., a therapeutic polynucleotide, a therapeutic DNA, a therapeutic RNA, a therapeutic mRNA, a therapeutic miRNA, a therapeutic tRNA, a therapeutic rRNA, a therapeutic siRNA, a therapeutic shRNA, a therapeutic SRP RNA, a therapeutic tmRNA, a therapeutic gRNA, or a therapeutic crRNA). In some cases, the therapeutic agent delivered by the extracellular vesicles may include a therapeutic non-coding polynucleotide (e.g., a non-coding RNA, a lncRNA, a piRNA, a snoRNA, a snRNA, an exRNA, or a scaRNA), a therapeutic polypeptide, a therapeutic compound, or an anti-cancer drug. In some cases, the extracellular vesicles may carry a non-therapeutic compound (e.g., a non-therapeutic polynucleotide).

[0110] The extracellular vesicles provided herein can be produced by several methods and approaches. One approach provided herein involves introducing at least one heterologous polynucleotide, such as a vector (e.g., a plasmid, DNA), into extracellular vesicle donor cells, where the at least one heterologous polynucleotide encodes extracellular matrix RNA (e.g., collagen) or VEGF RNA (e.g., VEGFA). Introduction of the vector (e.g., a DNA plasmid) can be via various methods, including transfection, calcium phosphate transfection, electroporation, nanoelectroporation, lipofection, cell nanoelectroporation, viral delivery, or other methods. In some cases, the extracellular vesicle donor cells are primary cells (e.g., primary adherent cells). In some cases, the extracellular vesicle donor cells are derived from cell lines (e.g., human cells, human fibroblast cell lines, human dermal fibroblast cell lines, neonatal human dermal fibroblast cell lines). In some cases, the extracellular vesicle donor cells are not genetically modified before nanoelectroporation. In some cases, the extracellular vesicle donor cells are genetically modified prior to nanoelectroporation. In some cases, the present disclosure provides methods for producing large numbers of exosomes containing large amounts of mRNA transcripts, even from cells that otherwise have low basal exosome secretion.

[0111] Described herein are methods for treating skin damage (e.g., caused by sun damage, aging, or other processes), comprising administering at least one extracellular vesicle to a subject. The methods provided herein are particularly useful for reversing, reducing, or alleviating the appearance of fine lines and / or wrinkles. In some cases, the methods provided herein reduce skin damage (e.g., wrinkles, fine lines, etc.) for a period of at least 30, 60, or 90 days, etc., after administration of the extracellular vesicles provided herein. In some cases, the methods provided herein can be used for wound healing. In some cases, the extracellular vesicles comprise at least one therapeutic polynucleotide (e.g., a therapeutic mRNA, miRNA, etc.). In some cases, the extracellular vesicles comprising the therapeutic polynucleotide can be obtained by nanoelectroporating at least one extracellular vesicle donor cell with at least a first vector (e.g., a plasmid), wherein the first vector encodes a therapeutic polypeptide. In some embodiments, the therapeutic polynucleotide comprises an extracellular matrix RNA (e.g., collagen). In some cases, the first vector can be expressed in extracellular vesicle donor cells to obtain therapeutic polynucleotides. In some embodiments, the extracellular vesicles released from the extracellular vesicle donor cells contain therapeutic polynucleotides. In some cases, the extracellular vesicles are collected and administered to the subject systemically. In some cases, the extracellular vesicles are collected and administered to the subject locally (for example, via subcutaneous injection).

[0112] Described herein are methods for treating blood flow disorders (e.g., ischemia), comprising administering at least one extracellular vesicle to a subject. In some cases, the extracellular vesicles comprise at least one therapeutic polynucleotide (e.g., therapeutic mRNA, miRNA, etc.). In some cases, the extracellular vesicles comprising the therapeutic polynucleotide can be obtained by nanoelectroporating at least one extracellular vesicle donor cell with at least a first vector (e.g., a plasmid), wherein the first vector encodes a therapeutic polypeptide. In some embodiments, the therapeutic polynucleotide comprises VEGF RNA (e.g., VEGFA). In some cases, the first vector can be expressed in the extracellular vesicle donor cell to obtain the therapeutic polynucleotide. In some embodiments, the extracellular vesicles released from the extracellular vesicle donor cell comprise the therapeutic polynucleotide. In some cases, the extracellular vesicles are collected and administered systemically to the subject. In some cases, the extracellular vesicles are collected and administered locally to the subject (e.g., via intramuscular injection).

[0113] Also described herein are hydrogel microneedles containing a plurality of extracellular vesicles.In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof.In some cases, the hydrogel comprises hyaluronic acid.For example, in some cases, the hydrogel comprises at least 5%, 10%, 15%, or 20% hyaluronic acid.

[0114] Also described herein is a needle containing a plurality of extracellular vesicles, wherein the extracellular vesicles contain at least one extracellular matrix mRNA or VEGF mRNA. Also described is a syringe containing a plurality of extracellular vesicles, wherein the extracellular vesicles contain at least one extracellular matrix mRNA or VEGF mRNA.

[0115] The present specification also describes a microneedle device.In some cases, the microneedle device comprises a substrate and a plurality of microneedles, wherein the plurality of microneedles protrude from the substrate, and at least one microneedle of the plurality of microneedles comprises at least one EV.The present specification also describes a method for manufacturing such a microneedle device. Extracellular vesicle donor cells

[0116] In some cases, extracellular vesicle donor cells that produce the extracellular vesicles described herein are described herein. Extracellular vesicle donor cells can be any cells that can be genetically modified or engineered to secrete extracellular vesicles at a level higher than the basal level of extracellular vesicle secretion, or to secrete extracellular vesicles containing exogenous nucleic acids (e.g., exogenous collagen RNA or VEGF RNA). Therefore, cells that secrete low or negligible basal levels of extracellular vesicles can also be extracellular vesicle donor cells. In some cases, extracellular vesicle donor cells can be nucleated cells. In some cases, extracellular vesicle donor cells can be autologous cells. In such cases, extracellular vesicle donor cells can be obtained from a subject, and then, following modification of the extracellular vesicle donor cells (e.g., introduction of a vector), the secreted extracellular vesicles are collected and then administered to the same subject. In some cases, the extracellular vesicle donor cells are allogeneic cells. In such cases, the extracellular vesicle donor cells are cells obtained from a source that is genetically different from the subject that will subsequently receive the extracellular vesicles secreted by the extracellular vesicle donor cells. Often, in the case of allogeneic extracellular vesicle donor cells, the extracellular vesicle donor cells are of the same species but genetically different from the subject that will subsequently receive the extracellular vesicles produced and secreted by the extracellular vesicle donor cells.

[0117] Extracellular vesicle donor cells can be any type of cell. In some cases, the extracellular vesicle donor cells are eukaryotic cells (e.g., mammalian cells, human cells, non-human mammalian cells, rodent cells, mouse cells, etc.). In some cases, the extracellular vesicle donor cells are cells from a cell line, stem cells, primary cells, or differentiated cells. In some embodiments, the extracellular vesicle donor cells are primary cells. In some cases, the extracellular vesicle donor cells are mouse embryonic fibroblasts (MEFs), human embryonic fibroblasts (HEFs), human dermal fibroblasts (HDFs), dendritic cells, mesenchymal stem cells, bone marrow-derived dendritic cells, bone marrow-derived stromal cells, adipose stromal cells, enucleated cells, neural stem cells, immature dendritic cells, or immune cells. In some cases, the extracellular vesicle donor cells are neonatal human primary dermal fibroblasts. In some cases, the neonatal human primary dermal fibroblasts are ATCC strain PCS-201-010. In some cases, the extracellular vesicle donor cells are primary dermal fibroblasts. In some cases, the primary dermal fibroblasts are ATCC strain PCS-201-012. The extracellular vesicle donor cells can be adherent cells. In some cases, the extracellular vesicle donor cells are adherent cells. In some cases, the extracellular vesicle donor cells are suspension cells.

[0118] In some cases, the extracellular vesicle donor cells comprise at least one heterologous polynucleotide. In some cases, the at least one heterologous polynucleotide is introduced into the extracellular vesicle donor cells by transfection. The at least one heterologous polynucleotide can be transfected into the extracellular vesicle donor cells by any one of the biological, chemical, or physical methods described herein, or by any other biological, chemical, or physical method. In some cases, the at least one heterologous polynucleotide is transfected into the extracellular vesicle donor cells by electroporation (e.g., nanoelectroporation). In some cases, the electroporation is microchannel electroporation or nanochannel electroporation. In some cases, the at least one heterologous polynucleotide is transfected into the extracellular vesicle donor cells by nanochannel electroporation. In some cases, the extracellular vesicle donor cells comprise genetically modified cells. Examples of genetically modified cells include induced pluripotent stem cells or cells genetically modified by nucleic acid-guided nucleases (e.g., CRISPR-Cas). In some cases, the extracellular vesicle donor cells are not genetically modified. For example, in some cases, the extracellular vesicle donor cells are not genetically modified before electroporation (e.g., nanoelectroporation). In some cases, the heterologous polynucleotide transfected into the extracellular vesicle donor cells is integrated into the chromosome of the extracellular vesicle donor cells. In some cases, the heterologous polynucleotide transfected into the extracellular vesicle donor cells is not integrated into the chromosome of the extracellular vesicle donor cells. In some cases, the extracellular vesicle donor cells are stably transfected with the heterologous polynucleotide. In some cases, the extracellular vesicle donor cells are transiently transfected with the heterologous polynucleotide. In some cases, the transfected extracellular vesicle donor cells are cells derived from a cell line. In some cases, at least one heterologous polynucleotide is a vector (e.g., a plasmid).

[0119] In some cases, extracellular vesicle donor cells can be electroporated with multiple vectors to produce and secrete extracellular vesicles. In some cases, extracellular vesicle donor cells can be nanoelectroporated with multiple vectors to produce and secrete extracellular vesicles. In some cases, the multiple vectors include at least a first vector, at least a second vector, or any additional vectors. In some cases, the first vector and the second vector can be nanoelectroporated into the extracellular vesicle donor cells simultaneously. In some cases, the first vector and the second vector can be nanoelectroporated into the extracellular vesicle donor cells at different times. In some cases, the time difference between nanoelectroporating the first vector and the second vector can be at least 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 5 hours, 12 hours, 1 day, 2 days, 5 days, 10 days, 30 days, or longer.

[0120] In some cases, the first vector can encode at least one therapeutic polynucleotide (e.g., ECM or VEGF mRNA). In some cases, the extracellular vesicle donor cells can transcribe the first vector to obtain the therapeutic polynucleotide when nanoelectroporated with the first vector. In some cases, the extracellular vesicle donor cells produce and secrete extracellular vesicles or exosomes containing the therapeutic polynucleotide encoded by the first vector.

[0121] In some cases, the second vector can encode a targeting polypeptide (e.g., a cell-specific targeting peptide that can increase the targeting and accumulation of extracellular vesicles in targeted cells). In some cases, when the second vector is nanoelectroporated, the extracellular vesicle donor cell can translate the second vector to obtain the targeting polypeptide. In some cases, the extracellular vesicle donor cell can produce extracellular vesicles or exosomes containing the targeting polypeptide. In some cases, the extracellular vesicle donor cell can secrete extracellular vesicles or exosomes containing the targeting polypeptide. In some cases, the extracellular vesicle donor cell can produce and secrete extracellular vesicles or exosomes containing the targeting peptide and the therapeutic polynucleotide encoded by the first vector.

[0122] In some cases, when the first vector is nanoelectroporated, the extracellular vesicle donor cells can transcribe or translate the first vector to obtain a therapeutic polynucleotide or therapeutic polypeptide. In some cases, the extracellular vesicle donor cells can produce and secrete extracellular vesicles or exosomes containing the therapeutic polynucleotide or therapeutic polypeptide encoded by the first vector. In some cases, the extracellular vesicle donor cells can produce and secrete extracellular vesicles containing a targeting peptide and the therapeutic polynucleotide or therapeutic polypeptide encoded by the first vector. In some cases, the therapeutic polynucleotide and the therapeutic polypeptide can be encapsulated in the same extracellular vesicles or exosomes. In some cases, the therapeutic polynucleotide and the therapeutic polypeptide can be encapsulated in different extracellular vesicles or exosomes.

[0123] In some cases, extracellular vesicle donor cells continuously produce and secrete extracellular vesicles at a steady or basal rate. Extracellular vesicle donor cells can be any cell type, including cells with a low or negligible basal rate or production and secretion of extracellular vesicles. For example, extracellular vesicle donor cells can be primary cells or non-cancerous cells that generally do not secrete or secrete a small amount of extracellular vesicles. In some cases, extracellular vesicle donor cells produce and secrete extracellular vesicles at a basal rate. In some cases, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate. For example, extracellular vesicle donor cells can be heat shocked or extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate. 2+By contacting the extracellular vesicle donor cells with ions, the extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate. In some cases, the extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by activating a stress response signaling pathway, such as the p53-TSAP6 signaling pathway. In some cases, the extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by electroporating at least one heterologous polynucleotide into the extracellular vesicle donor cells. In some cases, the extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by microchannel or nanochannel electroporation of at least one heterologous polynucleotide into the extracellular vesicle donor cells. In some cases, extracellular vesicle donor cells can be stimulated to produce and secrete extracellular vesicles at a rate faster than the basal rate by electroporating (e.g., nanochannel electroporation) at least one heterologous polynucleotide into the extracellular vesicle donor cells. In some cases, the extracellular vesicle donor cells stimulated by electroporation (e.g., nanochannel electroporation) can produce and secrete extracellular vesicles at a rate that is at least 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, 2 times, 5 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, 10,000 times, 50,000 times, 100,000 times, or more than the basal rate of the extracellular vesicle donor cells producing and secreting extracellular vesicles.In some cases, extracellular vesicle donor cells stimulated by nanochannel electroporation may produce and secrete extracellular vesicles at a rate that is at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 0.6-fold, 0.7-fold, 0.8-fold, 0.9-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, 50,000-fold, 100,000-fold, or more than the rate of extracellular vesicle donor cells stimulated by methods other than nanoelectroporation to produce and secrete extracellular vesicles.

[0124] In some cases, the heterologous polynucleotide transfected into the extracellular vesicle donor cell encodes at least one therapeutic agent described herein. In some cases, the therapeutic agent is a therapeutic polynucleotide. In some cases, the therapeutic agent is a therapeutic polypeptide. In some cases, the extracellular vesicle donor cell transfected with at least one heterologous polynucleotide produces and secretes extracellular vesicles comprising at least one targeting polypeptide. In some cases, the extracellular vesicle donor cell transfected with at least one heterologous polynucleotide produces and secretes extracellular vesicles comprising at least one therapeutic agent. extracellular vesicles

[0125] In some cases, compositions comprising extracellular vesicles and methods for producing extracellular vesicles are provided herein. In some cases, extracellular vesicles are any membrane-bound particles (e.g., vesicles with lipid bilayers). In many cases, the extracellular vesicles provided herein are secreted by cells. In some cases, extracellular vesicles can be any membrane-bound particles produced in vitro. In some cases, extracellular vesicles are produced and secreted by extracellular vesicle donor cells transfected with at least one heterologous polynucleotide. In some cases, extracellular vesicles are exosomes, microvesicles, retrovirus-like particles, apoptotic bodies, apoptosomes, oncosomes, exosomes, enveloped viruses, exomers, or very large extracellular vesicles, such as large oncosomes. In some cases, extracellular vesicles are exosomes.

[0126] In some cases, the extracellular vesicles can have a diameter of about 10 nm to about 50,000 nm. In some cases, the extracellular vesicles can have a diameter of about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 200 nm, about 10 nm to about 500 nm, about 10 nm to about 1,000 nm, about 10 nm to about 2,000 nm, about 10 nm to about 5,000 nm, about 10 nm to about 10,000 nm, about 10 nm to about 50,000 nm, about 20 nm to about 30 nm, about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 20 nm to about 200 nm, about 20 nm to about 500 nm, about 20 nm to about 1, 000nm, about 20nm to about 2,000nm, about 20nm to about 5,000nm, about 20nm to about 10,000nm, about 20nm to about 50,000nm, about 30nm to about 50nm, about 30nm to about 100nm, about 30nm to about 200nm, about 30nm to about 500nm, Approx. 30nm ~ approx. 1,000nm, approx. 30nm ~ approx. 2,000nm, approx. 30nm ~ approx. 5,000nm, approx. 30nm ~ approx. 10,000n m, about 30nm to about 50,000nm, about 50nm to about 100nm, about 50nm to about 200nm, about 50nm to about 500nm, about 50 nm ~ approx. 1,000 nm, approx. 50 nm ~ approx. 2,000 nm, approx. 50 nm ~ approx. 5,000 nm, approx. 50 nm ~ approx. 10,000 nm, approx. 50nm~about 50,000nm, about 100nm~about 200nm, about 100nm~about 500nm, about 100nm~about 1,000nm, about 100nm to approx. 2,000nm, approx. 100nm to approx. 5,000nm, approx. 100nm to approx. 10,000nm, approx. 100nm to approx. 50, 000nm, approximately 200nm to approximately 500nm, approximately 200nm to approximately 1,000nm, approximately 200nm to approximately 2,000nm, approximately 200nm to approximately 5,000nm, approx. 200nm ~ approx. 10,000nm, approx. 200nm ~ approx. 50,000nm, approx. 500nm ~ approx. 1,000nm, approx. 500nm to approx. 2,000nm, approx. 500nm to approx. 5,000nm, approx. 500nm to approx. 10,000nm, approx. 500nm to approx. 00nm, about 1,000nm to about 2,000nm, about 1,000nm to about 5,000nm, about 1,000nm to about 10,000nm , about 1,000nm to about 50,000nm, about 2,000nm to about 5,000nm, about 2,000nm to about 10,000nm, about 2,The extracellular vesicles may have a diameter of about 10,000 nm to about 50,000 nm, about 5,000 nm to about 10,000 nm, about 5,000 nm to about 50,000 nm, or about 10,000 nm to about 50,000 nm. In some cases, the extracellular vesicles have a diameter of about 10 nm, about 20 nm, about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, about 10,000 nm, or about 50,000 nm. In some cases, the extracellular vesicles can have a diameter of at least about 10 nm, about 20 nm, about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, or about 10,000 nm. In some cases, the extracellular vesicles can have a diameter of at most about 20 nm, about 30 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1,000 nm, about 2,000 nm, about 5,000 nm, about 10,000 nm, or about 50,000 nm.

[0127] Extracellular vesicle surface proteins are generally proteins associated with extracellular vesicles. In some cases, the extracellular vesicle surface protein may be expressed by extracellular vesicle donor cells and incorporated into and secreted as part of the extracellular vesicles produced and secreted by the extracellular donor cells. In some cases, the extracellular vesicle surface protein comprises at least one extracellular domain, which may comprise the N-terminus, C-terminus, or both the N-terminus and C-terminus of the extracellular vesicle surface protein. In some cases, the extracellular vesicle surface protein may be encoded by at least one heterologous polynucleotide or vector described herein. In some cases, the extracellular vesicle surface protein may be a member of the immunoglobulin superfamily. Members of the immunoglobulin superfamily may include antigen receptors, antigen-presenting molecules, co-receptors, antigen receptor accessory molecules, costimulatory or inhibitory molecules, receptors on natural killer cells, receptors on leukocytes, immunoglobulin-like cell adhesion molecules, cytokine receptors, growth factor receptors, receptor tyrosine kinases, receptor tyrosine phosphatases, immunoglobulin-binding receptors, cytoskeleton, or other members. In some cases, the extracellular vesicle surface protein comprising a member of the immunoglobulin superfamily comprises a variable immunoglobulin domain (IgV) or a constant immunoglobulin domain (IgC). In some cases, the extracellular vesicle surface protein comprising a member of the immunoglobulin superfamily comprises an IgV domain. Examples of members of the immunoglobulin superfamily that include IgV include cluster of differentiation proteins (e.g., CD2, CD4, CD47, CD80, or CD86), myelin membrane adhesion molecules, junctional adhesion molecules (JAM), tyrosine-protein kinase receptors, programmed cell death protein 1 (PD1), or T cell antigen receptors.

[0128] In some cases, the extracellular vesicles contain components other than therapeutic polynucleotides. In some cases, the extracellular vesicles contain one or more preservatives. In some cases, the extracellular vesicles contain one or more stabilizers. In some cases, the extracellular vesicles contain one or more DNAses. In some cases, the extracellular vesicles contain one or more RNase inhibitors. In some cases, the extracellular vesicles contain one or more DNAses and / or RNase inhibitors. In some cases, the extracellular vesicles contain one or more DNAses, DNAse inhibitors, RNAses and / or RNAse inhibitors.

[0129] In some cases, extracellular vesicles comprising at least one targeting polypeptide exhibit an increased half-life in circulation compared to the half-life of extracellular vesicles that do not contain a targeting polypeptide.In some cases, the half-life of extracellular vesicles comprising at least one targeting polypeptide is increased by at least 0.1 times, 0.2 times, 0.5 times, 1 times, 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 1000 times or more compared to the half-life of extracellular vesicles that do not contain a targeting polypeptide. In some cases, the half-life of extracellular vesicles comprising at least one targeting polypeptide is increased by at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or more, compared to the half-life of extracellular vesicles that do not contain a targeting polypeptide.

[0130] In some cases, extracellular vesicles comprising at least one targeting polypeptide exhibit a half-life in the circulation of a mammal (e.g., a human, a rodent, a mouse) of at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 5 minutes, or at least 10 minutes. In some cases, extracellular vesicles comprising a targeting polypeptide exhibit a half-life in the circulation of a mammal of less than 5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes.

[0131] In some cases, the targeting polypeptide comprises a heterologous targeting domain. In some cases, the heterologous targeting domain is a tumor targeting domain, a tissue targeting domain, a cell-penetrating peptide, a viral membrane protein, or a combination thereof. The heterologous targeting domain can target a cell surface marker expressed on the surface of the targeted cell. The cell surface marker can be any macromolecule or protein expressed on the surface of the targeted cell. Non-limiting examples of cell surface markers include vascular receptors, fibronectin receptors, A2B5, CD44, CD24, ESA, SSEA1, CD133, CD34, CD19, CD38, CD26, CD166, or CD90.

[0132] In some cases, the accumulation of extracellular vesicles comprising at least one targeting polypeptide in targeted cells expressing a cell surface marker is higher than the accumulation of extracellular vesicles not comprising at least one targeting polypeptide in the same targeted cells expressing the same cell surface marker. In some cases, the accumulation of extracellular vesicles comprising at least one targeting polypeptide in targeted cells expressing a cell surface marker is at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more, compared to the accumulation of extracellular vesicles not comprising a targeting polypeptide in the same targeted cells expressing the same cell surface marker.

[0133] In some cases, the accumulation of extracellular vesicles containing at least one targeting polypeptide in the liver and spleen of targeted cells expressing a cell surface marker, for example, the accumulation of extracellular vesicles in non-targeted cells, is reduced compared to the accumulation of extracellular vesicles not containing at least one targeting polypeptide in the liver and spleen of the same targeted cells. In some cases, the accumulation of extracellular vesicles containing at least one targeting polypeptide in the liver and spleen is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, or 10,000-fold compared to the accumulation of extracellular vesicles not containing a targeting polypeptide in the liver and spleen.

[0134] In some cases, the targeting polypeptide comprises at least one heterologous targeting domain.In some cases, at least one targeting domain is a tumor targeting domain, a tissue targeting domain, a cell-penetrating peptide, a viral membrane protein, or a combination thereof.In some cases, at least one heterologous targeting domain is a tumor targeting domain, and the tumor targeting domain targets cancerous cells.In some cases, at least one heterologous targeting domain is a tumor targeting domain, and the tumor targeting domain targets non-cancerous lesion cells.

[0135] In some cases, the targeting polypeptide comprises at least one, two, three, four, five, or more heterologous targeting domains. In some cases, at least two targeting domains can be identical. In some cases, at least two targeting domains can be different.

[0136] In some cases, the targeting polypeptide comprises at least one tissue targeting domain that targets the extracellular vesicles comprising the targeting polypeptide and directs the extracellular vesicles comprising the targeting polypeptide to a specific tissue. In some cases, the adaptor polypeptide comprises at least 2, 3, 4, 5 or more tissue targeting peptides. In some cases, at least two tissue targeting peptides are identical. In some cases, at least two tissue targeting peptides are different. In some cases, the tissue targeting peptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 100 amino acids. Exemplary tissue targeting domains that target endothelial tissue or cardiac tissue include SIGYPLP, ​​LSIPPKA, FQTPPQL, LTPATAI, CNIWGVVLSWIGVFPEC, NTTTH, VHPKQHR (tetramer), CRKRLDRNCCRTLTVRKC, CLWTVGGGC, QPWLEQAYYSTF, YPHIDSLGHWRR, LLADTTHHRPWT, SAHGTSTGVPWP, VPWMEPAYQRFL, TLPWLEESYWRP, HWRR, CSTSMLKAC, DDTRHWG, CARPAR, CKRAVR, CRSTRANPC, CPKTRRVPC, CSGMARTKC, or CRPPR. Exemplary tissue targeting domains that target pancreatic tissue include CRVASVLPC, SWCEPGWCR, LSGTPERSGQAVKVKLKAIP, CHVLWSTRCCVSNPRWKC, or LSALPRT. Exemplary tissue-targeting domains that target kidney tissue include CLPVASC, ELRGD(R / M)AX(W / L), GV(K / R)GX3(T / S)RDXR, HITSLLSHTTHREP, or ANTPCGPYTHDCPVKR. Exemplary tissue-targeting domains that target lung tissue include CGFELETCCGFECVRQCPERC, QPFMQCLCLIYDASCRNVPPIFNDVYWIAF, VNTANST, CTSGTHPRC, or SGEWVIKEARGWKHW-VFYSCCPTTPYLDITYH.Exemplary tissue-targeting domains that target intestinal tissue include YSGKWGW, LETTCASLCYPSYQCSYTMPHPPVVPPHPMTYSCQY, YPRLLTP, CSQSHPRHC, CSKSSDYQC, CKSTHPLSC, CTGKSCLRVG, SFKPSGLPAQSL, or CTANSSAQC. Exemplary tissue-targeting domains that target brain tissue include CLSSRLDAC, GHKAKGPRK, HAIYPRH, THRPPMWSPVWP, HLNILSTLWKYRC, CAGALCY, CLEVSRKNC, RPRTRLHTHRNR(D-aa), ACTTPHAWLCG, GLAHSFSDFARDFV, GYRPVHNIRGHWAPG, TGNYKALHPHNG, CRTIGPSVC, CTSTSAPYC, CSYTSSTMC, CMPRLRGC, TPSYDTYAAELR, RLSSVDSDLSGC, CAQK, or SGVYKVAYDWQH. Additional exemplary tissue targeting domains that target various tissues include LMLPRAD (targets the adrenal gland), CSCFRDVCC (targets the retina), CRDVVSVIC (targets the retina), CVALCREACGEGC (targets the skin and subcutaneous vasculature), GLSGGRS (targets the uterus), WYRGRL (targets cartilage), CPGPEGAGC (targets the breast vasculature), SMSIARLVSFLEYR (targets the prostate), GPEDTSRAPENQQKTGC (targets the skin and subcutaneous vasculature), and GLSGGRS (targets the uterus). and CKGGRAKDC (targets the thymus), CKGGRAKDC (targets white fat vasculature), CARSKNKDC (targets wound or damaged tissue), CHAQGSAEC (targets the thymus), LEPRWGFGWWLKLSTHTTESRSMV (targets ear or cochlear tissue), ACSTEALRHCGGGS (targets retinal blood vessels), ASSLNIA (targets muscle tissue), CSTSMLKAC (targets ischemia), or CSTSMLKAC (targets ischemic myocardium).

[0137] In some cases, the targeting polypeptide comprises at least 2, 3, 4, 5 or more cell-penetrating peptides. In some cases, the targeting polypeptide comprising a cell-penetrating peptide increases the rate at which extracellular vesicles are fused or endocytosed by the targeted cell. In some cases, at least two cell-penetrating peptides are identical. In some cases, at least two cell-penetrating peptides are different. In some cases, the cell-penetrating peptide comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. Non-limiting examples of cell penetrating peptides include DSLKSYWYLQKFSWR, DWLKAFYDKVAEKLKEAF, KSKTEYYNAWAVWERNAP, GNGEQREMAVSRLRDCLDRQA, HTPGNSNKWKHLQENKKGRPRR, DWLKAFYDKVAEKLKEAF, R9GPLGLAGE8, Ac-GAFSWGSLWSGIKNFGSTVKNYG, RLRWR, LGQQQPFPPQQPY, ILGKLLSTAAGLLSNL, TFFYGGSRGKRNNFKTEEY, Ac-LRKLRKRLLRX-Bpg-G, Ac-LRKLRKRLLR, or MVRRFLVTLRIRRACGPPRVRV.

[0138] In some cases, the targeting polypeptide comprises at least two, three, four, five, or more viral membrane proteins or fragments thereof. In some cases, the targeting polypeptide comprising a viral membrane protein increases the rate at which extracellular vesicles are fused or endocytosed by the targeted cell. In some cases, at least two viral membrane proteins are identical. In some cases, at least two viral membrane proteins are different. In some cases, the viral membrane protein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids. Non-limiting examples of viral membrane proteins include hemagglutinin, glycoprotein 41, envelope protein, VSV G, HSV01 gB, Ebola virus glycoprotein, or fusion-associated small transmembrane (FAST) protein.

[0139] In some cases, the extracellular vesicles described herein comprise at least one therapeutic agent. In some cases, the at least one therapeutic agent is within (e.g., encapsulated in) the extracellular vesicle. In some cases, the therapeutic agent is a therapeutic polynucleotide. In some cases, the therapeutic agent is a therapeutic polypeptide. In some cases, the therapeutic agent is a therapeutic compound. In some cases, the therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, or a combination thereof. In some cases, the extracellular vesicles comprise multiple therapeutic agents, and the multiple therapeutic agents comprise a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, or a combination thereof.

[0140] In some cases, the extracellular vesicles described herein comprise at least one targeting polypeptide.In some cases, the targeting polypeptide is a skin-targeting polypeptide comprising a skin-targeting domain.In some cases, the accumulation of the extracellular vesicles comprising the skin-targeting polypeptide comprising the skin-targeting domain in skin is higher than the accumulation of the extracellular vesicles that do not comprise a skin-targeting polypeptide.In some cases, the accumulation of the extracellular vesicles comprising the skin-targeting polypeptide in skin is at least 2 times, 5 times, 10 times, 50 times, 100 times, 200 times, 500 times, 1,000 times, 5,000 times, or 10,000 times higher than the accumulation of the extracellular vesicles that do not comprise a skin-targeting polypeptide.In some cases, the accumulation of the extracellular vesicles comprising the skin-targeting polypeptide in skin is at least 100 times higher than the accumulation of the extracellular vesicles that do not comprise a skin-targeting polypeptide.

[0141] In some cases, the skin-targeting polypeptide comprises at least one skin-targeting domain. In some cases, the skin-targeting domain can be at the N-terminus of the skin-targeting polypeptide. In some cases, the skin-targeting domain can be at the C-terminus of the skin-targeting polypeptide. In some cases, the skin-targeting domain can be at any peptide position of the skin-targeting polypeptide. In some cases, at least two targeting domains can be on the same skin-targeting polypeptide. In some cases, at least two targeting domains on the same skin-targeting polypeptide can be the same. In some cases, at least two targeting domains on the same skin-targeting polypeptide can be different. In some cases, the targeting domain comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 100 amino acids.

[0142] In some cases, extracellular vesicles containing an extracellular vesicle surface protein have an increased half-life in circulation compared to the half-life of extracellular vesicles that do not contain the extracellular vesicle surface protein. In some cases, the half-life of the extracellular vesicles increased by the extracellular vesicle surface protein is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the half-life of extracellular vesicles that do not contain the extracellular vesicle surface protein.

[0143] In some cases, extracellular vesicles that contain extracellular vesicle surface proteins have reduced toxicity compared to extracellular vesicles that do not contain extracellular vesicle surface proteins.In such cases, the extracellular vesicle surface proteins often specifically bind to targets and do not have significant off-target binding.In some cases, toxicity includes toxicity to cells that are not targeted by tumor targeting polypeptides.In some cases, extracellular vesicles that contain extracellular vesicle surface proteins have at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or more reduced toxicity compared to extracellular vesicles that do not contain extracellular vesicle surface proteins. In some cases, the reduced toxicity of extracellular vesicles comprising an extracellular vesicle surface protein is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more, compared to extracellular vesicles that do not contain the extracellular vesicle surface protein.

[0144] In some cases, the extracellular vesicles (e.g., exosomes) are tolerated by the subject after administration of the extracellular vesicles. For example, in some cases, the extracellular vesicles do not induce an immune response or are not immunogenic.

[0145] Therapeutic Polynucleotides In some cases, the extracellular vesicle described herein comprises at least one therapeutic polynucleotide.In some cases, the at least one therapeutic polynucleotide is encoded by at least one heterologous polynucleotide or vector that is transfected into the extracellular vesicle donor cell.In some cases, the at least one therapeutic polynucleotide comprises a peptide sequence that can be translated into a therapeutic polypeptide by the cell that is targeted and bound by the adaptor polypeptide described herein.

[0146] In some cases, the extracellular vesicles contain at least one therapeutic polynucleotide. In some cases, each extracellular vesicle contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000 copies or more of a therapeutic polynucleotide. In some cases, each extracellular vesicle contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000 copies or more of a therapeutic mRNA described herein. In some cases, the extracellular vesicles contain at least two therapeutic polynucleotides. In some cases, the extracellular vesicles contain at least two therapeutic polynucleotides, and the at least two therapeutic polynucleotides are different. In some cases, the at least two different therapeutic polynucleotides encapsulated by the extracellular vesicles contain different ratios. For example, the ratio of the first to the second of the two different therapeutic polynucleotides can be 1:1,000,000, 1:500,000, 1:100,000, 1:50,000, 1:10,000, 1:5,000, 1:1,000, 1:500, 1:100, 1:50, 1:10, 1:5, 1:4, 1:3, 1:2, or 1:1. In some cases, the extracellular vesicles contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more therapeutic polynucleotides encapsulated in the same extracellular vesicle. In some cases, the extracellular vesicles may be exosomes.

[0147] In some cases, the therapeutic polynucleotide comprises mRNA, rRNA, SRP RNA, tRNA, tmRNA, snRNA, snoRNA, gRNA, aRNA, crRNA, lncRNA, miRNA, ncRNA, piRNA, siRNA, and shRNA. In some cases, the therapeutic polynucleotide comprises mRNA. In some cases, the mRNA is fully intact or substantially intact. In some cases, the mRNA encodes a portion of a protein. In some cases, the mRNA comprises at least 50, 100, 200, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 or more RNA nucleotides. In some cases, the therapeutic polynucleotide comprises DNA. In some cases, the therapeutic polynucleotide comprises DNA, such as a vector encoding a therapeutic polypeptide. The therapeutic polynucleotide can encode a therapeutic polypeptide, including, but not limited to, an extracellular matrix (ECM) protein or peptide or an angiogenic protein or peptide.Examples of therapeutic polypeptides that can be encoded by therapeutic polynucleotides (e.g., messenger RNA therapeutics) include fibrillar collagens (e.g., type I, type II, type III, type V, type XI collagen), facit collagens (e.g., type IX collagen, type XII collagen, type XIV collagen), short-chain collagens (e.g., type VIII collagen, type X collagen), basement membrane collagens (e.g., type IV collagen), other collagens (e.g., type VI, type VII, type XIII, type XV, type XVII, type XVIIII collagen), elastin (ELN), fibronectin (FN), and the like. 1), laminin (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332 / laminin-3A32, laminin-3B32, laminin-311 / laminin-3A11, laminin-321 / laminin-3A21, laminin-411, laminin-421, laminin-511, laminin-521, laminin-213, laminin-423, laminin-522, laminin-523) or mixtures thereof, or VEGF (e.g., VEGFA, VEGFB, VEGFC, VEGFD, placenta growth factor (PlGF), or mixtures thereof). In some cases, the therapeutic polynucleotide described herein may encode type I collagen (Col1). In some cases, a therapeutic polypeptide encoded from a therapeutic polynucleotide described herein can be type I collagen (Col1). In some cases, a therapeutic polynucleotide described herein can encode VEGF (VEGFA). In some cases, a therapeutic polypeptide encoded from a therapeutic polynucleotide described herein can be VEGF (VEGFA).

[0148] In the EV production method disclosed herein, COL1A1 mRNA is present at a certain copy number per EV. In some cases, the plurality of extracellular vesicles contains an average of at least one copy of COL1A1 mRNA per 2000 EVs, per 1000 EVs, per 500 EVs, per 400 EVs, per 300 EVs, per 200 EVs, per 150 EVs, per 100 EVs, per 90 EVs, per 80 EVs, per 70 EVs, per 60 EVs, per 50 EVs, per 40 EVs, per 30 EVs, per 20 EVs, per 15 EVs, per 10 EVs, per 5 EVs, per 2 EVs, or per 1 EV. In some cases, the plurality of extracellular vesicles contains an average of at least 1, 2, 5, 10, 50, 100, 200, 300, 500, or 1000 copies of COL1A1 mRNA per EV. In some cases, multiple extracellular vesicles contain an average of at least one copy of COL1A1 mRNA per approximately 0.1–100 EVs.

[0149] In the EV production method disclosed herein, VEGF mRNA is present at a certain copy number per EV. In some cases, a plurality of extracellular vesicles comprises an average of at least one copy of VEGF mRNA per 2000 EVs, per 1000 EVs, per 500 EVs, per 400 EVs, per 300 EVs, per 200 EVs, per 150 EVs, per 100 EVs, per 90 EVs, per 80 EVs, per 70 EVs, per 60 EVs, per 50 EVs, per 40 EVs, per 30 EVs, per 20 EVs, per 15 EVs, per 10 EVs, per 5 EVs, per 2 EVs, or per 1 EV. In some cases, a plurality of extracellular vesicles comprises an average of at least 1, 2, 5, 10, 50, 100, 200, 300, 500, or 1000 copies of VEGF mRNA per EV. In some cases, multiple extracellular vesicles contain an average of at least one copy of VEGF mRNA per approximately 0.02–50 EVs.

[0150] In some cases, the copy number of the therapeutic polynucleotide (e.g., RNA therapeutic, mRNA therapeutic) encapsulated in the extracellular vesicles is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, or more copies of the therapeutic polynucleotide per extracellular vesicle. In some cases, the copy number of the therapeutic polynucleotide (e.g., RNA therapeutic, mRNA therapeutic) encapsulated in each extracellular vesicle or exosome described herein is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, or more copies.

[0151] In some cases, the copy number of a therapeutic polynucleotide (e.g., RNA therapeutic, mRNA therapeutic) encapsulated in a plurality of extracellular vesicles (e.g., a plurality of EVs secreted by donor cells in a culture vessel) is an average of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, or more copies of therapeutic polynucleotide per extracellular vesicle. In some cases, the copy number of a therapeutic polynucleotide (e.g., RNA therapeutic, mRNA therapeutic) encapsulated in each extracellular vesicle or exosome described herein is, on average, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1,000, at least 10,000, at least 100,000, or more copies.

[0152] In some cases, the copy number of a therapeutic polynucleotide (e.g., an RNA therapeutic or a messenger RNA therapeutic) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected (e.g., by microchannel electroporation or nanochannel electroporation) is increased by at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more, compared to the copy number of a therapeutic polynucleotide encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene guns, lipofectamine transfection, etc.). In some cases, the copy number of a therapeutic polynucleotide (e.g., an RNA therapeutic or a messenger RNA therapeutic) encapsulated in extracellular vesicles produced from a microchannel electroporated or nanochannel electroporated extracellular vesicle donor is increased by at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more, compared to the copy number of a therapeutic polynucleotide encapsulated in extracellular vesicles produced by directly introducing the therapeutic polynucleotide into the extracellular vesicles (e.g., directly transfecting the therapeutic polynucleotide into the extracellular vesicles).

[0153] In some cases, the therapeutic polynucleotide (e.g., RNA therapeutic or messenger RNA therapeutic) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is completely or substantially intact, with at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the encapsulated copies of the therapeutic polynucleotide being completely or substantially intact. In some cases, the percentage of complete or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel or nanochannel electroporation is increased compared to the percentage of complete or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene gun, lipofectamine transfection, etc.). In some cases, the number of complete or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased by at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more, compared to the number of complete or substantially intact therapeutic polynucleotides encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by other transfection methods (e.g., conventional bulk electroporation, gene guns, lipofectamine transfection, etc.).In some cases, the number of fully intact or substantially intact therapeutic polynucleotides (e.g., RNA therapeutics or messenger RNA therapeutics) encapsulated in extracellular vesicles produced from extracellular vesicle donor cells transfected by microchannel electroporation or nanochannel electroporation is increased by at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more, compared to the number of fully intact or substantially intact therapeutic polynucleotides encapsulated in extracellular vesicles produced from directly introducing the therapeutic polynucleotide into the extracellular vesicles (e.g., directly transfecting the therapeutic polynucleotide into the extracellular vesicles).

[0154] In some cases, the therapeutic polynucleotide comprises at least one modified nucleic acid or nucleic acid analog. Exemplary modified nucleic acids include uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil ... These include, but are not limited to, uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain modified nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that enhance the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl, and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil,5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidines ([5,4-b][l,4]benzoxazin-2(3H)-ones), phenothiazine cytidines (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-ones), G-clamps, phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-ones), carbazole cytidines (2H-pyrimido[4,5-b]indol-2-ones), pyridoindoles Cytidine (2H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), purine or pyrimidine bases replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine Tosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thioguanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine modified nucleic acids containing a variety of heterocyclic bases and a variety of sugar moieties (and sugar analogs) are available in the art, and in some casesNucleic acids contain one or more heterocyclic bases other than the five main base components of naturally occurring nucleic acids. For example, heterocyclic bases include, in some cases, uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-5-yl, and 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-3-yl groups, and purines are linked to the sugar moiety of nucleic acids via the 9-position, pyrimidines via the 1-position, pyrrolopyrimidines via the 7-position, and pyrazolopyrimidines via the 1-position.

[0155] In some cases, nucleotide analogs are also modified at the phosphate moiety. Modified phosphate moieties include those with modifications in the linkage between two nucleotides, such as, but not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate linkages or modified phosphate linkages between two nucleotides are via a 3'-5' or 2'-5' linkage, and it is understood that this linkage includes reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0156] In some cases, modified nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleoside 5'-substituted DNA and RNA derivatives or 5'-substituted monomers made as monophosphates with modified bases.

[0157] In some cases, modified nucleic acids contain modifications at the 5' and 2' positions of the sugar ring (e.g., 5'-CH2-substituted 2'-O-protected nucleosides). In some cases, modified nucleic acids contain amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleosides in the dimer (5'-3') contain 2'-OCH3 and 5'-(S)-CH3. Modified nucleic acids can contain 2'-substituted 5'-CH2 (or O) modified nucleosides. Modified nucleic acids can contain 5'-methylene phosphonate DNA and RNA monomers and dimers. Modified nucleic acids can contain 5'-phosphonate monomers with 2' substitutions and other modified 5'-phosphonate monomers. Modified nucleic acids can contain 5'-modified methylene phosphonate monomers. Modified nucleic acids may include 5'- or 6'-phosphonate ribonucleoside analogs containing hydroxyl groups at the 5' and / or 6' positions. Modified nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers with a 5'-phosphate group. Modified nucleic acids may include nucleosides with a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and its analogs), a methyleneamino group (CH2NH2) (and its analogs), or a cyano group (CN) (and its analogs).

[0158] In some cases, modified nucleic acids also include modifications to the sugar moiety. In some cases, nucleic acids include one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer improved nuclease stability, increased binding affinity, or some other beneficial biological property. In certain cases, nucleic acids include chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include the addition of substituents (including 5' and / or 2' substituents); bridging two ring atoms to form bicyclic nucleic acids (BNAs); and replacing the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C 12alkyl or protecting groups); and combinations thereof.

[0159] In some cases, modified nucleic acids contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be linked to the respective heterocyclic base in either the [α] or [β] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs, and the respective "nucleosides" in which such sugars or analogs are attached to heterocyclic bases (nucleobases), is known. Sugar modifications can also be made and combined with other modifications.

[0160] Modifications to the sugar moiety include natural and modified modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 The 2' sugar modifications can be alkenyl and alkynyl of the formula: O[(CH2) n O] m CH3, -O(CH2)n OCH3, -O(CH2) n NH2, -O(CH2) n CH 3、 -O(CH2) n ONH2, and -O(CH2) n ON[(CH2) n CH3)]2 (where n and m are from 1 to about 10).

[0161] Other modifications at the 2-position include C1-C 10 Modifications include, but are not limited to, lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic or pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in 2'-5'-linked oligonucleotides, and the 5' position of the 5'-terminal nucleotide. Modified sugars also include those containing modifications on the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0162] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH)OCH substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-(C1-C2). 10 alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n) can also be selected from each R m and R n are independently H or substituted or unsubstituted C1-C 10 It is alkyl.

[0163] In certain cases, the nucleic acids described herein include one or more bicyclic nucleic acids. In certain such cases, the bicyclic nucleic acid includes a bridge between the 4' and 2' ribosyl ring atoms. In certain cases, the nucleic acids provided herein include one or more bicyclic nucleic acids in which the bridge includes a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2', and analogs thereof; 4'-C(CH3)(CH3)-O-2' and analogs thereof.

[0164] In certain cases, nucleic acids include linked nucleic acids. Nucleic acids can be linked together using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkages include methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N * Examples of suitable chiral linkages include, but are not limited to, -dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linkages having chiral atoms can be prepared as separate enantiomers, e.g., alkylphosphonates and phosphorothioates, as racemic mixtures. Modified nucleic acids can contain a single modification. Modified nucleic acids can contain multiple modifications within one moiety or between different moieties.

[0165] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

[0166] In some cases, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acids and / or enhance their stability in vivo.

[0167] In some cases, the phosphorus derivative (or modified phosphonate group) is attached to a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc.

[0168] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include anionic internucleoside linkages; N3'-P5' phosphoramidate modifications; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8:1157-1179). Modified nucleic acids can contain chimeric or mixed backbones containing one or more modifications, such as combinations of phosphate linkages, e.g., combinations of phosphodiester and phosphorothioate linkages.

[0169] Phosphate substituents include, for example, short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbone; sulfide, sulfoxide and sulfone backbone; formacetyl and thioformacetyl backbone; methyleneformacetyl and thioformacetyl backbone; alkene-containing backbone; sulfamate backbone; methyleneimino backbone and methylenehydrazino backbone; sulfonate and sulfonamide backbone; amide backbone; and others with mixed N, O, S and CH components. It is also understood that in nucleotide substitutes, both the sugar portion and the phosphate portion of the nucleotide can be replaced, for example, with an amide-type linkage (aminoethylglycine) (PNA). United States Patent No. 5,539,082; United States Patent No. 5,714,331; and United States Patent No. 5,719,262 teach the preparation and use of PNA molecules, each of which is incorporated herein by reference.See Nielsen et al., Science, 1991,254,1497-1500.Other types of molecules (conjugates) can also be attached to nucleotide or nucleotide analogues, for example, to enhance cellular uptake.Conjugates can be chemically linked to nucleotide or nucleotide analogues. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties, cholic acid, thioethers, such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains, such as dodecanediol or undecyl residues (phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonat), polyamine or polyethylene glycol chains, or adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0170] In some cases, at least one modified nucleotide or nucleotide analog described herein may be resistant to nucleases, such as ribonucleases, e.g., RNase H, deoxyribonucleases, e.g., DNase, or exonucleases, e.g., 5'-3' exonucleases and 3'-5' exonucleases, as compared to naturally occurring nucleic acid molecules. In some cases, at least one modified nucleotide or nucleotide analog is a 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleotide, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or a combination thereof, which is resistant to nucleases, such as ribonucleases, e.g., RNases In some cases, 2'-O-methyl modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, 2'-O-methoxyethyl (2'-O-MOE) modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, 2'-O-aminopropyl modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant).In some cases, 2'-deoxy modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, T-deoxy-2'-fluoro modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-aminopropyl (2'-O-AP) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified nucleic acid molecules are nuclease resistant (e.g., RNase H, DNase, 5'-3' exonuclease or 3'-5' exonuclease resistant). In some cases, 2'-ON-methylacetamide (2'-O-NMA) modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, LNA modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, ENA modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, HNA modified nucleic acid molecules are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant).In some cases, the morpholino is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the PNA-modified nucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the methylphosphonate nucleotide-modified nucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the thiolphosphonate nucleotide-modified nucleic acid molecule is nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, nucleic acid molecules comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease-resistant (e.g., RNase H, DNase, 5'-3' exonuclease, or 3'-5' exonuclease-resistant). In some cases, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some cases, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0171] In further cases, the modified nucleotides or nucleotide analogs described herein are modified to enhance their stability. In some embodiments, the nucleic acid molecule is RNA (e.g., mRNA). In some cases, the mRNA can be modified by one or more modifications to enhance its stability. In some cases, the mRNA can be modified at the 2' hydroxyl position, for example, by 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), T-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modification, or by a locked or bridged ribose conformation (e.g., LNA or ENA). In some cases, at least one modified nucleotide or nucleotide analog is modified with 2'-O-methyl and / or 2'-O-methoxyethyl ribose. In some cases, at least one modified nucleotide or nucleotide analog also includes morpholino, PNA, HNA, methylphosphonate nucleotide, thiolphosphonate nucleotide, and / or 2'-fluoro N3-P5'-phosphoramidite to enhance its stability. In some cases, at least one modified nucleotide or nucleotide analog is a chirality-pure (or stereochemically pure) nucleic acid molecule. In some cases, the chirality-pure (or stereochemically pure) nucleic acid molecule is modified to enhance its stability.

[0172] In some cases, the extracellular vesicles described herein comprise at least one of the therapeutic polypeptides described herein. In some cases, the at least one therapeutic polypeptide is encoded by at least one heterologous polynucleotide or vector transfected into the extracellular vesicle donor cell.

[0173] In some cases, the therapeutic polynucleotide can be translated by the extracellular vesicle donor cell to obtain at least one therapeutic polypeptide.In some cases, the therapeutic polypeptide encoded by the therapeutic polynucleotide can be encapsulated in the extracellular vesicles produced and secreted by the extracellular vesicle donor cell.In some cases, the extracellular vesicle can encapsulate both the therapeutic polynucleotide and the therapeutic polypeptide encoded by the nanoelectroporated vector.In some cases, the extracellular vesicle can be exosomes.

[0174] In some cases, the extracellular vesicles described herein can contain at least one therapeutic compound. In some cases, the at least one therapeutic compound is complexed or fixed by any one of the extracellular vesicle surface proteins described herein. In some cases, the at least one therapeutic compound is present in the extracellular vesicle. Exemplary therapeutic compounds include cancer drugs. Exemplary therapeutic compounds for use in the compositions and methods described herein include therapeutic compounds for treating skin damage (e.g., due to aging or sun damage) and blood flow disorders (e.g., ischemia).

[0175] In some embodiments, the present disclosure provides a plurality of extracellular vesicles comprising an average of at least one copy of exogenous VEGF mRNA. In some embodiments, the exogenous VEGF mRNA is selected from the group consisting of VEGFA, VEGFB, VEGFC, VEGFD, PIGF, and any combination thereof. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles comprising a targeting polypeptide.

[0176] In some embodiments, a mixture of extracellular vesicles is described, the mixture comprising at least two extracellular vesicles containing 1 to 6 copies of exogenous VEGF mRNA, the mixture comprising exosomes, microvesicles, and apoptotic bodies. In some embodiments, the mixture is formulated for injection via intravenous, intramuscular, or subcutaneous routes. In some embodiments, the mixture is formulated for injection via a coronary artery catheter.

[0177] In some aspects, methods for treating a blood flow disorder in a subject are described herein, comprising administering at least one extracellular vesicle comprising VEGF mRNA to the subject, thereby treating the blood flow disorder. In some embodiments, treating the blood flow disorder results in at least a 5% increase in revascularization. In some embodiments, the at least 5% increase in revascularization occurs within 14 days of administering the at least one extracellular vesicle comprising VEGF mRNA. In some embodiments, administering the at least one extracellular vesicle comprising VEGF mRNA to the subject via intravenous, intramuscular, or subcutaneous injection, or via a coronary artery catheter. In some embodiments, the intravenous, intramuscular, or subcutaneous injection is performed using a needle having a gauge of at least 14. In some embodiments, the blood flow disorder is ischemia. In some embodiments, the at least one extracellular vesicle comprising VEGF mRNA is administered in at least one dose. In some embodiments, the dose comprises at least 1,000 extracellular vesicles comprising VEGF mRNA. In some embodiments, at least one extracellular vesicle comprising VEGF mRNA is administered in at least two doses. In some embodiments, administering at least one extracellular vesicle comprising VEGF mRNA to a subject comprises administering at least one extracellular vesicle comprising VEGF mRNA to a tissue of the subject.

[0178] In some aspects, a plurality of extracellular vesicles are described herein, each containing an average of at least one copy of exogenous extracellular matrix mRNA. In some embodiments, the exogenous extracellular matrix mRNA is selected from the group consisting of collagen, COL1, type I collagen, type II collagen, type III collagen, type V collagen, type XI collagen, type IX collagen, type XII collagen, type XIV collagen, type VIII collagen, type X collagen, type IV collagen, type VI collagen, type VII collagen, type XIII collagen, type XV collagen, type XVII collagen, and type XVIIII collagen. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, or any combination thereof. In some embodiments, the plurality of extracellular vesicles comprises extracellular vesicles comprising a targeting polypeptide.

[0179] In some aspects, a mixture of extracellular vesicles is described, the mixture comprising at least two extracellular vesicles containing one to two copies of exogenous extracellular matrix mRNA, the mixture comprising exosomes, microvesicles, and apoptotic bodies. In some embodiments, the mixture is formulated for injection via intravenous, intramuscular, or subcutaneous routes.

[0180] In some aspects, methods for treating skin damage in a subject are described herein, the methods comprising administering at least one extracellular vesicle comprising extracellular matrix mRNA to the subject, thereby treating the skin damage in the subject. In some embodiments, treating the skin damage results in at least a 10% reduction in the appearance of wrinkles. In some embodiments, the at least 10% reduction in the appearance of wrinkles occurs within 14 days of administering the at least one extracellular vesicle comprising extracellular matrix mRNA. In some embodiments, administering the at least one extracellular vesicle comprising extracellular matrix mRNA comprises administering the at least one extracellular vesicle comprising extracellular matrix mRNA to the subject via subcutaneous injection. In some embodiments, the subcutaneous injection is performed using a needle having at least 14 gauge. In some embodiments, the skin damage is caused by aging or sun damage. In some embodiments, the at least one extracellular vesicle comprising extracellular matrix mRNA is administered in at least one dose. In some embodiments, the dose comprises at least 1,000 extracellular vesicles comprising extracellular matrix mRNA. In some embodiments, at least one extracellular vesicle comprising extracellular matrix mRNA is administered in at least two doses. In some embodiments, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject comprises administering at least one extracellular vesicle comprising VEGF mRNA to the subject's tissue. In some embodiments, within 72 hours after administering at least one extracellular vesicle comprising extracellular matrix mRNA to the subject, the tissue has an extracellular matrix protein concentration of at least 6000 pg / ml.

[0181] In some embodiments, described herein are methods for producing extracellular vesicles containing VEGF mRNA, the methods comprising introducing a vector or plasmid encoding VEGF into donor cells via transfection, incubating the donor cells for a sufficient time to produce extracellular vesicles encapsulating the VEGF mRNA transcribed from the vector or plasmid, and recovering the extracellular vesicles encapsulating the VEGF mRNA transcribed from the vector or plasmid.

[0182] In some embodiments, described herein are methods for producing extracellular vesicles containing extracellular matrix mRNA, the methods comprising introducing a vector or plasmid encoding an extracellular matrix protein into donor cells via transfection, incubating the donor cells for a sufficient time to produce extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid, and recovering the extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid.

[0183] In some embodiments, a plurality of extracellular vesicles comprising exogenous extracellular matrix messenger RNA (mRNA) are described herein. In some cases, the exogenous extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is type I collagen alpha 1 chain (Col1A1). In some cases, the collagen is Col1A1 and the plurality of extracellular vesicles do not contain the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2. In some cases, the exogenous extracellular matrix mRNA is mRNA encoding the pro-alpha 1(I) chain.

[0184] In some cases, the plurality of extracellular vesicles comprises an average of at least one copy of exogenous extracellular matrix mRNA per 450 EVs. In some cases, the plurality of extracellular vesicles comprises an average of at least one copy of exogenous extracellular matrix mRNA per 200 EVs. In some cases, the plurality of extracellular vesicles comprises an average of at least one copy of exogenous extracellular matrix mRNA per about 0.1-100 EVs. In some cases, the plurality of extracellular vesicles comprises an average of at least 1 copy of exogenous extracellular matrix mRNA per 2000 EVs, per 1000 EVs, per 500 EVs, per 400 EVs, per 300 EVs, per 200 EVs, per 150 EVs, per 100 EVs, per 90 EVs, per 80 EVs, per 70 EVs, per 60 EVs, 50 EVs, per 40 EVs, per 30 EVs, per 20 EVs, per 15 EVs, per 10 EVs, per 5 EVs, per 2 EVs, or per 1 EV. In some cases, the plurality of extracellular vesicles comprises an average of at least 1, 2, 5, 10, 50, 100, 200, 300, 500, or 1000 copies of exogenous extracellular matrix mRNA per EV. In some cases, the plurality of extracellular vesicles comprises an average of about 1-10 copies of exogenous extracellular matrix mRNA per EV. In some cases, the plurality of extracellular vesicles comprises an average of about 1 copy of exogenous extracellular matrix mRNA per EV. In some cases, the plurality of extracellular vesicles comprises an average of at least 1 copy of exogenous extracellular matrix mRNA per 30 EVs.

[0185] In some cases, the plurality of extracellular vesicles are produced by transfecting cells with a plasmid encoding an extracellular matrix protein. In some cases, transfecting the cells is performed by cell nanoporation. In some cases, transfecting the cells is performed by electroporation. In some cases, the cells are human cells. In some cases, the cells are fibroblasts selected from the group consisting of dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, and neonatal human dermal fibroblasts. In some cases, the cells are adherent cells. In some cases, the cells are human dermal fibroblasts (e.g., neonatal human dermal fibroblasts).

[0186] In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the plurality of extracellular vesicles comprises at least one exosome. In some cases, the plurality of extracellular vesicles comprises at least one apoptotic body. In some cases, the plurality of extracellular vesicles comprises at least one microvesicle. In some cases, the plurality of extracellular vesicles comprises a mixture of at least two of exosomes, microvesicles, or apoptotic bodies.

[0187] In some cases, the plurality of extracellular vesicles are formulated for injection via intravenous route, intramuscular route, subcutaneous route, or any combination thereof.In some cases, the formulation comprises one or more stabilizers and / or one or more preservatives.In some cases, the formulation comprises one or more DNA and / or RNase inhibitors.In some cases, the formulation comprises one or more DNAse, DNAse inhibitor, RNAse and / or RNAse inhibitor.

[0188] In some cases, multiple extracellular vesicles are present, at least 1 x 10 6 , 5×10 6 , 1×107 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 13 In some cases, the number of extracellular vesicles is 1 x 10 14 In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 9 extracellular vesicles and up to 1 × 10 20 In some cases, the number of extracellular vesicles may be up to 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 1 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 6 of extracellular vesicles and up to 1 × 10 30In some cases, the plurality of extracellular vesicles comprises at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of extracellular matrix mRNA. In some cases, the plurality of extracellular vesicles comprises at most 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of extracellular matrix mRNA. In some cases, the plurality of extracellular vesicles comprises at least 1 ng and at most 20 μg of extracellular matrix mRNA. In some cases, the plurality of extracellular vesicles comprises at least 5 ng and at most 30 μg of extracellular matrix mRNA.

[0189] In some cases, the plurality of extracellular vesicles includes extracellular vesicles comprising a targeting polypeptide. In some cases, the targeting polypeptide targets dermal cells. In some cases, the targeting polypeptide targets aminopeptidase N, CD26 / DPP4, fibroblast activation protein alpha, or any combination thereof. In some cases, the plurality of extracellular vesicles does not contain one or more of the following miRNAs: hsa-miR-29c-3p, hsa-miR-29a-3p, hsa-miR-378a-3p, hsa-miR-125b-5p, hsa-miR-23a-3p, hsa-miR-449a, hsa-miR-196a-5p, hsa-miR-744-5p, hsa-miR-223-3p, hsa-miR-23a-3p, hsa-miR-133a-3p, hsa-miR-223-3p, hsa-miR-5011-5p, hsa-miR-325, or hsa-miR-199b-5p.

[0190] In some cases, the plurality of extracellular vesicles comprises a size distribution having a peak at a diameter of 50 nm to 200 nm. In some cases, the plurality of extracellular vesicles comprises a size distribution having a peak at a diameter of about 100 nm. In some cases, the plurality of extracellular vesicles comprises a size distribution having a peak at a diameter of about 75 to 130 nm. In some cases, the extracellular vesicles have a diameter of more than 20 nm, 30 nm, 50 nm, 75 nm, or 100 nm. In some cases, at least 90% of the extracellular vesicles have a diameter of more than 20 nm, 30 nm, 50 nm, 75 nm, or 100 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 50 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 30 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 20 nm. In some cases, the extracellular vesicles do not include extracellular vesicles with a diameter of less than 10 nm. In some cases, the extracellular vesicles do not include extracellular vesicles less than 5 nm in diameter.

[0191] In some cases, the exogenous extracellular matrix mRNA is present at a level at least two times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level at least three times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 10 times, at least 15 times, at least 20 times, at least 50 times, at least 75 times, at least 100 times, at least 500 times, at least 1000 times, at least 1500 times, or at least 2000 times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level about 3000 times higher than the level of the exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the exogenous extracellular matrix mRNA is present at a level about 2000 times higher than the level of exogenous extracellular matrix mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the plurality of extracellular vesicles expresses a higher level of at least one of the following markers: CD9, CD63, TSG101, or ARF6, compared to a comparable number of naturally occurring extracellular vesicles.

[0192] In some embodiments, the present invention provides a container comprising a plurality of extracellular vesicles according to any one of the preceding claims, wherein the container further comprises cells.In some cases, the cells comprise human cells, human fibrocytes, fibroblasts, dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, neonatal human dermal fibroblasts, or any combination thereof.In some cases, the plurality of extracellular vesicles are present in the container at a ratio of at least 1000, 2000, 5000, 10000, or 12000 extracellular vesicles per cell.

[0193] In some embodiments, needles comprising a plurality of extracellular vesicles disclosed herein are described herein. In some cases, the needles are microneedles. In some cases, the needles are solid. In some cases, the needles are hydrogel needles. In some cases, at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the needles comprise hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid. In some cases, the hydrogel comprises at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel contains up to 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid. In some cases, the hydrogel contains about 15% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0194] In some aspects, described herein is a syringe containing a plurality of extracellular vesicles disclosed herein. In some cases, the plurality of extracellular vesicles are suspended in hyaluronic acid within the syringe.

[0195] In some embodiments, hydrogel microneedles containing a plurality of extracellular vesicles are described herein. In some cases, the hydrogel contains at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel contains hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel contains hyaluronic acid. In some cases, the hydrogel contains at least 5%, 7%, 10%, 15%, or 20% hyaluronic acid. In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the plurality of extracellular vesicles comprises at least one exosome. In some cases, the plurality of extracellular vesicles comprises at least one apoptotic body. In some cases, the plurality of extracellular vesicles comprises at least one microvesicle. In some cases, the plurality of extracellular vesicles comprises at least 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the number of extracellular vesicles is 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8, 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , 1×10 30 , 1×10 30 , 1×10 40 , 1×10 50 , 1×10 60 , 1×10 70 , 1×10 80 , 1×10 90 , or 1 × 10 100 In some cases, the number of extracellular vesicles is about 1 x 10 6 ~1×10 30 In some cases, the plurality of extracellular vesicles comprises about 1 x 10 10 ~1×10 14 In some cases, the plurality of extracellular vesicles comprises about 1 x 10 13 ~1×10 14 Contains extracellular vesicles.

[0196] In some cases, the hydrogel microneedles have a length of less than 2 mm. In some cases, the hydrogel microneedles have a length of less than 1 mm. In some cases, the hydrogel microneedles have a length of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 μm. In some cases, the hydrogel microneedles have a length of 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, 500 μm or less, 600 μm or less, 700 μm or less, 800 μm or less, 900 μm or less, 1000 μm or less, 1100 μm or less, 1200 μm or less, 1300 μm or less, 1400 μm or less, 1500 μm or less, 1600 μm or less, 1700 μm or less, 1800 μm or less, 1900 μm or less, or 2000 μm or less. In other cases, the hydrogel microneedles have a length of 100 μm to 3000 μm, 500 μm to 2500 μm, 1000 μm to 2500 μm, or 1000 μm to 2000 μm. In certain embodiments, the hydrogel microneedles have a length of about 1000 μm. In certain embodiments, the hydrogel microneedles have a length of about 2000 μm.

[0197] In some cases, the hydrogel microneedles have a base with a diameter of 10,000 μm or less, 9,000 μm, 8,000 μm, 7,000 μm, 6,000 μm, 5,000 μm, 4,000 μm, 3,000 μm, 2,000 μm, less than 1,800 μm, less than 1,500 μm, less than 1,000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, or less than 100 μm. In some cases, the hydrogel microneedles contain extracellular vesicles loaded with one or more mRNA cargos.

[0198] In some embodiments, needles comprising a plurality of extracellular vesicles are described herein, wherein the extracellular vesicles comprise at least one extracellular matrix messenger RNA (mRNA). In some cases, the extracellular matrix mRNA encodes a collagen. In some cases, the extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is the alpha 1 chain of type I collagen (Col1A1). In some cases, the collagen is Col1A1 and the extracellular vesicles do not contain the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2. In some cases, the extracellular matrix mRNA is mRNA encoding the pro-alpha 1 (I) chain.

[0199] In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof. In some cases, the plurality of extracellular vesicles comprises at least one exosome. In some cases, the plurality of extracellular vesicles comprises at least one apoptotic body. In some cases, the plurality of extracellular vesicles comprises at least one microvesicle. In some cases, the plurality of extracellular vesicles comprises a mixture of any two of exosomes, microvesicles, or apoptotic bodies.

[0200] In some cases, the needle is a microneedle. In some cases, the needle is solid. In some cases, the needle is a hydrogel needle, and optionally, at least 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the needle comprises a hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel comprises hyaluronic acid. In some cases, the hydrogel comprises at least 1%, 5%, 7%, 10%, 12%, 15%, 20%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% hyaluronic acid. In some cases, the hydrogel comprises at least 5% hyaluronic acid. In some cases, the hydrogel contains about 15% hyaluronic acid. In some cases, the hydrogel contains at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel contains more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0201] In some cases, the needle has a length of less than 2 mm. In some cases, the needle has a length of less than 1 mm. In some cases, the needle has a length of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 μm. In some cases, the needle has a length of 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, 500 μm or less, 600 μm or less, 700 μm or less, 800 μm or less, 900 μm or less, 1000 μm or less, 1100 μm or less, 1200 μm or less, 1300 μm or less, 1400 μm or less, 1500 μm or less, 1600 μm or less, 1700 μm or less, 1800 μm or less, 1900 μm or less, or 2000 μm or less. In other cases, the needle has a length of 100 μm to 3000 μm, 500 μm to 2500 μm, 1000 μm to 2500 μm, or 1000 μm to 2000 μm. In certain embodiments, the needle has a length of about 1000 μm. In certain embodiments, the needle has a length of about 2000 μm.

[0202] In some cases, the needle has a base with a diameter of 10,000 μm or less, 9,000 μm, 8,000 μm, 7,000 μm, 6,000 μm, 5,000 μm, 4,000 μm, 3,000 μm, 2,000 μm, less than 1,800 μm, less than 1,500 μm, less than 1,000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, or less than 100 μm.

[0203] The COL1A1 gene produces a component of type I collagen called the proalpha1(I) chain. This chain also binds to other proalpha1(I) and proalpha2(I) chains (produced by the COL1A2 gene) to create type I procollagen molecules. These triple-stranded, rope-like procollagen molecules can be processed enzymatically. Once processed, they arrange into elongated fibrils that cross-link with each other in the space surrounding the cell. This cross-linking results in the formation of very strong, mature type I collagen fibers. Thus, in some cases, at least one copy of exogenous extracellular matrix mRNA is transcribed and assembled into the proalpha1(I) chain in cells exposed to multiple extracellular vesicles. In some cases, at least one copy of exogenous extracellular matrix mRNA is transcribed and further assembled into type I procollagen in cells exposed to multiple extracellular vesicles.

[0204] In some cases, the plurality of extracellular vesicles comprises an average of at least 10, 100, 1000, 2000, 3000, 4000, 5000, 6000, 10,000, 15,000, 30,000, 50,000, 80,000, 100,000, 150,000, 300,000, 500,000, 700,000, or 1,000,000 copies of exogenous extracellular matrix mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises an average of about 10, 100, 1000, 2000, 3000, 4000, 5000, 6000, 10,000, 15,000, 30,000, 50,000, 80,000, 100,000, 150,000, 300,000, 500,000, 700,000, or 1,000,000 copies of exogenous extracellular matrix mRNA per extracellular vesicle.

[0205] In some cases, the extracellular vesicles disclosed herein are exosomes.In other cases, the extracellular vesicles disclosed herein are microvesicles.In other cases, the extracellular vesicles disclosed herein are apoptotic bodies.In other cases, the extracellular vesicles disclosed herein are any two or three types of combinations mentioned in this paragraph.

[0206] In some cases, the plurality of extracellular vesicles comprises extracellular vesicles comprising a targeting polypeptide disclosed herein.

[0207] Further provided herein is a needle comprising a plurality of extracellular vesicles comprising, on average, at least one copy of exogenous extracellular matrix mRNA. Further provided herein is a needle comprising a plurality of extracellular vesicles comprising, on average, at least one copy of endogenous extracellular matrix mRNA. Further provided herein is a needle comprising a plurality of extracellular vesicles comprising, on average, at least one copy of endogenous extracellular matrix mRNA and at least one copy of exogenous extracellular matrix mRNA.

[0208] In some cases, the needles are microneedles. In some cases, the needles include a hydrogel. In specific cases, the hydrogel includes hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In specific cases, the hydrogel includes hyaluronic acid.

[0209] Further provided herein is a syringe containing a plurality of extracellular vesicles containing, on average, at least one copy of exogenous extracellular matrix mRNA. Further provided herein is a syringe containing a plurality of extracellular vesicles containing, on average, at least one copy of endogenous extracellular matrix mRNA. Further provided herein is a syringe containing a plurality of extracellular vesicles containing, on average, at least one copy of endogenous extracellular matrix mRNA and at least one copy of exogenous extracellular matrix mRNA.

[0210] In some cases, the syringe has a gauge of at least 34. In some cases, the syringe has a gauge of less than 10. In some cases, the syringe has a gauge of 34, 33, 32, 31, 30, 29, 28, 27, 26, 26s, 25, 25s, 24, 23s, 23, 22s, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10.

[0211] In some cases, multiple extracellular vesicles within the syringe are suspended in hyaluronic acid.

[0212] Further provided herein is a mixture of extracellular vesicles, comprising at least two extracellular vesicles containing one or two copies of exogenous extracellular matrix mRNA.In some cases, the mixture comprises exosomes, microvesicles, apoptotic bodies, or any combination thereof.

[0213] In some cases, the mixtures disclosed herein are formulated for injection by intravenous, intramuscular, or subcutaneous routes.

[0214] Further provided herein is a method for producing extracellular vesicles comprising extracellular matrix mRNA, wherein producing the extracellular vesicles comprising extracellular matrix mRNA comprises: (a) introducing a vector or plasmid encoding an extracellular matrix protein into donor cells via transfection; (b) incubating the donor cells for a sufficient time to produce extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid; and (c) recovering the extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid.

[0215] In some cases, in step (a), transfection is performed using cell electroporation, as shown in FIG. 7A. In some specific cases, in step (a), transfection involves plating a monolayer of donor cells on the surface of a silicon chip as shown in FIG. 7A, and applying an electric field of a certain voltage with a certain pulse and duration to electroporate the vector or plasmid into the donor cells. In some further specific cases, plating requires overnight incubation as described above. In some further specific cases, the voltage of the electric field is at least 10V, 50V, 100V, 150V, 200V, 250V, 300V, 500V, or 1000V. In some further specific cases, the voltage of the electric field is about 10V, 50V, 100V, 150V, 200V, 250V, 300V, 500V, or 1000V. In some more specific cases, the voltage of the electric field is at most 500V, 600V, 700V, 800V, 900V, 1000V, 1500V, 2000V, 5000V, or 10,000V. In some more specific cases, the pulses of the electric field are at least 5, 10, 15, 20, 25, 30, 40, 50, or 60. In some more specific cases, the pulses of the electric field are no more than 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some more specific cases, the pulses of the electric field are about 5, 10, 15, 20, 25, 30, 40, 50, or 60. In some more specific cases, the interval between pulses of the electric field is at least 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, or 1 s. In some more specific cases, the interval between pulses of the electric field is about 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, or 1 s.

[0216] In some cases, in step (c), the recovery of extracellular vesicles is performed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after transfection. In some cases, in step (c), the recovery of extracellular vesicles is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after transfection.

[0217] In some embodiments, methods of fabricating a microneedle device are described herein, the methods comprising: (a) mixing extracellular vesicles (EVs) with a first batch of a polymerizable solution; and (b) casting the mixture from (a) into a polydimethylsiloxane (PDMS) mold having at least one needle-like shape. In some cases, the mixing in (a) is performed under vacuum.

[0218] In some cases, the method further includes further concentrating the EVs at the tip of at least one needle-like shape of the PDMS mold. In some cases, the concentrating is by maintaining the PDMS mold at a temperature of at most 10° C. In some cases, the concentrating is by maintaining the PDMS mold at about 4° C. In some cases, the concentrating lasts for about 2 to about 6 hours.

[0219] In some cases, the method further includes adding a second batch of polymerizable solution onto the PDMS mold. In some cases, the polymerizable solution includes a hydrogel. In some cases, the hydrogel includes hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, the hydrogel includes hyaluronic acid. In some cases, the hydrogel includes at least 5% hyaluronic acid. In some cases, the hydrogel includes at least 5% hyaluronic acid. In some cases, the hydrogel includes about 15% hyaluronic acid. In some cases, the hydrogel includes at least 5% hyaluronic acid and up to 30% hyaluronic acid. In some cases, the hydrogel includes more than 10% hyaluronic acid and up to 20% hyaluronic acid. In some cases, the hydrogel includes more than 10% hyaluronic acid and up to 15% hyaluronic acid. In some cases, the hydrogel comprises more than 10% hyaluronic acid and up to 18% hyaluronic acid.

[0220] In some cases, the final ratio of EVs to polymerizable solution is at least 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:80, 1:90, or 1:100. In some cases, the final ratio of EVs to polymerizable solution is no greater than 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50. In some cases, the final ratio of EVs to polymerizable solution is about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, or 1:40.

[0221] In some cases, the EV comprises exogenous extracellular matrix mRNA. In some cases, the exogenous extracellular matrix mRNA comprises type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the EV comprises exogenous VEGF mRNA. In some cases, the exogenous VEGF mRNA comprises VEGFA, VEGFB, VEGFC, VEGFD, PIGF, or any combination thereof.

[0222] In some embodiments, the present invention provides a method for producing a type I collagen heterodimer or heterotrimer in a tissue, the method comprising administering a plurality of extracellular vesicles disclosed herein to the tissue. In some cases, the heterodimer or heterotrimer comprises at least one type I collagen alpha chain (Col1A1) and at least one type I collagen alpha chain (Col1A2), wherein Col1A1 is exogenously delivered by a plurality of extracellular vesicles disclosed herein. In some cases, Col1A2 is endogenous to the tissue.

[0223] In some embodiments, the present invention provides a plurality of extracellular vesicles, each containing at least one copy of exogenous VEGF mRNA per 400 extracellular vesicles on average.In some cases, the exogenous VEGF mRNA is selected from the group consisting of VEGFA, VEGFB, VEGFC, VEGFD, PIGF, and any combination thereof.In some cases, the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, or any combination thereof.In some cases, the plurality of extracellular vesicles comprises extracellular vesicles containing targeting polypeptides.

[0224] In some cases, the plurality of extracellular vesicles comprises at least one copy of exogenous VEGF mRNA per up to 1, 5, 10, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, or 350 extracellular vesicles. In some cases, the plurality of extracellular vesicles comprises at least 1, 2, 5, 10, 20, 25, 30, 35, 30, 50, 60, 70, 90, or 100 copies of VEGF mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises at least 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 copies of VEGF mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises up to 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 copies of VEGF mRNA per extracellular vesicle. In some cases, the plurality of extracellular vesicles comprises at least one copy of exogenous VEGF mRNA per about 0.001-100, 0.01-100, or 0.01-50 extracellular vesicles. In some cases, the plurality of extracellular vesicles comprises at least one copy of exogenous VEGF mRNA per about 0.02-50 extracellular vesicles.

[0225] In some cases, multiple extracellular vesicles are present, at least 1 x 10 6 , 5×10 6 , 1×107 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, the number of extracellular vesicles may be up to 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 Contains extracellular vesicles.

[0226] In some cases, the extracellular vesicles are formulated for intravenous injection, intramuscular injection, subcutaneous injection, or injection via a coronary catheter.

[0227] In some cases, the mixture includes at least two extracellular vesicles containing 1 to 6 copies of exogenous VEGF mRNA, and the mixture includes exosomes, microvesicles, and apoptotic bodies. In some cases, the mixture is formulated for injection via intravenous, intramuscular, or subcutaneous routes. In some cases, the mixture is formulated for injection via a coronary artery catheter. In some cases, the VEGF mRNA is present at a level at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 500-fold, at least 1000-fold, at least 1500-fold, or at least 2000-fold higher than the level of VEGF mRNA in the same amount of naturally occurring extracellular vesicles. In some cases, the plurality of extracellular vesicles comprises at least 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, or 200 μg of VEGF mRNA. In some cases, the plurality of extracellular vesicles contains VEGF mRNA at or below 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg. In some cases, the plurality of extracellular vesicles contains between about 1 ng and 200 μg of VEGF mRNA.

[0228] Treatment with extracellular vesicles In some embodiments, the present disclosure provides a method for treating a skin condition in a subject, comprising administering at least one extracellular matrix mRNA to a subject in need of such treatment, thereby treating the skin condition. In some cases, the exogenous extracellular matrix mRNA encodes a collagen. In some cases, the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, the collagen is type I collagen. In some cases, the collagen is type II collagen. In some cases, the collagen is the alpha 1 chain of type I collagen (Col1A1). In some cases, the collagen is Col1A1 but not the alpha 2 chain of type I collagen (Col1A2). In some cases, the collagen is Col1A2. In some cases, the exogenous extracellular matrix mRNA is mRNA encoding the pro-alpha 1 (I) chain.

[0229] In some cases, the skin condition is a skin lesion. In some cases, the skin lesion is caused by aging or sun damage. In some cases, the skin condition is a wound.

[0230] In some cases, administering comprises administering to the subject at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of extracellular matrix mRNA. In some cases, administering comprises administering to the subject no more than 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, or 500 μg of extracellular matrix mRNA. In some cases, administering comprises administering to the subject between 1 ng and 20 μg of extracellular matrix mRNA. In some cases, administering comprises administering 1 ng to 10 μg of extracellular matrix mRNA to the subject. In some cases, administering comprises administering 10 ng to 10 μg, 10 ng to 1 μg, 50 ng to 1 μg, 100 ng to 1 μg, or 500 ng to 1 μg of extracellular matrix mRNA to the subject. In some cases, administering comprises administering about 50 ng of extracellular matrix mRNA to the subject.

[0231] In some cases, the extracellular matrix mRNA is encapsulated in at least one extracellular vesicle (EV). In some cases, the EV does not contain one or more of the following miRNAs: hsa-miR-29c-3p, hsa-miR-29a-3p, hsa-miR-378a-3p, hsa-miR-125b-5p, hsa-miR-23a-3p, hsa-miR-449a, hsa-miR-196a-5p, hsa-miR-744-5p, hsa-miR-223-3p, hsa-miR-23a-3p, hsa-miR-133a-3p, hsa-miR-223-3p, hsa-miR-5011-5p, hsa-miR-325, or hsa-miR-199b-5p.

[0232] In some cases, at least one EV is an exosome. In some cases, at least one EV is an apoptotic body or a microvesicle. In some cases, at least one EV comprises a mixture of at least two of exosomes, microvesicles and / or apoptotic bodies.

[0233] In some cases, treating skin damage results in at least a 10% reduction in the appearance of wrinkles. In some cases, at least a 10% reduction in the appearance of wrinkles occurs within 7 to 14 days after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject. In some cases, treating skin damage results in at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the appearance of wrinkles. In some cases, at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduction in the appearance of wrinkles occurs within 7 to 14 days after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject. In some cases, treating skin damage results in at least a 30%, 40%, 50%, 60%, 70%, or 80% reduction in the total number of wrinkles. In some cases, treating the skin damage results in a reduction of about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in total wrinkle count. In some cases, treating the skin damage results in a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in total wrinkle area. In some cases, treating the skin damage results in a reduction of about 70%, 80%, or 90% in total wrinkle area.

[0234] In some cases, treating the skin condition includes treating skin damage, and treating the skin damage results in a reduction in skin damage that persists for at least 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days after treating the skin condition. In some cases, the reduction in skin damage is one or more of a reduction in total wrinkle number, a reduction in total wrinkle area, a reduction in the appearance of wrinkles, or any combination thereof.

[0235] In some cases, the administering is by subcutaneous injection. In some cases, the subcutaneous injection is performed at or near the site of a skin injury or wound. In some cases, the subcutaneous injection is performed using a needle with at least a 14 gauge. In some cases, the subcutaneous injection is performed using a needle with a gauge of 34, 33, 32, 31, 30, 29, 28, 27, 26s, 26, 25s, 25, 24, 23s, 23, 22s, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10. In some cases, the subcutaneous injection is performed using a 28 gauge needle.

[0236] In some cases, administering comprises administering at least 1 x 10 extracellular vesicles to a subject in need thereof. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, administering includes administering up to 1 x 10 extracellular vesicles to a subject in need thereof, wherein at least some of the extracellular vesicles comprise at least one extracellular matrix mRNA. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12, 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, administering comprises administering about 1 x 10 extracellular vesicles to a subject in need thereof, at least some of the extracellular vesicles comprising at least one extracellular matrix mRNA. 7 ~1×10 17 , 1×10 8 ~1×10 16 , 1×10 9 ~1×10 15 In some cases, administering comprises administering about 1 x 10 extracellular vesicles to a subject in need thereof, at least some of the extracellular vesicles comprising at least one extracellular matrix mRNA. 10 ~1×10 14 The method includes administering extracellular vesicles, at least some of which contain at least one extracellular matrix mRNA.

[0237] In some cases, extracellular vesicles are administered in multiple doses or as a single dose. For example, extracellular vesicles can be administered to a subject multiple times until the skin condition is treated. In other cases, a single dose is sufficient to achieve a therapeutic effect, and therefore the subject can only receive a single dose of extracellular vesicles. In some cases, the single dose is repeated at a later time to maintain the therapeutic effect on the skin condition. In some cases, the multiple dose regimen is repeated at a later time to maintain the therapeutic effect on the skin condition. In some cases, the therapeutic effect decreases over time. For example, the therapeutic effect may decrease after at least 30 days, at least 45 days, at least 60 days, at least 75 days, at least 90 days, etc. In such cases, a single dose regimen or a multiple dose regimen can be administered to the subject again to treat the skin condition. In some cases, the single dose is administered using a single container, such as a single needle or syringe. In some cases, the single dose is administered using a microneedle device, and multiple microneedles on the device deliver the single dose. In some cases, at least one microneedle of the microneedle device contains a dose of extracellular vesicles sufficient to treat the skin condition. In some cases, the extracellular vesicles are administered to a subject at intervals of at least once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks. In some cases, the extracellular vesicles are administered to a subject at most once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks. In some cases, the administration is performed once and at most 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×1011 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 extracellular vesicles are administered to a subject. In some cases, the administration is a single dose and comprises at least 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 or 1×10 30 Extracellular vesicles are administered to a subject. In some cases, the extracellular vesicles are administered in at least one dose containing at least 1,000 extracellular vesicles, and at least a portion of the extracellular vesicles contain at least one extracellular matrix mRNA. In some cases, the extracellular vesicles are administered in at least one dose containing at least 5,000 extracellular vesicles, and at least a portion of the extracellular vesicles contain at least one extracellular matrix mRNA. In some cases, the extracellular vesicles are administered in up to one dose containing at least 1,000 extracellular vesicles, and at least a portion of the extracellular vesicles contain at least one extracellular matrix mRNA. In some cases, the extracellular vesicles are administered in up to one dose containing at least 5,000 extracellular vesicles, and at least a portion of the extracellular vesicles contain at least one extracellular matrix mRNA. In some cases, administering comprises administering at least one extracellular matrix mRNA to a tissue of a subject.

[0238] In some cases, the tissue is subcutaneous tissue. In some cases, the tissue is dermis. In some cases, the tissue is epidermis. In some cases, the tissue is gum. In some cases, the tissue is lip.

[0239] In some cases, within 72 hours after administering at least one extracellular vesicle containing extracellular matrix mRNA to a subject, the tissue has an extracellular matrix protein concentration of at least 6000 pg / ml. In some cases, after administering the extracellular matrix mRNA to a subject's tissue, the subject's tissue has an extracellular matrix protein concentration of at least 200, 300, 500, 750, 1000, 2000, 3000, 4000, or 5000 pg / ml. In some cases, after administering the extracellular vesicles containing extracellular matrix mRNA to a subject's tissue, the subject's tissue has an extracellular matrix protein concentration of at least 200, 300, 500, 750, 1000, 2000, 3000, 4000, or 5000 pg / ml. In some cases, within 72 hours after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject, the tissue has an extracellular matrix protein concentration of at least 200, 300, 500, 750, 1000, 2000, 3000, 4000, or 5000 pg / ml. In some cases, within about 4 days after administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject, the tissue has a peak extracellular matrix protein concentration.

[0240] In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated at least every day, every 3 days, every 5 days, every 7 days, every 10 days, every 20 days, every 30 days, every 40 days, or every 50 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 10 days, every 20 days, every 30 days, every 40 days, or every 50 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 30 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 60 days. In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated about every 90 days.

[0241] In some cases, the method further includes producing extracellular vesicles containing at least one extracellular matrix mRNA by (a) introducing a vector or plasmid corresponding to the extracellular matrix mRNA into donor cells via transfection, (b) culturing the donor cells in culture medium for a sufficient time to produce extracellular vesicles encapsulating at least one extracellular matrix mRNA transcribed from the vector or plasmid, and (c) recovering the extracellular vesicles from the culture medium. In some cases, in step (c), the recovery of the extracellular vesicles is performed about 8 to 24 hours after transfection. In some cases, the extracellular vesicles are present at a ratio of at least 1,000, 2,000, 5,000, 10,000, or 12,000 extracellular vesicles per donor cell. In some cases, the donor cells comprise human cells, human fibrocytes, fibroblasts, dermal fibroblasts, human fibroblasts, adult fibroblasts, human adult fibroblasts, neonatal fibroblasts, neonatal human fibroblasts, neonatal human dermal fibroblasts, or any combination thereof.

[0242] The present invention provides a method for treating a disease in a subject by administering a therapeutically effective amount of a composition or pharmaceutical composition comprising the extracellular vesicles described herein.In some cases, the extracellular vesicles comprise at least one therapeutic agent described herein.In some cases, the therapeutic agent comprises a therapeutic polynucleotide, a therapeutic polypeptide, a therapeutic compound, or a combination thereof.In some cases, the therapeutic polynucleotide comprises extracellular matrix RNA (for example, collagen or elastin, or a mixture of collagen and elastin) or VEGF RNA (for example, VEGFA, or a mixture of VEGF).

[0243] In some cases, the extracellular vesicle comprises at least one targeting polypeptide and at least one therapeutic agent as described herein.In some cases, the targeting polypeptide comprises a heterologous targeting domain, including a tumor targeting domain, a tissue targeting domain, a cell-penetrating peptide, a viral membrane protein, or any combination or fragment thereof.In some cases, each targeting domain binds to a cell surface marker associated with diseased cells, and when bound to diseased cells, the extracellular vesicle delivers at least one therapeutic agent to diseased cells.

[0244] In some cases, the targeted cellular uptake of a therapeutic agent delivered by extracellular vesicles comprising at least one targeting polypeptide is increased by at least 0.1-fold, 0.2-fold, 0.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1,000-fold, 5,000-fold, 10,000-fold, or more compared to the targeted cellular uptake of a therapeutic agent delivered by extracellular vesicles not comprising a targeting polypeptide. In some cases, the targeted cells exhibiting increased uptake of a therapeutic agent delivered by extracellular vesicles comprising at least one targeting polypeptide are cells that are part of a tissue (e.g., skin cells such as melanocytes).

[0245] In some cases, described herein are methods for treating blood flow disorders by administering extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) described in the present disclosure to a subject with a blood flow disorder. In some cases, described herein are methods for treating ischemia in a subject using extracellular vesicles containing a therapeutic polynucleotide, such as VEGF mRNA. In some cases, the ischemia is selected from the group consisting of cerebral ischemia, ischemic heart disease, myocardial ischemia, cardiac ischemia, critical limb ischemia, renal ischemia, acute mesenteric ischemia, intestinal ischemia, cyanosis, and gangrene. In some cases, the therapeutic polynucleotide delivered to the ischemic tissue comprises an mRNA encoding a full-length or truncated protein. In some cases, the therapeutic polynucleotide delivered to the ischemic tissue encodes a VEGF protein. In some cases, delivery of the therapeutic polynucleotide stimulates revascularization of the ischemic tissue.

[0246] In some cases, the method includes administering to the subject a single dose of extracellular vesicles containing VEGF mRNA. In some cases, the single dose is at least 1 x 10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 In some cases, a single dose is 1 x 10 EVs. 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×1015 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , 1×10 21 , 1×10 22 , 1×10 23 , 1×10 24 , 1×10 25 , 1×10 30 , 1×10 40 , 1×10 50 , or 1 × 10 60 In some cases, a single dose is about 1 x 10 EV or less. 10 ~1×10 16 In some cases, the single dose is at least 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, or 200 μg of VEGF mRNA. In some cases, the single dose is no more than 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg of VEGF mRNA. In some cases, the single dose is about 1 ng to 200 μg of VEGF mRNA.

[0247] In some cases, the method includes administering multiple doses of extracellular vesicles containing VEGF mRNA to a subject. In some cases, the multiple doses are administered at least once a day, once a week, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every ten weeks, once every twelve weeks, or once every sixteen weeks over a period of time. In some cases, the multiple doses are administered for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, or at least 14 weeks. In some cases, at least one of the multiple doses is administered for at least 1×10 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 In some cases, at least one of the multiple doses contains 1 x 10 EVs. 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 , 1×10 18 , 1×10 19 , 1×10 20 , 1×10 21 , 1×10 22 , 1×1023 , 1×10 24 , 1×10 25 , 1×10 30 , 1×10 40 , 1×10 50 , or 1 × 10 60 In some cases, at least one of the multiple doses contains about 1 x 10 EVs or less. 10 ~1×10 16 In some cases, at least one of the multiple doses is at least 10 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, or 200 μg of VEGF mRNA. In some cases, at least one of the multiple doses is 1 ng, 100 ng, 500 ng, 1000 ng, 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, or 1000 μg or less of VEGF mRNA. In some cases, a single dose is about 1 ng to 200 μg of VEGF mRNA.

[0248] In some cases, described herein are methods for treating skin damage by administering to a subject extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) described herein. In some cases, described herein are methods for treating sun damage or age-related skin damage in a subject using extracellular vesicles containing a therapeutic polynucleotide. In some cases, the skin damage includes wrinkles. In some cases, described herein are methods for treating wrinkles with a therapeutic polynucleotide delivered to the skin by an extracellular vesicle described herein. In some cases, the therapeutic polynucleotide delivered to the skin includes mRNA encoding a full-length or truncated protein. In some cases, the therapeutic polynucleotide delivered to the skin encodes an ECM protein. In some cases, the therapeutic polynucleotide delivered to the skin encodes type I collagen. In some cases, delivery of the therapeutic polynucleotide reduces the appearance of wrinkles in damaged skin.

[0249] In some cases, the present invention provides a method for treating diseases or conditions by administering extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) to a subject who needs to treat the disease or condition.In some cases, the extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) are administered systemically, for example, via intravenous injection.In some cases, the extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) are administered locally, for example, via intramuscular or subcutaneous injection.

[0250] In some cases, the intravenous, intramuscular, or subcutaneous injection is performed using a hypodermic needle. In some cases, the intravenous, intramuscular, or subcutaneous injection is performed using a needle with a gauge of at least 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14. In some cases, the intravenous, intramuscular, or subcutaneous injection is performed using a needle with a gauge of at most 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32. In some cases, the injection is performed through a coronary catheter, such as a coronary artery catheter or a coronary vein catheter. In some cases, the injection is performed through a large vessel catheter. In some cases, the intravenous, intramuscular, or subcutaneous injection is performed using a 28-gauge needle. In some cases, intravenous, intramuscular, or subcutaneous injection is performed using a 14-gauge or larger needle. In some cases, delivery to the heart (e.g., delivery to the heart for treating myocardial infarction) is achieved through a coronary catheter (e.g., a coronary artery catheter or a coronary vein catheter). In some cases, delivery for the treatment of stroke (e.g., delivery for the treatment of ischemic stroke or hemorrhagic stroke) is achieved through a catheter in one of the cerebral blood vessels or through direct open-heart injection into the muscle. In some cases, delivery for the treatment of peripheral arterial disease is achieved through a catheter into a blood vessel or direct injection into the muscle. In some cases, delivery for the treatment of skin diseases or disorders (e.g., delivery for skin ulcers) is achieved by subcutaneous injection.

[0251] In some cases, the extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) are formulated for administration by injection in a solution or suspension containing an injection buffer.The injection buffer can include saline, phosphate buffered saline (PBS), and / or 25 mM trehalose PBS.In some cases, the dose is measured by the number of extracellular vesicles administered per dose. In some cases, the doses are about 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000, 1,000,000,000,000, 10,000,000,000,000, 100,000,000,000,000, 1,000,000,000 ,000,000, 10,000,000,000,000,000, 100,000,000,000,000,000, 1,000,000,000,000,000,000, 10,000,000,000,000,000,000, 100,000,000,000,000,000, or 1,000,000,000,000,000,000,000,000 therapeutic polypeptides.In some cases, the dose is about 100 to 1,000,000,000,000,000,000,000 extracellular vesicles comprising a therapeutic polypeptide per dose, or about 1,000 to 100,000,000,000,000,000,000 per dose, or about 10,000 to 10,000,000,000,000,000,000, is between about 100,000 and 1,000,000,000,000,000,000, or between about 1,000,000 and 1,000,000,000,000,000,000, or between about 10,000,000 and 1,000,000,000,000,000,000, or between about 100,000,000 and 1,000,000,000,000,000 ,000, or about 1,000,000,000 to 1,000,000,000,000,000,000, or about 10,000,000,000 to 1,000,000,000,000,000,000, or about 100,000,000,000 to 1,000,000,000,000,000,000, or about 1,000,000, The therapeutic polypeptide may comprise extracellular vesicles comprising about 10,000,000 to 1,000,000,000,000,000,000, or about 10,000,000,000,000 to 1,000,000,000,000,000, or about 100,000,000,000,000 to 1,000,000,000,000,000,000,000 therapeutic polypeptides. In some cases, the dose is measured by the amount of therapeutic polypeptide administered per dose. In some cases, the dose comprises about 1 ng to 500 μg of therapeutic polypeptide per dose. In some cases, the dose comprises about 10 ng to 100 μg of therapeutic polypeptide per dose. In some cases, the dose comprises about 100 ng to 100 μg, or about 1 μg to 100 μg, or about 10 μg to 100 μg of therapeutic polypeptide per dose.

[0252] In some cases, methods for treating diseases or conditions are described herein by administering extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) to a subject in need of such treatment. In some cases, the extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) are administered daily, every day, every other day, 5 days a week, once a week, every other week, 2 weeks a month, 3 weeks a month, once a month, twice a month, 3 times a month, or more. The extracellular vesicles containing therapeutic polynucleotides (and optionally targeting polypeptides) can be administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or longer.

[0253] If the subject's condition improves, the dose of the administered extracellular vesicles containing the therapeutic polynucleotide (and optionally the targeting polypeptide) can be temporarily reduced or temporarily suspended for a period of time (a "drug holiday"). In some cases, the length of the drug holiday varies from 2 days to 1 year, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during the drug holiday can be, by way of example, 10% to 100%, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0254] In some cases, a subject in need of an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) can be administered an effective amount of extracellular vesicles comprising an adaptor polypeptide and a therapeutic polynucleotide once per week, once per 2 weeks, once per 3 weeks, once per 4 weeks, once per 5 weeks, once per 6 weeks, once per 7 weeks, once per 8 weeks, once per 9 weeks, once per 10 weeks, once per 11 weeks, once per 12 weeks, once per 13 weeks, once per 14 weeks, once per 15 weeks, once per 16 weeks, once per 17 weeks, once per 18 weeks, once per 19 weeks, once per 20 weeks, once per 21 weeks, once per 22 weeks, once per 23 weeks, once per 24 weeks, once per 25 weeks, once per 26 weeks, once per 27 weeks, or once per 28 weeks.

[0255] Once improvement of the subject's disease or condition has occurred, a maintenance dose of extracellular vesicles is administered as needed. Thereafter, the dosage or frequency of administration, or both, can be reduced as a function of symptoms to a level at which the improved disease, disorder, or condition is maintained.

[0256] In some cases, the amount of extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) corresponding to such an amount will vary depending on factors such as the severity of the disease or condition, the identity (e.g., weight) of the subject or host requiring treatment, etc., but will nevertheless be routinely determined by methods known in the art according to the specific circumstances surrounding the case, including, for example, the specific extracellular vesicles to be administered, the route of administration, and the subject or host to be treated. In some cases, the desired dose is conveniently presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example, as 2, 3, 4 or more partial doses per day. In some cases, the desired dose is administered in a single dose. In some cases, a relatively high dose of extracellular vesicles (e.g., at least 1 × 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×108 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 extracellular vesicles) are administered in a single dose. In some cases, a relatively high dose of exosomes (e.g., 1 x 10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 exosomes) are administered in a single dose.

[0257] In some cases, dosage may be determined at least in part by the occurrence or severity of grade 3 or grade 4 adverse events in a subject. Non-limiting examples of adverse events include hypothermia; shock; bradycardia; ventricular extrasystoles; myocardial ischemia; syncope; bleeding; atrial arrhythmia; phlebitis; second-degree atrioventricular (AV) block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disease; stomatitis; nausea and vomiting; abnormal liver function tests; gastrointestinal bleeding; hematemesis; bloody diarrhea; gastrointestinal disorders; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; increased alkaline phosphatase; increased blood urea nitrogen (BUN); hyperuricemia; increased non-protein nitrogen (NPN); respiratory acidosis; somnolence; agitation; neuropathy; paranoid reactions; convulsions; grand mal seizures; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; pupillary disturbances; abnormal renal function; renal failure; acute These include: renal tubular necrosis; duodenal ulcer; intestinal necrosis; myocarditis; supraventricular tachycardia; permanent or transient blindness secondary to optic neuritis; transient ischemic attack; meningitis; cerebral edema; pericarditis; allergic interstitial nephritis; tracheoesophageal fistula; malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; hepatic or renal failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure; leukopenia, thrombocytopenia, increased alanine aminotransferase (ALT), anorexia, joint pain, back pain, chills, diarrhea, dyslipidemia, fatigue, fever, flu-like symptoms, hypoalbuminemia, increased lipase, injection site reactions, myalgia, nausea, night sweats, pruritus, rash, erythematous rash, maculopapular rash, transaminitis, vomiting, and weakness.

[0258] The above ranges are merely suggestive, as the number of variables regarding any particular treatment regimen is large and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on a number of variables, including but not limited to, the activity of the compound used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0259] In some cases, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that includes the ED50 with minimal toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized.

[0260] In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in at least one dose. In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in at least two doses. In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in at least two doses. Extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide), including mRNA, at least 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000, 1,000,000,000, 10,000,000,000, 100,000 Contains ,000,000,000,000, 1,000,000,000,000,000, 10,000,000,000,000,000, 100,000,000,000,000,000, 1,000,000,000,000,000,000, 10,000,000,000,000,000,000, 100,000,000,000,000,000, or 1,000,000,000,000,000,000,000.

[0261] In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject disclosed herein at at least 3 doses / week, at least 4 doses / week, at least 5 doses / week, at least 6 doses / week, at least 7 doses / week, at least 8 doses / week, at least 9 doses / week, or at least 10 doses / week. In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject disclosed herein at less than 20 doses / week, less than 19 doses / week, less than 18 doses / week, less than 17 doses / week, less than 16 doses / week, less than 15 doses / week, less than 14 doses / week, less than 13 doses / week, less than 12 doses / week, less than 11 doses / week, or less than 10 doses / week. In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising an ECM mRNA is administered to a subject as disclosed herein in 1 to 10 doses per week, 2 to 9 doses per week, or 3 to 8 doses per week.

[0262] In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in one dose, wherein the effective amount is at least 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, , 100,000,000,000,000, 1,000,000,000,000,000, 10,000,000,000,000,000, 100,000,000,000,000,000, 1,000,000,000,000,000,000, 10,000,000,000,000,000,000, 100,000,000,000,000,000,000, or 1,000,000,000,000,000,000,000,000 extracellular vesicles. In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optional targeting polypeptide) comprising ECM mRNA is administered to a subject in one dose, wherein the effective amount is at least 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7 ng, 0.8 ng, 0.9 ng, 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1 μg of ECM mRNA. In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optional targeting polypeptide) comprising ECM mRNA is administered to a subject in a single dose, wherein the effective amount is up to 50 μg, 40 μg, 30 μg, 20 μg, 10 μg, 1 μg, 900 ng, 800 ng, 700 ng, 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, or 100 ng of ECM mRNA.In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in a single dose, wherein the effective amount is 0.1 ng to 50 μg, 0.1 ng to 30 μg, 1 ng to 20 μg, 1 ng to 10 μg, 1 ng to 1 μg, 1 ng to 500 ng, 1 ng to 100 ng, 10 ng to 10 μg, 10 ng to 1 μg, 10 ng to 500 ng, 10 ng to 100 ng, 50 ng to 10 μg, 50 ng to 1 μg, 50 ng to 500 ng, or 50 ng to 100 ng.

[0263] In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in multiple doses, wherein in each dose, the effective amount is at least 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, ,000, 100,000,000,000,000, 1,000,000,000,000,000, 10,000,000,000,000,000, 100,000,000,000,000,000, 1,000,000,000,000,000,000, 10,000,000,000,000,000,000, 100,000,000,000,000,000, or 1,000,000,000,000,000,000,000,000 extracellular vesicles. In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optional targeting polypeptide) comprising ECM mRNA is administered to a subject in multiple doses, wherein in each dose the effective amount is at least 0.1 ng, 0.2 ng, 0.3 ng, 0.4 ng, 0.5 ng, 0.6 ng, 0.7 ng, 0.8 ng, 0.9 ng, 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, or 1 μg of ECM mRNA. In other cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optional targeting polypeptide) comprising ECM mRNA is administered to a subject in multiple doses, with each dose having an effective amount of up to 50 μg, 40 μg, 30 μg, 20 μg, 10 μg, 1 μg, 900 ng, 800 ng, 700 ng, 600 ng, 500 ng, 400 ng, 300 ng, 200 ng, or 100 ng of ECM mRNA.In other cases, an effective amount of extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to a subject in multiple doses, wherein the effective amount in each dose is 0.1 ng to 50 μg, 0.1 ng to 30 μg, 1 ng to 20 μg, 1 ng to 10 μg, 1 ng to 1 μg, 1 ng to 500 ng, 1 ng to 100 ng, 10 ng to 10 μg, 10 ng to 1 μg, 10 ng to 500 ng, 10 ng to 100 ng, 50 ng to 10 μg, 50 ng to 1 μg, 50 ng to 500 ng, or 50 ng to 100 ng. When an effective amount of extracellular vesicles is administered multiple times, the interval between administrations is at least 1 day, 5 days, 10 days, 15 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, or 120 days.

[0264] In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject comprises administering at least one extracellular vesicle comprising extracellular matrix mRNA to the tissue of the subject.In some specific cases, the tissue is subcutaneous tissue.In some specific cases, the tissue is dermis.

[0265] In some cases, to treat skin damage of a subject, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to the subject.In some cases, the effective amount is sufficient to cause at least a 50% reduction in the appearance of wrinkles.In some cases, the effective amount is sufficient to cause at least a 10%, 20%, 30%, 40% or 50% reduction in the appearance of wrinkles.In some cases, the effective amount is sufficient to cause at least a 50% reduction in the appearance of wrinkles within about 1, 2, 3, 4 or 5 days after administration.In some cases, the effective amount is sufficient to cause at least a 50% reduction in the appearance of wrinkles within about 6, 7, 8, 9 or 10 days after administration.In some cases, the effective amount is sufficient to cause at least a 10%, 20%, 30%, 40% or 50% reduction in the appearance of wrinkles within about 14 days after administration. In some cases, the effective amount is sufficient to cause at least a 10%, 20%, 30%, 40%, or 50% reduction in the appearance of wrinkles within about 5 days of administration.

[0266] In some cases, treating the skin damage results in a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total number of wrinkles. In some cases, treating the skin damage results in a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total number of wrinkles. In some cases, treating the skin damage results in a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total wrinkle area. In some cases, treating the skin damage results in a reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total wrinkle area.

[0267] In some cases, to treat the skin damage of a subject, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA is administered to the subject.In some cases, the effective amount is sufficient to produce a tissue concentration of at least 6000 pg / ml of extracellular matrix protein (e.g., collagen) within about 72 hours after administration.

[0268] In some cases, the tissue has a peak concentration of the extracellular matrix protein within about four days after administering to the subject at least one extracellular vesicle comprising extracellular matrix mRNA.

[0269] In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated approximately every day, every day, every 5 days, every 10 days, every 20 days, every 30 days, every 40 days, every 50 days, every 60 days, every 70 days, every 80 days, every 90 days, or every 100 days. In specific cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject is repeated approximately every 30 days.

[0270] In some cases, administering at least one extracellular vesicle comprising extracellular matrix mRNA comprises administering at least one extracellular vesicle comprising extracellular matrix mRNA to a subject via subcutaneous injection.In some specific cases, the subcutaneous injection is carried out at or near the site of skin injury.In some cases, the subcutaneous injection is carried out using a needle with a gauge of about 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, or 14.In some cases, the subcutaneous injection is carried out using a needle with a gauge of at least 20, 19, 18, 17, 16, 15, or 14.

[0271] In some cases, skin damage is caused by aging. In other cases, skin damage is caused by sun damage. In other cases, skin damage is caused by autoimmune diseases. In other cases, skin damage is caused by burns. In other cases, skin damage is caused by scarring. In other cases, skin damage is caused by skin diseases. In some specific cases, skin damage is caused by cutis laxa Ehlers-Danlos syndrome. In some specific cases, skin damage is caused by epidermolysis bullosa. In some specific cases, skin damage is caused by pseudosyndactyly. In some specific cases, skin damage is caused by poor wound healing. In some specific cases, skin damage is caused by contractures of the fingers and hands. In some specific cases, skin damage is caused by keratosis. In some specific cases, skin damage is caused by systemic sclerosis. In some specific cases, skin damage is caused by scleroderma. In some specific cases, skin damage is caused by atopic dermatitis. In some specific cases, skin damage is caused by periodontal disease.

[0272] In some cases, the method for treating skin damage in a subject includes producing extracellular vesicles comprising extracellular matrix mRNA, wherein producing the extracellular vesicles comprising extracellular matrix mRNA includes (a) introducing a vector or plasmid encoding an extracellular matrix protein into donor cells via transfection; (b) incubating the donor cells for a period of time sufficient to produce extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid; and (c) recovering the extracellular vesicles encapsulating the extracellular matrix mRNA transcribed from the vector or plasmid.

[0273] In some cases, in step (a), transfection is performed using cell electroporation, as shown in FIG. 7A. In some specific cases, in step (a), transfection involves plating a monolayer of donor cells on the surface of a silicon chip as shown in FIG. 7A, and applying an electric field of a certain voltage with a certain pulse and duration to electroporate the vector or plasmid into the donor cells. In some further specific cases, plating requires overnight incubation as described above. In some further specific cases, the voltage of the electric field is at least 10V, 50V, 100V, 150V, 200V, 250V, 300V, 500V, or 1000V. In some further specific cases, the voltage of the electric field is about 10V, 50V, 100V, 150V, 200V, 250V, 300V, 500V, or 1000V. In some more specific cases, the voltage of the electric field is at most 500V, 600V, 700V, 800V, 900V, 1000V, 1500V, 2000V, 5000V, or 10,000V. In some more specific cases, the pulses of the electric field are at least 5, 10, 15, 20, 25, 30, 40, 50, or 60. In some more specific cases, the pulses of the electric field are about 5, 10, 15, 20, 25, 30, 40, 50, or 60. In some more specific cases, the interval between pulses of the electric field is at least 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, or 1 s. In some more specific cases, the interval between pulses of the electric field is about 0.1 s, 0.2 s, 0.3 s, 0.4 s, 0.5 s, 0.6 s, 0.7 s, 0.8 s, 0.9 s, or 1 s.

[0274] In some cases, in step (c), the recovery of extracellular vesicles is performed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after transfection. In some cases, in step (c), the recovery of extracellular vesicles is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after transfection.

[0275] In some cases, extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising ECM mRNA are administered to a subject via subcutaneous injection to treat the subject's skin damage (e.g., sun damage or age-related).

[0276] In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising VEGF mRNA is administered to a subject in at least one dose. In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising VEGF mRNA is administered to a subject in at least two doses. In some cases, an effective amount of extracellular vesicles comprising a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising VEGF mRNA is administered to a subject in at least two doses. Extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide), including mRNA, at least 100, 1,000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, 10,000,000,000, 100,000,000,000, 1,000,000,000, 10,000,000,000, 100,000 Contains ,000,000,000,000, 1,000,000,000,000,000, 10,000,000,000,000,000, 100,000,000,000,000,000, 1,000,000,000,000,000,000, 10,000,000,000,000,000,000, 100,000,000,000,000,000, or 1,000,000,000,000,000,000,000.

[0277] In some cases, to treat the blood flow disorder of a subject, an effective amount of extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising VEGF mRNA is administered to the subject.In some cases, the effective amount is sufficient to cause at least a 50% increase in revascularization and / or angiogenesis.In some cases, the effective amount is sufficient to cause at least a 10%, 20%, 30%, 40%, or 50% increase in revascularization and / or angiogenesis.In some cases, the effective amount is sufficient to cause at least a 50% increase in revascularization and / or angiogenesis within about 1, 2, 3, 4, 5, 6, 7, or 8 days after administration.In some cases, the effective amount is sufficient to cause at least a 5%, 10%, 20%, 30%, 40%, or 50% increase in revascularization and / or angiogenesis within about 8 days after administration.In some cases, the effective amount is sufficient to cause at least a 5% increase in revascularization and / or angiogenesis within 8 days after administration. In some cases, the effective amount is sufficient to cause an increase in revascularization and / or neovascularization of about 5% to 10% within 8 days of administration. In some cases, the effective amount is sufficient to cause an increase in revascularization and / or neovascularization of about 5% to 15%, or about 5% to 20%, or about 5% to 25%, or about 5% to 30%, or about 5% to 40%, or about 5% to 50%, or about 5% to 60%, or about 5% to 70%, or about 5% to 75% within 8 days of administration. In some cases, the effective amount is sufficient to cause at least a 5% increase in revascularization and / or neovascularization within 14 days of administration. In some cases, the effective amount is sufficient to cause an increase in revascularization and / or neovascularization of about 5% to 10% within 14 days of administration. In some cases, the effective amount is sufficient to cause an increase in revascularization and / or neovascularization of about 5% to 15%, or about 5% to 20%, or about 5% to 25%, or about 5% to 30%, or about 5% to 40%, or about 5% to 50%, or about 5% to 60%, or about 5% to 70%, or about 5% to 75% within 14 days of administration.

[0278] In some cases, to treat the blood flow disorder of the subject, an effective amount of extracellular vesicles comprising the therapeutic polynucleotide (and optionally targeting polypeptide) comprising VEGF mRNA is administered to the subject.In some cases, the effective amount is sufficient to produce the tissue concentration of VEGF protein (e.g., VEGFA) of at least 6000pg / ml within about 72 hours after administration.

[0279] In some cases, extracellular vesicles containing a therapeutic polynucleotide (and optionally a targeting polypeptide) comprising VEGF mRNA are administered to a subject via intravenous or intramuscular injection to treat the subject's blood flow disorder (e.g., ischemia).

[0280] Further provided herein is a method for reducing the risk of pseudosyndactyly, poor wound healing, or contractures of fingers and limbs in a subject in need thereof, the method comprising administering an effective amount of EVs loaded with COL7A1 mRNA to the subject. In some cases, the subject is a patient with dystrophic epidermolysis bullosa. In some cases, the patient is a pediatric patient. In some cases, the administering is to the subject's hands and / or feet. In some cases, the effective amount of Col7A1 mRNA is at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of Col7A1 mRNA. In some cases, the effective amount of Col7A1 mRNA is incorporated into at least one extracellular vesicle. In some cases, the administering comprises administering at least 1×10 extracellular vesicles to the subject in need thereof. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×1013 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 The method includes administering extracellular vesicles, at least some of which contain at least one Col7A1 mRNA. In some cases, the at least one extracellular vesicle is injected via a needle, hydrogel needle, microneedle, or microneedle device described herein.

[0281] Further provided herein is a method for preventing, alleviating, or treating hair loss in a subject in need thereof, the method comprising administering an effective amount of Col17a1 mRNA to the subject. In some cases, the administration is to the affected scalp of the subject. In some cases, the effective amount of Col17a1 mRNA is at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of Col17a1 mRNA. In some cases, the effective amount of Col17a1 mRNA is 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, or 150 μg or less of Col17a1 mRNA. In some cases, the effective amount of Col17a1 mRNA is incorporated into at least one extracellular vesicle. In some cases, administering comprises administering at least 1×10 extracellular vesicles to a subject in need thereof. 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×1012 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 The method includes administering extracellular vesicles, at least some of which contain at least one Col17a1 mRNA. In some cases, the at least one extracellular vesicle is injected via a needle, hydrogel needle, microneedle, or microneedle device described herein.

[0282] Further provided herein are methods for preventing, alleviating, or treating palmoplantar keratosis in a subject in need thereof, the methods comprising administering to the subject an effective amount of viable keratin. In some cases, the administering is to the affected skin of the subject.

[0283] Further provided herein is a method for preventing, alleviating or treating keloid scars in a subject in need thereof, the method comprising administering to the subject an effective amount of an inhibitor that reduces TGF-β1, SMAD3 and EPHB2.

[0284] Further provided herein is a method for injecting into a subject's lips, the method comprising administering to the subject an effective amount of EVs comprising at least one collagen mRNA.In some cases, the at least one collagen mRNA is type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, an effective amount of EVs comprises at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of collagen mRNA. In some cases, an effective amount of EVs comprising at least one collagen mRNA comprises at least 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, an effective amount of EVs is injected through a needle, hydrogel needle, microneedle, or microneedle device described herein.

[0285] Further provided herein is a method for injecting into a subject's gums, the method comprising administering to the subject an effective amount of EVs comprising at least one collagen mRNA. In some cases, the at least one collagen mRNA is type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In some cases, an effective amount of EVs comprises at least 1 ng, 10 ng, 20 ng, 40 ng, 50 ng, 100 ng, 500 ng, 1 μg, 10 μg, 20 μg, 30 μg, 40 μg, or 50 μg of collagen mRNA. In some cases, an effective amount of EVs comprising at least one collagen mRNA comprises at least 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 3×10 8 , 5×10 8 , 1×10 9 , 3×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 20 , 1×10 25 , or 1 × 10 30 In some cases, an effective amount of EVs is injected through a needle, hydrogel needle, microneedle, or microneedle device described herein.

[0286] Further provided herein is a method for administering an effective amount of EVs of any cargo to a subject in need thereof. In some cases, the cargo is a microRNA (miRNA). In some cases, the cargo is a small interfering RNA (siRNA). In some cases, the cargo is a peptide. In some cases, the cargo is a protein. In some cases, the cargo is an inhibitory double-stranded RNA (dsRNA). In some cases, the cargo is a small or short hairpin RNA (shRNA). In some cases, the cargo is an antisense oligonucleotide (ASO). In some cases, the cargo is a piwi-interacting RNA (piRNA). In some cases, the cargo is a heterologous nuclear RNA (hnRNA). In some cases, the cargo is a small nuclear RNA (snRNA). In some cases, the cargo is an enzymatically prepared siRNA (esiRNA). In some cases, the cargo is a precursor of any of the above cargoes. In some cases, the cargo is any combination of two or more of the above cargoes.

[0287] Further described herein is a microneedle device, the microneedle device comprising a substrate and a plurality of microneedles, the plurality of microneedles protruding from the substrate, and at least one microneedle of the plurality of microneedles comprising at least one extracellular vesicle (EV).

[0288] In some cases, the substrate provides a surface onto which the plurality of microneedles are attached. In some cases, the microneedle devices disclosed herein include a substrate and other components other than the plurality of microneedles. In some specific cases, other components are added to improve skin penetration and depth, and drug delivery and / or distribution.

[0289] In some cases, the plurality of microneedles comprises one microneedle. In other cases, the plurality of microneedles comprises at least 1, at least 2, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 10,000, or at least 100,000 microneedles. In some cases, the area of ​​the substrate is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 mm 2 In some cases, the area of ​​the substrate is 1000 mm 2 Less than 900mm 2 Less than 800mm 2 Less than 700mm 2 Less than 600mm 2 Less than 500mm 2 Less than 400mm 2 Less than 300mm 2 Less than 200mm 2 In some specific cases, the area of ​​the substrate is less than about 169 mm 2 is.

[0290] In some cases, the density of the microneedles on the substrate is such that 2 In other cases, the density of the plurality of microneedles on the substrate is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1 microneedle per mm of substrate. 2In particular cases, the density of the plurality of microneedles on the substrate may be less than 100 microneedles, less than 90 microneedles, less than 80 microneedles, less than 70 microneedles, less than 60 microneedles, less than 50 microneedles, less than 40 microneedles, less than 30 microneedles, less than 20 microneedles, less than 10 microneedles, less than 9 microneedles, less than 8 microneedles, less than 7 microneedles, less than 6 microneedles, less than 5 microneedles, less than 4 microneedles, less than 3 microneedles, less than 2 microneedles, or less than 1 microneedle per mm of the substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.5 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.51 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.52 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.53 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.54 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.55 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.56 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.57 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.58 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.59 microneedles per mm of substrate. 2In a specific case, the density of the microneedles on the substrate is about 0.6 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.61 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.62 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.63 microneedles per mm of substrate. 2 In a specific case, the density of the microneedles on the substrate is about 0.64 microneedles per mm of substrate. 2 Approximately 0.65 microneedles per needle.

[0291] In some cases, the plurality of microneedles are arranged in a single area on the substrate. In other cases, the plurality of microneedles are arranged in multiple areas on the substrate. In some cases, the plurality of microneedles are arranged in a linear row. In some cases, the plurality of microneedles are arranged in a circular array. In some cases, the plurality of microneedles are arranged in at least two rows, with at least two microneedles in each row. In some cases, the plurality of microneedles are arranged in at least five rows, with at least two microneedles in each row. In some cases, the plurality of microneedles are arranged in at least ten rows, with at least two microneedles in each row. In some cases, the plurality of microneedles are arranged in at least two rows, with at least five microneedles in each row. In some cases, the plurality of microneedles are arranged in at least two rows, with at least ten microneedles in each row. In some specific embodiments, the plurality of microneedles are arranged in ten rows, with ten microneedles in each row.

[0292] In some cases, the substrate is made of the same material as the plurality of microneedles. In some cases, the substrate is made of hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In other cases, the substrate is made of a different material from the plurality of microneedles. In some cases, the substrate is made of a polymer (e.g., polycarbonate, polypropylene, or polyethylene), stainless steel, alloy, titanium, or any combination thereof. In some cases, the substrate is at least 100, at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, or at least 5000 mm thick. In some cases, the substrate is approximately circular, approximately a sector of a circle, approximately square, approximately rectangular, approximately triangular, approximately pentagonal, approximately parallelogram, approximately trapezoidal, or approximately polygonal. In some cases, the substrate has a Young's modulus of less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, less than 1, less than 0.9, less than 0.8, less than 0.7, less than 0.6, less than 0.5, less than 0.4, less than 0.3, less than 0.2, or less than 0.1.

[0293] In some cases, each microneedle of the plurality of microneedles has a base attached to a substrate. In some cases, at least one microneedle of the plurality of microneedles has a point capable of piercing a target. In particular cases, the point can pierce the stratum corneum of a subject. In particular cases, the point can pierce the epidermis of a subject. In particular cases, the point can pierce the dermis of a subject.

[0294] In some cases, the plurality of microneedles are solid. In some cases, the plurality of microneedles are hollow. In some cases, at least one microneedle of the hollow plurality of microneedles has an inner diameter of at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, or at least 3 mm. In some cases, at most one microneedle of the plurality of microneedles that is hollow has an inner diameter of at most 0.05, at most 0.1, at most 0.15, at most 0.2, at most 0.25, at most 0.3, at most 0.35, at most 0.4, at most 0.45, at most 0.5, at most 0.55, at most 0.6, at most 0.65, at most 0.7, at most 0.75, at most 0.8, at most 0.9, at most 1, at most 1.5, at most 2, at most 2.5, at most 3 mm, at most 4 mm, at most 5 mm, at most 6 mm, at most 7 mm, at most 8 mm, at most 9 mm, or at most 10 mm.

[0295] In some cases, at least one microneedle of the plurality of microneedles comprises a hydrogel, in particular, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof.

[0296] In some cases, at least one microneedle of the plurality of microneedles has a conical shape. In some cases, at least one microneedle of the plurality of microneedles has a funnel shape. In some cases, at least one microneedle of the plurality of microneedles has a pyramidal shape. In some cases, at least one microneedle of the plurality of microneedles has a tapered shape. In some cases, at least one microneedle of the plurality of microneedles has a shape as disclosed in Makvandi et al., Nano-Micro Letters volume 13, Article number: 93 (2021).

[0297] The plurality of microneedles provided herein generally have at least one microneedle with a base of a specific length. In some cases, at least one microneedle of the plurality of microneedles includes a base with a diameter of less than 2000 μm, less than 1800 μm, less than 1500 μm, less than 1000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than 200 μm, or less than 100 μm. In some cases, at least one microneedle of the plurality of microneedles comprises a base having a diameter of at least 2000 μm, at least 1800 μm, at least 1500 μm, at least 1000 μm, at least 900 μm, at least 800 μm, at least 700 μm, at least 600 μm, at least 500 μm, at least 400 μm, at least 300 μm, at least 200 μm, at least 100 μm, at least 10 μm, or at least 1 μm. In some cases, at least one microneedle of the plurality of microneedles comprises a base having a diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, about 300 μm to about 800 μm, about 400 μm to about 700 μm, or about 500 μm to about 600 μm. In certain embodiments, at least one microneedle of the plurality of microneedles comprises a base having a diameter of about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μm, hi certain embodiments, at least one microneedle of the plurality of microneedles comprises a base having a diameter of about 400 μm.

[0298] At least one microneedle of the microneedle device has a tip radius of a particular size. In some cases, the tip radius of at least one microneedle of the microneedle device is 4 mm, 3.5 mm, 3 mm, 2.5 mm, 2 mm, 1.5 mm, 1 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm or less.

[0299] In some cases, the length of at least one microneedle of the plurality of microneedles is at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 μm. In some cases, the length of at least one microneedle of the plurality of microneedles is 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, 500 μm or less, 600 μm or less, 700 μm or less, 800 μm or less, 900 μm or less, 1000 μm or less, 1100 μm or less, 1200 μm or less, 1300 μm or less, 1400 μm or less, 1500 μm or less, 1600 μm or less, 1700 μm or less, 1800 μm or less, 1900 μm or less, or 2000 μm or less. In other cases, the length of at least one microneedle of the plurality of microneedles is 100 μm to 3000 μm, 500 μm to 2500 μm, 1000 μm to 2500 μm, or 1000 μm to 2000 μm. In certain embodiments, at least one microneedle of the plurality of microneedles has a length of about 1000 μm. In certain embodiments, at least one microneedle of the plurality of microneedles has a length of about 2000 μm.

[0300] The length of at least one microneedle of the plurality of microneedles should be adjusted based on the thickness of the target tissue to be penetrated. In some cases, when the forehead is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.25-0.5 mm for thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 0.5-0.75 mm for thick skin. In some cases, when the glabella is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.25-0.5 mm for thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 0.5-0.75 mm for thick skin. In some cases, when the eye area is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.25 mm for thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 0.25-0.5 mm for thick skin. In some cases, when the nose is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.25 mm in thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 0.5 mm in thick skin. In some cases, when the cheekbone is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.5 mm in thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 0.5-1.0 mm in thick skin. In some cases, when the lip region is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.25 mm in thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 0.25-0.75 mm in thick skin.In some cases, when the cheek or chin is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.5-1.0 mm in thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 1.0-2.0 mm in thick skin. In some cases, when the neck is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.5-1.0 mm in thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 1.0-2.0 mm in thick skin. In some cases, when the chest is the target tissue, the length of at least one microneedle of the plurality of microneedles is at least 0.5-1.0 mm in thin skin, and the length of at least one microneedle of the plurality of microneedles is at least 1.0-2.0 mm in thick skin.

[0301] In some embodiments, the center-to-center distance between the microneedles of the plurality of microneedles is at least 100, 200, 300, 400, 500, 600, or 700 μm. In other embodiments, the center-to-center distance between the microneedles of the plurality of microneedles is about 100-2000 μm, 100-1500 μm, 100-1000 μm, 300-2000 μm, 300-1500 μm, 300-1000 μm, 500-2000 μm, 500-1500 μm, or 500-1000 μm. In specific cases, the center-to-center distance between the microneedles of the plurality of microneedles is about 700 μm. In other specific cases, the center-to-center distance between the microneedles of the plurality of microneedles is about 1000 μm.

[0302] In some cases, at least one microneedle of the plurality of microneedles includes a low aspect ratio, which provides stronger internal support and less breakage. In some cases, the plurality of microneedles distributes contents evenly within the target tissue.

[0303] In some cases, at least one microneedle of the plurality of microneedles has at least 3×10 2 , at least 3 × 10 3 , at least 3 × 10 4 , at least 3 × 10 5 , at least 3 × 10 6 , at least 3 × 10 7 , at least 3 × 10 8 , at least 3 × 10 9 , 3×10 10 , 3×10 11 , 3×10 12 , 3×10 15 , 3×10 20 , or 3 × 10 30 In some cases, at least one microneedle of the plurality of microneedles contains about 3 x 10 EVs. 2 ~3×10 12 , about 3×10 3 ~3×10 12 , about 3×10 4 ~3×10 12 , about 3×10 5 ~3×10 12 , about 3×10 6 ~3×10 12 , about 3×10 7 ~3×10 12 , about 3×10 6 ~3×10 12 , about 3×10 7 ~3×10 12 , about 3×10 8 ~3×10 12 , about 3×10 9 ~3×10 12 , about 3×10 10 ~3×10 12 , or approximately 3 × 10 11 ~3×10 12 In some cases, at least one microneedle of the plurality of microneedles contains about 3 x 10 EVs. 2 ~3×10 11 , about 3×10 3 ~3×10 11 , about 3×10 4 ~3×10 11 , about 3×105 ~3×10 11 , about 3×10 6 ~3×10 11 , about 3×10 7 ~3×10 11 , about 3×10 6 ~3×10 11 , about 3×10 7 ~3×10 11 , about 3×10 8 ~3×10 11 , about 3×10 9 ~3×10 11 , or approximately 3 × 10 10 ~3×10 11 In some cases, at least one microneedle of the plurality of microneedles contains about 3 x 10 EVs. 2 ~3×10 10 , about 3×10 3 ~3×10 10 , about 3×10 4 ~3×10 10 , about 3×10 5 ~3×10 10 , about 3×10 6 ~3×10 10 , about 3×10 7 ~3×10 10 , about 3×10 6 ~3×10 10 , about 3×10 7 ~3×10 10 , about 3×10 8 ~3×10 10 , or approximately 3 × 10 9 ~3×10 10 In other cases, at least one microneedle of the plurality of microneedles contains about 3 x 10 EVs. 2 ~3×10 9 , about 3×10 3 ~3×10 9 , about 3×10 4 ~3×10 9 , about 3×10 5 ~3×10 9 , about 3×10 6 ~3×10 9 , about 3×10 7 ~3×10 9 , about 3×10 6~3×10 9 , about 3×10 7 ~3×10 9 , or approximately 3 × 10 8 ~3×10 9 In some cases, at least one microneedle of the plurality of microneedles contains about 3 x 10 EVs. 2 ~3×10 8 , about 3×10 3 ~3×10 8 , about 3×10 4 ~3×10 8 , about 3×10 5 ~3×10 8 , about 3×10 6 ~3×10 8 , about 3×10 7 ~3×10 8 , about 3×10 6 ~3×10 8 , or approximately 3 × 10 7 ~3×10 8 Includes EVs.

[0304] In some cases, the EVs in at least one microneedle of the plurality of microneedles are suspended in a hydrogel. In some cases, the hydrogel comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In particular cases, the hydrogel comprises hyaluronic acid.

[0305] In some cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% at up to 30, 20, 15, 10, or 5 minutes. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% at about 30, 20, 15, 10, or 5 minutes. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% at up to 60, 50, or 40 minutes. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% at about 60, 50, or 40 minutes. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% at about 1, 3, 5, 10, 15, 20, or 24 hours. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% at about 1, 3, 5, 10, 15, 20, or 24 hours. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% for up to 1, 3, 5, 7, 9, 14, 21, or 30 days. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% for about 1, 3, 5, 7, 9, 14, 21, or 30 days. In some cases, at least one microneedle of the plurality of microneedles comprises a protein. In some cases, the dissolvable gel comprises a compound derived from an animal source. In some cases, at least one microneedle of the plurality of microneedles comprises a synthetic compound.In some cases, at least one microneedle of the plurality of microneedles comprises hyaluronic acid, sodium alginate, polylactic acid, polyglycolic acid, polylactic-glycolic acid, cartilage thioflavin, silk protein, maltose, chitosan, carboxymethylcellulose, or any combination thereof. In some cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% when exposed to tissue. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% when exposed to the epidermis. In some specific cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% when exposed to the dermis. In some specific examples, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% when exposed to subcutaneous tissue.

[0306] In some cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% when the local temperature is increased by at least 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. In some cases, at least one microneedle of the plurality of microneedles has a breaking strength retention of up to 50%, 40%, 30%, 20%, or 10% when the local temperature is increased by at least 15° C., 14° C., 13° C., 12° C., 11° C., or 10° C.

[0307] In some cases, the EVs in at least one microneedle of the plurality of microneedles comprise extracellular matrix mRNA. In particular cases, the EVs in at least one microneedle of the plurality of microneedles comprise exogenous extracellular matrix mRNA. In some cases, the extracellular matrix mRNA or exogenous extracellular matrix mRNA comprises type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof. In other cases, the EVs in at least one microneedle of the plurality of microneedles comprise VEGF mRNA. In particular cases, the EVs in at least one microneedle of the plurality of microneedles comprise exogenous VEGF mRNA. In some cases, the VEGF mRNA or exogenous VEGF mRNA comprises VEGFA, VEGFB, VEGFC, VEGFD, PIGF, or any combination thereof.

[0308] In some cases, the microneedle device comprises siRNA. In some cases, the microneedle device comprises miRNA. In some cases, the microneedle device comprises DNA, a plasmid, or other nucleic acid. In some cases, the microneedle device comprises a small molecule drug with or without EVs. In some cases, the microneedle device comprises a large macromolecule with or without EVs. In some cases, the microneedle device comprises insulin with or without EVs. In some cases, the microneedle device comprises a growth hormone with or without EVs. In some cases, the microneedle device comprises a vaccine or an active ingredient thereof. In some cases, the microneedle device comprises a receptor agonist with or without EVs. In some cases, the microneedle device comprises an anesthetic with or without EVs. In some cases, the microneedle device comprises a protein or peptide with or without EVs.

[0309] In some cases, all microneedles of the plurality of microneedles contain EVs disclosed herein. In other cases, some of the plurality of microneedles contain EVs disclosed herein, while other portions of the plurality of microneedles do not contain EVs. In specific cases, some of the plurality of microneedles contain EVs disclosed herein, while other portions of the plurality of microneedles contain a different substance that does not contain EVs. In some cases, the different substance is an anesthetic, a healing agent, an ingredient for improving skin conditions, aloe, or any combination thereof. In specific cases, the anesthetic is lidocaine, pramoxine, phenol, prilocaine, benzocaine, dibucaine, methyl salicylate, capsaicin, zinc acetate, tetracaine, hydrocortisone, menthol, or prilocaine. In particular cases, the healing agent is vitamin C, extract from Aloe vera, extract from Hippophae rhamnoides (sea buckthorn), extract from Angelica sinensis, extract from Catharanthus roseus (Vinca rosea), or green tea extract. In particular cases, the ingredients for improving skin conditions are glycerin, coenzyme Q10 (CoQ10), ceramide, cetylstearyl alcohol, petrolatum, squalene, and lactic acid.

[0310] Further provided herein is a method for fabricating a microneedle device, the method comprising: (a) mixing extracellular vesicles (EVs) with a first batch of a polymerizable solution; and (b) casting the mixture from step (a) into a polydimethylsiloxane (PDMS) mold having at least one needle-like shape. In some cases, the method further comprises step (c) after step (b), wherein step (c) comprises concentrating the EVs at the tip of the at least one needle-like shape of the PDMS mold. In some specific cases, the concentrating is performed by maintaining the PDMS mold at a temperature of at most 15°C, at most 14°C, at most 13°C, at most 12°C, at most 11°C, at most 10°C, at most 9°C, at most 8°C, at most 7°C, at most 6°C, at most 5°C, at most 4°C, at most 3°C, at most 2°C, at most 1°C, or at most 0°C. In some specific cases, the concentrating is by maintaining the PDMS mold at a temperature of at least 6° C., at least 5° C., at least 4° C., at least 3° C., at least 2° C., at least 1° C., at least 0° C., at least −1° C., at least −2° C., at least −3° C., at least −4° C., at least −5° C., at least −6° C., at least −7° C., at least −8° C., or at least −10° C. In specific cases, the concentrating is by maintaining the PDMS mold at about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., or about 10° C. In one specific case, the concentrating is by maintaining the PDMS mold at about 4° C. In some cases, the concentration lasts for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In some c...

Claims

1. A plurality of extracellular vesicles comprising an exogenous extracellular matrix messenger RNA (mRNA), wherein the plurality of extracellular vesicles comprises, on average, at least 1 copy of the exogenous extracellular matrix mRNA per 450 EVs.

2. 2. The plurality of extracellular vesicles of claim 1, wherein the exogenous extracellular matrix mRNA encodes a collagen.

3. 3. The plurality of extracellular vesicles of claim 2, wherein the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof.

4. The plurality of extracellular vesicles of claim 2, wherein the collagen is type I collagen.

5. 3. The plurality of extracellular vesicles of claim 2, wherein the collagen is type II collagen.

6. 3. The plurality of extracellular vesicles of claim 2, wherein the collagen is the alpha 1 chain of type I collagen (Col1A1) or the proalpha 1 (I) chain.

7. The plurality of extracellular vesicles of claim 6, wherein the collagen is Col1A1 and the plurality of extracellular vesicles does not contain the alpha 2 chain of type I collagen (Col1A2).

8. The plurality of extracellular vesicles of claim 2, wherein the collagen is Col1A1.

9. 2. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles is produced by transfecting cells with a plasmid encoding an extracellular matrix protein.

10. 10. The plurality of extracellular vesicles of claim 9, wherein said transfecting said cells is performed by cell nanoporation.

11. The plurality of extracellular vesicles of claim 9, wherein the cells are human cells.

12. 10. The plurality of extracellular vesicles of claim 9, wherein the cell is a fibroblast selected from the group consisting of a dermal fibroblast, a human fibroblast, an adult fibroblast, a human adult fibroblast, a neonatal fibroblast, a neonatal human fibroblast, and a neonatal human dermal fibroblast.

13. 2. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles comprises extracellular vesicles selected from the group consisting of exosomes, microvesicles, apoptotic bodies, and any combination thereof.

14. 2. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles comprises at least one exosome.

15. 2. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles is formulated for injection via an intravenous route, an intramuscular route, a subcutaneous route, or any combination thereof.

16. The plurality of extracellular vesicles of claim 1 , wherein the plurality of extracellular vesicles comprises at least 1×10 6 extracellular vesicles.

17. 2. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles comprises at least 10 ng of the extracellular matrix mRNA.

18. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles comprises a size distribution having a peak at a diameter of 50 nm to 200 nm.

19. 2. The plurality of extracellular vesicles of claim 1, wherein the exogenous extracellular matrix mRNA is present at a level at least two-fold higher than the level of the exogenous extracellular matrix mRNA in an identical amount of naturally occurring extracellular vesicles.

20. 2. The plurality of extracellular vesicles of claim 1, wherein the plurality of extracellular vesicles expresses higher levels of at least one of the following markers: CD9, CD63, TSG101, or ARF6, as compared to a comparable number of naturally occurring extracellular vesicles.

21. A composition comprising a plurality of extracellular vesicles according to any one of claims 1 to 20, for use in treating a skin condition in a subject, comprising administering to the subject at least one extracellular matrix messenger RNA (mRNA) of the plurality of extracellular vesicles comprising the exogenous extracellular matrix mRNA, thereby treating the skin condition.

22. The composition of claim 21 , wherein the at least one exogenous extracellular matrix mRNA encodes a collagen.

23. 23. The composition of claim 22, wherein the collagen is selected from the group consisting of type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVII collagen, type XXVIII collagen, or any combination thereof.

24. 24. The composition of claim 23, wherein the collagen is type I collagen.

25. 24. The composition of claim 23, wherein the collagen is type II collagen.

26. 24. The composition of claim 23, wherein the collagen is the alpha 1 chain of type I collagen (Col1A1) or the proalpha 1 (I) chain.

27. The composition of claim 23 , wherein the collagen is Col1A1.

28. 22. The composition of claim 21, wherein the skin condition is a skin lesion or wound.

29. The composition of claim 21 , wherein the EV is an exosome, an apoptotic body or a microvesicle.

30. A method for producing a plurality of extracellular vesicles comprising the exogenous extracellular matrix messenger RNA (mRNA) according to any one of claims 1 to 20, the method comprising: (a) introducing into donor cells via transfection a vector or plasmid corresponding to said exogenous extracellular matrix mRNA; (b) culturing the donor cells in a culture medium for a sufficient amount of time to produce extracellular vesicles encapsulating the exogenous extracellular matrix mRNA transcribed from the vector or plasmid; (c) recovering the plurality of extracellular vesicles from the culture medium. A method comprising:

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