Compositions and methods for treating peripheral vascular disease

Abstract

Methods for treating peripheral vascular disease, vascular disorders, or vascular dysfunction in a subject generally involve administering to the subject a therapeutic composition comprising a purified exosome product (PEP) and a pharmaceutically acceptable carrier. In one or more embodiments, the therapeutic composition further comprises a support matrix.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 392,335, filed July 26, 2022, which is incorporated by reference herein in its entirety. Summary of the Invention

[0002] In one aspect, the present disclosure describes a method for treating peripheral vascular disease, vascular disorders, or vascular dysfunction in a subject. Generally, the method includes administering to the subject a therapeutic composition comprising a purified exosome product (PEP) and a pharmaceutically acceptable carrier.

[0003] In one or more embodiments, the PEP contains 1% to 20% of CD63 - Exosomes and 80%-99% CD63 + Contains exosomes.

[0004] In one or more embodiments, the PEP has at least 50% CD63 - Contains exosomes.

[0005] In one or more embodiments, the therapeutic composition further comprises a support matrix.

[0006] In one or more embodiments, the therapeutic composition further comprises a tissue sealant, fibrin glue, or a hydrogel.

[0007] In one or more embodiments, the therapeutic composition is applied in an amount effective to enhance pro-angiogenic activity compared to peripheral vascular disease treated without the therapeutic composition.

[0008] In one or more embodiments, the therapeutic composition is applied in an amount effective to enhance in vivo perfusion following ischemia compared to peripheral vascular disease treated without the therapeutic composition.

[0009] In one or more embodiments, the therapeutic composition is applied in an amount effective to enhance driving of the MAPK pathway or the AKT pathway compared to peripheral vascular disease treated without the therapeutic composition.

[0010] In one or more embodiments, the therapeutic composition is delivered by intramuscular injection.

[0011] In one or more embodiments, the peripheral vascular disease, vascular disorder, or vascular dysfunction comprises peripheral arterial disease.

[0012] In one or more embodiments, the peripheral vascular disease, vascular disorder, or vascular insufficiency comprises atherosclerosis, ischemia, deep vein thrombosis, pulmonary embolism, varicose veins, chromic venous insufficiency, Buerger's disease, Raynaud's phenomenon, thrombophlebitis, or aneurysm.

[0013] In one or more embodiments, the subject is a human. In one or more embodiments, the subject suffers from an ischemic wound, and wherein the method enhances ischemic wound closure compared to a method without PEP.

[0014] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly exemplifies illustrative embodiments. In several places in this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief explanation of the drawings]

[0015] [Figure 1AB]PEP stimulates pro-angiogenic events in vitro. (A) Representative atomic force microscopy (AFM) images of PEP exosomes. (B) Size distribution of PEP exosomes measured by NANOSIGHT tracking analysis (Malvern Panalytical Ltd., Malvern, UK). [Figure 1CD] PEP stimulates pro-angiogenic events in vitro. (C) Zeta potential of exosomes measured using a ZETASIZER instrument (Malvern Panalytical Ltd., Malvern, UK). Data are shown as mean ± SD. (D) Stiffness of exosomes measured using AFM indentation. Young's modulus was used to indicate particle stiffness. Data are shown as ± SD. [Figure 1EF] PEP stimulates pro-angiogenic events in vitro. (E) Western blot analysis of exosomal marker proteins in fractionated samples. 10 μg of exosomal protein was loaded per gel lane. Three separate CGMP batches of PEP were analyzed. (F) Quantification of the INCUCYTE proliferation assay (Essen Bioscience, Inc. Ann Arbor, MI) using the INCUCYTE red channel to calculate the percentage of red nuclei. [Figure 1G] PEP stimulates pro-angiogenic events in vitro. (G) Scratch assay examining migration of HUVECs treated with FBS, PEP, or serum-free. Quantification of wound closure. [Figure 2] PEP stimulates pro-angiogenic events in vitro. (A) INCUCYTE proliferation assay (Essen Bioscience, Inc. Ann Arbor, MI). Representative fluorescence images of FBS vs. PEP vs. serum-free medium at 72 hours. (B) Representative fluorescence images of tube formation of HUVECs on Matrigel treated with PEP, VEGF, or suramin. [Figure 3]PEP stimulates pro-angiogenic events in vitro. Scratch assay examining migration of HUVECs treated with FBS, PEP, or serum-free medium. Representative images of proliferation of PEP vs. FBS vs. PBS (serum-free) at 72 hours. [Figure 4] PEP stimulates pro-angiogenic events in vitro. Tube formation of human umbilical vein endothelial cells (HUVECs) on MATRIGEL (Corning Life Sciences, Inc., Corning, NY) treated with PEP, VEGF, or suramin. (A) Quantification of tube length performed 1, 3, and 6 hours after treatment. (B) Quantification of tube junctions performed 1, 3, and 6 hours after treatment. [Figure 5] PEP stimulates pro-angiogenic events in vitro. The expression levels of 55 angiogenesis-related proteins in human umbilical vein endothelial cells (HUVECs) were quantified at 0, 6, 12, and 24 hours after PEP treatment (n = 4 per time point). [Figure 6] Administration of PEP biogel enhances perfusion in rat hind limbs after 3 weeks of ischemia. (A) Scheme of TISSEEL-PEP treatment for rat hind limb ischemia. (B) SEM image analysis of PEP biopotentiated TISSEEL (inset B) and TISSEEL without PEP. Exosomes are indicated by a wedge shape. [Figure 7] Representative SPY (Stryker Corp., Kalamazoo, MI) angiography images of rats treated with fibrin glue alone (TISSEEL, Baxter International, Inc., Deerfield, IL), fibrin glue with PEP (TISSEEL-PEP), or negative control (sham) at pre-op, post-op day 0, and post-op day 21. [Figure 8]Quantification of total blood perfusion in rats treated with fibrin glue alone (TISSEEL, Baxter International, Inc., Deerfield, IL), fibrin glue with PEP (TISSEEL-PEP), or negative control (Sham) at pre-op (Pre-lig), post-op day 0 (Post-lig D0), and post-op day 21 (Post-lig D21). ****=p<0.0001. [Figure 9] Quantification of blood perfusion in rats treated with fibrin glue alone (TISSEEL, Baxter International, Inc., Deerfield, IL), fibrin glue with PEP (TISSEEL-PEP), or negative control (sham). (A) Proximal limb section; (B) Mid-limb section; (C) Distal limb section. *=p<0.05; ***=p<0.005; ns.=not significant. [Figure 10] Immunofluorescence analysis of rat hindlimb tissue in transverse section. Rats were treated with fibrin glue alone (TISSEEL, Baxter International, Inc., Deerfield, IL), fibrin glue with PEP (TISSEEL-PEP), or negative control (Sham), and then stained for von Willebrand factor (vWF), smooth muscle actin (SMA), and the cell proliferation marker 5-ethynyl-2'-deoxyuridine (EDU). Scale bar: 200 μm. [Figure 11] Quantification of vWF+, SMA+, and EdU+ cells in the tissue sections shown in Figure 10. (A) vWF; (B) SMA; (C) EdU. *=p<0.05; **=p<0.01; ****=p<0.0001. [Figure 12]Quantification of vascular and fiber areas in tissue sections. (A) Hematoxylin and eosin staining of tissue sections taken from rat hind leg tissue. (B) Masson's trichrome staining of tissue sections taken from rat hind leg tissue. (C) Vascular area calculated from positive staining in hematoxylin and eosin stained sections. (D) Fiber area calculated from Masson's trichrome stained sections. *=p<0.05; ***=p<0.005; ****=p<0.0001; ns.=not significant. [Figure 13] Expression analysis of genes involved in the angiogenesis process. (A) Heatmap of the expression of 84 angiogenesis-related genes in the TISSEEL-PEP and TISSEEL groups (n=3 per group). (B) Volcano plot of differentially regulated genes between TISSEEL-PEP and TISSEEL (control). (C) Heatmap of differentially regulated genes involved in endothelial cell proliferation. (D) Heatmap of differentially regulated genes involved in the VEGFR signaling pathway. [Figure 14] TISSEEL-PEP biogel improved blood perfusion in ischemic wound tissue. (A) Representative wound images from untreated tissue (Sham), tissue treated with fibrin glue alone (TISSEEL, Baxter International Inc., Deerfield, IL), or fibrin glue + PEP (TISSEEL-PEP) at days 0 and 28. (B) Quantification of wound healing area. Scale bar: 20 μm. **=p<0.01; ****=p<0.0001. [Figure 15]TISSEEL-PEP biogel improved blood perfusion in ischemic wound tissue. (A) Representative SPY (Stryker Corp., Kalamazoo, MI) angiography images of ear wounds at 4 weeks post-op for wounds untreated (Sham), treated with fibrin glue (TISSEEL, Baxter International Inc., Deerfield, IL) alone, or fibrin glue + PEP (TISSEEL-PEP). (B) Quantification of total blood perfusion pre-op, post-op, and 4 weeks post-op. (C) Blood flow levels in the distal ear were quantified. (D) Blood flow levels in the mid-ear were quantified. (E) Blood flow levels in the proximal ear were quantified. [Figure 16] Immunofluorescence images stained for CD31 and smooth muscle actin (SMA) in wounds treated with fibrin glue (TISSEEL, Baxter International Inc., Deerfield, IL) alone, fibrin glue + PEP (TISSEEL-PEP), or untreated (Sham) wounds. Scale bar: 1 μm. [Figure 17] Immunofluorescence quantification. (A) CD31, normalized to control. (B) SMA, normalized to control. **=p<0.01; ***=p<0.005; ns.=not significant. [Figure 18] Quantification of rabbit tissue vascular area percentage in hematoxylin and eosin stained sections. *=p<0.05; ***=p<0.005. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Embodiments This disclosure describes compositions and methods for treating peripheral vascular disease, vascular disorders, or vascular dysfunction in a subject. Generally, the methods include administering to the subject a purified exosome product (PEP) in an amount to ameliorate at least one symptom or clinical sign of peripheral vascular disease, vascular disorders, or vascular dysfunction.

[0017] PEP has been thoroughly characterized, and methods for its preparation are described in International Patent Application No. PCT / US2018 / 065627 (published as WO 2019 / 118817), U.S. Patent Application Publication No. 2021 / 0169812 A1, and U.S. Patent No. 10,596,123, which are incorporated herein by reference in their entireties. Briefly, PEP is a purified exosome product prepared using a lyophilization process that produces a product with a structure different from exosomes prepared using conventional methods. For example, PEP typically has a spherical or spheroidal structure and an intact lipid bilayer, rather than the crystalline structure that results from lipid reaggregation of the exosome lipid bilayer after exosome disruption during conventional exosome preparation methods. Spherical or spheroidal exosome structures generally have diameters of 300 nm or less. Typically, PEP preparations contain spherical or spheroidal exosome structures with a relatively narrow size distribution. In some preparations, the PEP comprises spherical or spheroidal exosome structures having an average diameter of about 110 nm ± 90 nm, with the majority of the exosome structures having an average diameter of 110 nm ± 50 nm, such as 110 nm ± 30 nm.

[0018] Unmodified PEP preparations—i.e., PEP preparations whose characteristics have not been altered by sorting or separating the exosome population in the preparation—represent CD63 + Exosomes and CD63 - Naturally contains a mixture with exosomes. CD63 - Exosomes can inhibit unlimited cell proliferation, and CD63 + and CD63 - Unmodified PEP preparations that naturally contain exosomes can stimulate cell proliferation for wound repair and / or tissue regeneration, or limit uncontrolled cell proliferation.

[0019] Furthermore, CD63 + Exosome sorting revealed CD63 expression in naturally isolated PEP preparations. + Extract the exosomes and then lyse them with the desired amount of CD63.+ By returning exosomes, CD63 in the PEP product + Exosomal CD63 - In one or more embodiments, the PEP preparation contains CD63. - It may have only exosomes.

[0020] In one or more embodiments, the PEP preparation comprises CD63 + Exosomes and CD63 - Contains both CD63 and exosomes. + Exosomal CD63 - The ratio of CD63 to exosomes can vary, at least in part, depending on the amount of cell expansion desired in a particular application. + / CD63 - Exosome ratio is CD63 + Desired cell proliferation induced by exosomes and CD63 achieved through cell contact inhibition - In certain scenarios, such as tissues containing non-adherent cells (e.g., blood-derived components), this ratio can be adjusted to provide the appropriate balance of cell proliferation or cell inhibition for the tissue being treated. For example, in tissues with non-adherent cells, cell-to-cell contact is not a trigger, so CD63 + The ratio of exosomes can be reduced to avoid unlimited cell proliferation. Conversely, when one wishes to expand a clonal population of cells, such as in allogeneic cell-based therapies or immunotherapy, one can use CD63 to ensure that a large cell population can be obtained from a very small source. + The ratio of exosomes can be increased.

[0021] Thus, in one or more embodiments, CD63 in the PEP preparation + Exosomal CD63 -The ratio of CD63 to exosomes can be at least 1:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 11:1, at least 12:1, at least 13:1, at least 14:1, at least 15:1, or at least 16:1. + Exosomal CD63 - The ratio to exosomes can be at most 15:1, at most 16:1, at most 17:1, at most 18:1, at most 19:1, at most 20:1, at most 25:1, or at most 30:1. For example, CD63 + Exosomal CD63 - The ratio of PEP product to exosomes can be 1:1 to 30:1, 2:1 to 20:1, 4:1 to 15:1, or 8:1 to 10:1. In one or more specific embodiments, the PEP product is CD63 + In one or more specific embodiments, the exosomes are formulated to contain a 9:1 ratio of exosomes to CD63 exosomes. + Exosomal CD63 - An unmodified ratio of PEP to exosomes may also be used.

[0022] The production of purified exosome product (PEP) involves separating plasma from blood and isolating a solution of exosomes from the separated plasma by filtration and centrifugation. PEP has been thoroughly characterized, and methods for its preparation are described in International Patent Application No. PCT / US2018 / 065627 (published as WO 2019 / 118817), U.S. Patent Application Publication No. 2021 / 0169812 A1, and U.S. Patent No. 10,596,123, which are incorporated herein by reference in their entireties. Peripheral arterial disease (PAD) is a significant cause of morbidity and mortality. Therapeutic angiogenesis using extracellular vesicles to rescue ischemic tissue has yielded modest results. This disclosure describes an alternative approach, in which purified exosome product (PEP), derived from platelets and enriched in VEGFR2, was evaluated as an angiogenic therapy for PAD. By activating the VEGFR2-regulated program, PEP promoted angiogenic events in vitro. In vivo, local delivery of PEP triggered new blood vessel formation and resulted in increased tissue perfusion in rat ischemic hind limb and rabbit ischemic ear wound models. This study proposes a commercially available, translation-ready exosome-based regenerative strategy to promote angiogenesis for the treatment of ischemic diseases.

[0023] Although described herein with reference to peripheral arterial disease as a prototype of peripheral vascular disease, the compositions and methods described herein may relate to any form of peripheral vascular disease, vasculopathy, or vascular dysfunction. Peripheral vascular disease is a blood flow disorder that causes narrowing, blockage, or spasm of blood vessels in the peripheral vasculature. Peripheral vascular disease can occur in either arteries or veins. Functional peripheral vascular disease typically involves narrowing of blood vessels in response to factors including, but not limited to, brain signals or temperature changes. The narrowing causes reduced blood flow but without physical damage to the structural blood vessels. Structural peripheral vascular disease involves changes in vascular structure, such as inflammation, plaque, and / or tissue damage. Exemplary forms of peripheral vascular disease, vascular disorders, and vascular insufficiency that can be treated using the methods described herein include, but are not limited to, atherosclerosis, ischemia, deep vein thrombosis, pulmonary embolism, varicose veins, chromic venous insufficiency, Buerger's disease, Raynaud's phenomenon, thrombophlebitis, or aneurysm.

[0024] PEP vesicles have distinct exosome markers and biophysical properties PEP preparations were imaged by atomic force microscopy (AFM, Figure 1A) to assess vesicle integrity and morphology, and further measured by nanoparticle tracking analysis (NTA) to assess the mean particle size and concentration of the vesicle population (Figure 1B). NTA demonstrated that the PEP preparation had a mean particle size of 126.7 nm and a mode size of 108.5 nm (Figure 1B). Measured zeta potential and mean surface charge revealed that all particles were negatively charged (-4.94 mV to -6.91 mV, Figure 1C). PEP particles demonstrated stiffness ranging from 428 to 890 MPa (Figure 1D). Batch-to-batch uniformity in the CGMP product was confirmed by cholesterol concentration, moisture level, and protein concentration, along with constitutive expression of CD63, CD9, and Alix (Figure 1E). Human umbilical vein endothelial cells (HUVECs) exposed to PKH-labeled PEP and tracked by live-cell imaging showed efficient vesicle uptake in response to inhibitors of clathrin-dependent endocytosis, macropinocytosis, or membrane fusion.

[0025] PEP enhances pro-angiogenic cellular activity in vitro The effect of PEP on angiogenesis events was examined by assessing HUVEC proliferation, tube formation, and migration. HUVEC proliferation assays were performed by culturing HUVECs with PEP using a predefined optimal concentration. Compared to FBS-supplemented medium, PEP-treated (2.5 × 10 11 HUVECs (100 ng / mL, 100 μM vesicles / mL) achieved similar proliferation potential with higher expression of Ki-67 (Figure 1F, Figure 2A). Angiogenic activity on Matrigel substrates was significantly enhanced by the treatment with VEGF (100 ng / mL), suramin (100 μM), or PEP (2.5 × 10 11Scratch wound assays on HUVEC monolayers were performed using basal medium supplemented with 2.5 × 10 vesicles / mL (Figure 2B). Scratch wound assays on HUVEC monolayers showed that PEP treatment promoted wound closure, with a higher percentage of cell confluency in the PEP-treated group compared with that of the control (Figure 1G, Figure 3). Quantification of the total branch length per field (3053.68 vs. 2459.59 pixels, PEP vs. VEGF) and the number of junctions (70.75 vs. 59.75, PEP vs. VEGF) were greater in the PEP-treated group (Figure 2B, Figure 4). 11 HUVECs treated with vesicles / mL and screened using an angiogenic antibody array displayed a significant increase in pro-angiogenic paracrine factors 24 hours after treatment with PEP, demonstrating the in vitro angiogenic activity induced by PEP (Figure 5).

[0026] TISSEEL-PEP biogel promoted angiogenesis in hindlimb ischemia Crosslinking allows for sustained release of bioactive components and enhances therapeutic efficacy. Here, we incorporated PEP into TISSEEL (Baxter International, Deerfield, IL), a clinical-grade fibrin glue, and investigated whether sustained PEP release could aid in a hindlimb ischemia (HLI) model (Figure 6A). 12 PEP vesicles / mL were 2 × 10 11 3 x 10 vesicles / mL 11 This resulted in consistent exosome release of vesicles / mL. Following induction of hindlimb ischemia, rats were randomly assigned to receive saline, TISSEEL, or TISSEEL-PEP injections at the ligation site. Perfusion was tracked preoperatively, immediately after surgery, and 21 days postoperatively. Vascular occlusion halved perfusion to the ischemic limb, as measured by SPY angiography (Figure 7). In rats treated with TISSEEL-PEP, perfusion recovered to non-ischemic control limb values by 21 days postoperatively, whereas there was virtually no recovery in the distal, mid, and proximal limb regions in the sham or TISSEEL-treated groups (Figures 8 and 9).

[0027] To evaluate the angiogenic effect, vascular cells were immunohistochemically stained and quantified using the endothelial marker von Willebrand factor (vWF), the smooth muscle marker smooth muscle actin (SMA), and the cell proliferation marker EdU (Fig. 10). All three were significantly higher in the TISSEEL-PEP group compared with sham or TISSEEL alone (Figs. 10 and 11). Closer examination revealed a substantial increase in the vascularized area for the TISSEEL-PEP group only (Figs. 12A, C). Furthermore, sham- or TISSEEL-treated rats had muscle fibers that appeared more fibrous, with a characteristic collagen distribution (Figs. 12B, D). In contrast, the TISSEEL-PEP group demonstrated collagen levels similar to non-ischemic tissue (Fig. 12B). Thus, treatment with TISSEEL-PEP biogel appeared to restore perfusion and rescue ischemic tissue injury. To address the underlying mechanism of PEP action, muscle samples obtained on day 21 were profiled for the expression of 84 angiogenesis-related genes (Fig. S13A) and noted 29 that were significantly upregulated (Fig. S13B). Pathway analysis suggested PEP-mediated activation of endothelial cell proliferation and VEGFR signaling events (Fig. S13C, D).

[0028] TISSEEL-PEP biogel aids perfusion in a rabbit ischemic wound model To further evaluate the efficacy and translational value of TISSEEL-PEP biogel, we tested this approach in a large animal model, where impaired blood flow can lead to irreversible tissue necrosis and damage. We utilized a well-established rabbit ear ischemic wound model, in which less than 20% of the blood supply is maintained postoperatively, as previously described (Chien, S., Wound Rep Reg. 15:928-935, 2007). TISSEEL-PEP biogel was placed on the wound bed, whereas intact, sham- and TISSEEL-treated ears served as baseline and treatment controls. After 4 weeks of treatment, TISSEEL-PEP significantly improved ischemic wound closure compared with sham or TISSEEL treatment alone (Figure 14A, B). At 4 weeks, both SPY angiography and histological analysis revealed that the TISSEEL-PEP-treated group restored blood flow in the distal, middle, and proximal portions of the ear, whereas the controls did not (Figure 15). Although immunohistochemical staining did not demonstrate angiogenesis in the sham or TISSEEL arms compared with healthy controls, a significant increase in angiogenesis in the TISSEEL-PEP group was documented by increased CD31 and SMA expression (Figures 16 and 17). Histological analysis of hematoxylin and eosin (HE)-stained tissues quantified the increased vascularity in the TISSEEL-PEP-treated cohort (Figure 18). Taken together, these results demonstrate the angiogenic effects of PEP in a large animal model of ischemic wounding.

[0029] This disclosure therefore describes a mechanism by which PEP exosomes drive pro-angiogenic events through pVEGFR-2 delivery. Protein profiling documenting vascular polarization 24 hours after treatment demonstrated that PEP induced endothelial cell proliferation, migration, and angiogenesis. MAPK and AKT were activated in intracellular pathways, revealing that PEP provided bioactive pVEGFR-2 and eliminated the need for a growth factor-rich environment. Incorporation of PEP into a fibrin glue-based composition mediated sustained release of PEP and resulted in pro-angiogenic cellular events and promoted angiogenesis in a separate model of peripheral vascular occlusion.

[0030] Ischemic disease is prevalent worldwide, and few treatments exist to restore blood flow beyond manual revascularization. Platelets are proven promoters of angiogenic events following tissue injury. PEP, purified exosomes derived from activated platelets, provides a platform to assess the underlying mechanisms of angiogenesis and the treatment of peripheral vascular disease.

[0031] VEGFR-2 signaling is a well-characterized pathway critical in the cellular processes that support angiogenesis. During angiogenesis, phosphorylated VEGFR-2 activates multiple downstream pathways through signaling intermediates, including MAPK, AKT, and GTPases. Beyond cell proliferation, migration, and tube formation, VEGFR-2 also regulates vascular permeability and is a potent survival factor. Early efforts to develop pro-angiogenic therapies focused on the direct delivery of growth factors (GFs) into the ischemic region. However, short half-lives and initial burst release profiles led to limited efficacy and significant side effects associated with treatment with these growth factors. Advances in materials science have led to the use of various biomatrices, including fibrin, alginate, and hyaluronic acid hydrogels, as delivery vehicles and have demonstrated localized, sustained, and degradable capabilities in angiogenesis studies.

[0032] This study establishes exosomes capable of delivering bioactive proteins to drive angiogenic events. PEP-mediated delivery of pVEGFR-2 initiated a fine-tuned signaling network in endothelial cells associated with angiogenesis both in vitro and in vivo. Pro-angiogenic cellular activity was observed in PEP-treated HUVECs. Furthermore, increased VEGFR-2 expression led to rapid induction of downstream MAPK and AKT pathways, suggesting protein delivery, rather than transcription or translation, as a novel mode by which these events can be driven. Following growth factor stimulation, VEGFR-2 is internalized from the cell surface via clathrin- and dynamin-mediated endocytosis, macropinocytosis, and membrane fusion. Here, we demonstrate that PEP mimics this process in a growth factor-independent manner, as cellular internalization was suppressed by inhibitors of these pathways.

[0033] PEP biopotentiation of a fibrin-based biogel (TISSEEL, Baxter International, Deerfield, IL) achieved controlled release of exosomes over a prolonged period to drive a desired biological event. Purification of exosomes using an intact lipid bilayer allowed for stability and guaranteed compatibility with a sustained-release strategy, something not possible with recombinant proteins due to rapid degradation. Utilizing this platform, this study evaluated the benefits in a small-animal rodent model and further validated it in a large-animal rabbit model to demonstrate the cross-species feasibility of the observed findings and ensure sufficient preclinical evidence for clinical translation.

[0034] Therefore, the present disclosure describes compositions and methods for treating peripheral vascular disease in a subject. Generally, the compositions include PEP and a pharmaceutically acceptable carrier. In a surgical setting, the PEP may be combined with a suitable carrier, such as, for example, a surgical adhesive, a tissue adhesive, and / or a support matrix (e.g., a collagen scaffold).

[0035] Thus, the method includes administering to a subject an effective amount of a composition. In this embodiment, an "effective amount" is an amount effective to alleviate (e.g., at least partially improve) at least one symptom or clinical sign of peripheral vascular disease, vascular disorder, or vascular dysfunction. As used herein, the term "symptom" refers to any subjective evidence of a disease or patient's condition, whereas the term "sign" or "clinical sign" refers to an objective physical observation of a specific condition that can be detected by a person other than the patient. Thus, for example, the method can include administering to a subject an amount of a composition effective to enhance pro-angiogenic activity compared to a subject treated similarly but without PEP (e.g., with or without a suitable carrier, as described in more detail below), to enhance post-ischemic in vivo perfusion compared to a subject treated similarly but without PEP, and / or to enhance driving of the MAPK and / or AKT pathway compared to a subject treated similarly but without PEP.

[0036] Exemplary indicators of enhanced pro-angiogenic cellular activity include, but are not limited to, enhanced cell proliferation, enhanced tube formation (e.g., increased branch length, increased number of junctions, etc.), enhanced cell migration (e.g., decreased time to cell confluence in vitro or in vivo), or enhanced presence of pro-angiogenic factors. In all cases, the particular indicator of enhanced pro-angiogenic activity is compared to an identical subject treated without PEP.

[0037] Exemplary indicators of enhanced in vivo perfusion after ischemia include, but are not limited to, a shortened time to perfusion (e.g., in the proximal, mid, and / or distal regions), increased expression of endothelial markers (e.g., von Willebrand factor, smooth muscle actin, 5-ethynyl-2'-deoxyuridine (EdU)), an increased area of neovascularization, and / or a decreased degree of fibrosis. In all cases, the particular indicator of enhanced in vivo perfusion after ischemia is compared to an identical subject treated without PEP.

[0038] As used herein, a "subject" can be a human or any non-human animal. Exemplary non-human animal subjects include, but are not limited to, livestock animals or companion animals. Exemplary non-human animal subjects include, but are not limited to, members of the family Hominidae (e.g., including chimpanzees, gorillas, or orangutans), bovidae (e.g., including cattle), caprineae (e.g., including goats), ovines (e.g., including sheep), porcinidae (e.g., including pigs), equidae (e.g., including horses), members of the family Cervidae (e.g., including deer, elk, moose, caribou, reindeer, etc.), members of the family Bisonidae (e.g., including bison), felidae (e.g., including domestic cats, tigers, lions, etc.), canidae (e.g., including domestic dogs, wolves, etc.), birds (e.g., including turkeys, chickens, ducks, geese, etc.), rodents (e.g., including mice, rats, etc.), members of the family Leporidae (e.g., including rabbits or hares), members of the family Mustelidae (e.g., including ferrets), or members of the order Chiroptera (e.g., including bats).

[0039] PEP can be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, hydrogel, carrier solution, suspension, colloid, etc. The use of such media and / or agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a substance that is not biologically or otherwise undesirable; i.e., the substance can be administered to an individual together with PEP without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is included. As noted above, in surgical settings, exemplary suitable carriers include surgical adhesives, tissue adhesives, or support matrices (e.g., collagen scaffolds).

[0040] Pharmaceutical compositions containing PEP can be formulated in various forms suitable for the preferred route of administration. Thus, pharmaceutical compositions can be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., application to exposed nerve tissue during surgery, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transdermal, rectal, etc.) administration. Pharmaceutical compositions can be administered to mucosal surfaces, such as by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). Pharmaceutical compositions can also be administered via sustained or delayed release. Thus, for example, in one or more embodiments, pharmaceutical compositions can be formulated for intramuscular injection, intravenous administration, or subcutaneous administration.

[0041] Therefore, the pharmaceutical composition may be provided in any suitable form, including, but not limited to, a solution, a suspension, an emulsion, a spray, an aerosol, or any mixture. The pharmaceutical composition may be provided in a formulation containing any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be provided in a conventional topical dosage form, such as a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, or a lotion. The formulation may further include one or more additives, including, for example, an adjuvant, a skin penetration enhancer, a colorant, an aroma, a flavoring, a moisturizer, a thickener, etc.

[0042] The formulations may be conveniently presented in unit dosage form and may be prepared by methods known in the art of pharmacy. Methods of preparing compositions containing pharmaceutically acceptable carriers include the step of bringing the PEP into association with the carrier, which constitutes one or more accessory ingredients. In general, the formulations may be prepared by uniformly and / or intimately bringing the PEP into association with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0043] The amount of PEP administered can vary depending on various factors, including but not limited to the content and / or source of the administered PEP, the subject's weight, health condition, and / or age, and / or administration route. Thus, the absolute weight of PEP contained in a given unit dosage form can vary widely and depends on factors such as the subject's species, age, weight, and health condition, and / or administration method. Therefore, it is not practical to generally describe the amount that constitutes an effective amount of PEP for all possible uses. However, those skilled in the art can easily determine the appropriate amount by fully considering such factors.

[0044] In one or more embodiments, the dose of PEP can be measured in terms of PEP exosomes delivered per dose. Thus, in one or more embodiments, the method can be carried out using, for example, about 1×10 6 PEP exosomes ~ approx. 1 x 10 15This may include administering sufficient PEP to provide a dose of PEP exosomes to the subject, although in one or more embodiments, the method may be practiced by administering PEP at a dose outside this range.

[0045] Thus, in one or more embodiments, the method comprises: 6 PEP exosomes, at least 1 × 10 7 PEP exosomes, at least 1 × 10 8 PEP exosomes, at least 1 × 10 9 PEP exosomes, at least 1 × 10 10 PEP exosomes, at least 1 × 10 11 PEP exosomes, at least 2 × 10 11 PEP exosomes, at least 3 × 10 11 PEP exosomes, at least 4 × 10 11 PEP exosomes, at least 5 × 10 11 PEP exosomes, at least 6 × 10 11 PEP exosomes, at least 7 × 10 11 PEP exosomes, at least 8 × 10 11 PEP exosomes, at least 9 × 10 11 PEP exosomes, at least 1 × 10 12 PEP exosomes, 2 × 10 12 PEP exosomes, at least 3 × 10 12 PEP exosomes, at least 4 × 10 12 PEP exosomes, at least 5 × 10 12 PEP exosomes, at least 1 × 10 13 PEP exosomes, or at least 1 × 10 14 This may include administering sufficient PEP to provide a minimal dose of PEP exosomes.

[0046] In one or more embodiments, the method comprises: 15 The following PEP exosomes, 1 × 10 14 The following PEP exosomes, 1 × 10 13The following PEP exosomes, 1 × 10 12 The following PEP exosomes, 1 × 10 11 or 1 × 10 PEP exosomes 10 This may include administering sufficient PEP to provide a maximum dose of PEP exosomes of:

[0047] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method may include administering 1×10 PEP to provide a dose of ... 11 ~1×10 13 Dose of PEP exosomes, e.g., 1 x 10 11 ~5×10 12 PEP exosome dose, 1 × 10 12 ~1×10 13 PEP exosome dose, or 5 × 10 12 ~1×10 13 In certain embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method may include administering 1×10 PEP exosomes. 10 PEP exosomes, 1 × 10 11 PEP exosomes, 5 × 10 11 PEP exosomes, 1 × 10 12 PEP exosomes, 5 × 10 12 PEP exosomes, 1 × 10 13 PEP exosomes, 1 × 10 14 The method may include administering a dose of PEP exosomes.

[0048] Alternatively, the dose of PEP can be measured in terms of the concentration of PEP upon reconstitution from a lyophilized state. Thus, in one or more embodiments, the method can include administering PEP to a subject to provide PEP in a dose of, for example, about 0.01% to 100% solution, although in one or more embodiments, the method can be practiced by administering PEP at a dose outside this range. As used herein, a 100% solution of PEP refers to a concentration of PEP (about 2×10) solubilized in 1 mL of a liquid or gel carrier (e.g., water, phosphate buffered saline, serum-free culture medium, surgical glue, tissue adhesive, etc.). 11 For comparison, a dose of 0.01% PEP is approximately equivalent to a standard dose of exosomes prepared using conventional methods for obtaining exosomes, such as isolating exosomes from cells in vitro using standard cell-conditioned medium.

[0049] Thus, in one or more embodiments, the method may include administering sufficient PEP to provide a minimum dose of at least 0.01%, at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, or at least 70%.

[0050] In one or more embodiments, the method may include administering sufficient PEP to provide a maximum dose of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

[0051] In one or more embodiments, the method can include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method can include administering sufficient PEP to provide 1% to 50% of the dose, e.g., 5% to 20% of the dose. In certain embodiments, the method can include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method can include administering 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100% of the dose.

[0052] A single dose can be administered as a single dose, continuously over a predetermined time, or in multiple separate doses.When multiple doses are used, the amount of each dose can be the same or different.For example, a prescribed daily dose can be administered as a single dose, or as two equal or unequal doses continuously over 24 hours.When multiple doses are used to deliver a single dose, the interval between doses can be the same or different.In certain embodiments, PEP can be administered by only one dose, for example, during surgical procedures.

[0053] In certain embodiments in which multiple doses of a PEP composition are administered to a subject, the PEP composition comprises: It can be administered as needed to treat peripheral vascular disease. Alternatively, the PEP composition can be administered two, three, four, five, six, seven, eight, nine, or at least ten times. The interval between doses can be at least one day, such as at least three days, at least five days, at least seven days, at least 10 days, at least 14 days, or at least 21 days. The interval between doses can be up to six months, such as three months, two months, one month, 21 days, or 14 days.

[0054] In one or more embodiments, the method can include multiple administrations of PEP to a subject at intervals (in the case of two administrations) or multiple intervals (in the case of three or more administrations) characterized by a range having endpoints defined by any minimum interval and any maximum interval greater than the minimum interval identified above. For example, in one or more embodiments, the method can include multiple administrations of PEP at intervals of 1 day to 6 months, e.g., 3 days to 10 days. In certain embodiments, the method can include multiple administrations of PEP at intervals equal to any minimum interval or any maximum interval listed above. Thus, for example, the method can include multiple administrations of PEP at intervals of 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 1 month, 2 months, 3 months, or 6 months.

[0055] In one or more embodiments, the method may include administering a cocktail of PEPs prepared from various cell types, where each cell type has a unique pro-angiogenic, pro-perfusion restoration, or APK / AKT-driven pro-profile, e.g., protein composition and / or gene expression. In this manner, the PEP composition can provide the activity needed to treat a broader spectrum of peripheral vascular diseases than if the PEP composition were prepared from a single cell type.

[0056] In the foregoing description and in the claims that follow, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements; the terms "comprises," "comprising," and variations thereof are to be construed as open-ended, i.e., that additional elements or steps are optional and may or may not be present; unless otherwise specified, "one," "one," "the," "the," and "at least one" are used interchangeably to mean one or more; and the recitation of numerical ranges by endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0057] In the foregoing description, particular embodiments may be described in isolation for clarity. A particular embodiment may include any non-inconsistent combination of features described herein with respect to one or more embodiments, unless expressly specified otherwise that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0058] In any method disclosed herein that includes separate steps, the steps may be performed in any practicable order, and, if desired, any combination of two or more steps may be performed simultaneously.

[0059] The present invention is illustrated by the following examples, it being understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention described herein.

[0060] Illustrative Embodiments Embodiment 1 is a method of treating peripheral vascular disease, vascular disorders, or vascular dysfunction in a subject, comprising administering to the subject a therapeutic composition comprising a purified exosome product (PEP) and a pharmaceutically acceptable carrier.

[0061] Embodiment 2 is the method of embodiment 1, wherein the PEP comprises spherical or spheroidal exosomes having a diameter of 300 nm or less.

[0062] Embodiment 3 is the method of embodiment 1, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±90 nm.

[0063] Embodiment 4 is the method of embodiment 3, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±50 nm.

[0064] Embodiment 5 is the method of embodiment 4, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm±30 nm.

[0065] In a sixth embodiment, the PEP comprises 1% to 20% of CD63. - Exosomes and 80% to 99% CD63 + and exosomes.

[0066] Embodiment 7 is a method for treating a leukemia, the method comprising: - The method according to any one of embodiments 1 to 5, comprising exosomes.

[0067] Embodiment 8 is a method for treating a pulmonary artery disease, the method comprising: 11 PEP exosomes ~ 1 x 10 13 The method of any one of embodiments 1 to 7, comprising PEP exosomes.

[0068] Embodiment 9 is a method for treating a pulmonary artery disease, the method comprising: 12 PEP exosomes ~ 1 x 10 13 9. The method of embodiment 8, comprising PEP exosomes.

[0069] Embodiment 10 is the method of any one of embodiments 1 to 9, wherein the therapeutic composition further comprises a support matrix.

[0070] Embodiment 11 is the method of embodiment 10, wherein the support matrix comprises a collagen scaffold.

[0071] Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the therapeutic composition further comprises a tissue sealant, fibrin glue, or hydrogel.

[0072] Embodiment 13 is the method of any one of embodiments 1-12, wherein the therapeutic composition is applied in an amount effective to enhance pro-angiogenic activity compared to peripheral vascular disease treated without the therapeutic composition.

[0073] Embodiment 14 is the method of any one of Embodiments 1-13, wherein the therapeutic composition is applied in an amount effective to enhance in vivo perfusion after ischemia compared to peripheral vascular disease treated without the therapeutic composition.

[0074] Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the therapeutic composition is administered in an amount effective to enhance driving of the MAPK pathway or the AKT pathway compared to peripheral vascular disease treated without the therapeutic composition.

[0075] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the therapeutic composition is delivered by intramuscular injection.

[0076] Embodiment 17 is the method of any one of embodiments 1 to 16, wherein the peripheral vascular disease, vascular disorder, or vascular insufficiency comprises peripheral arterial disease.

[0077] Embodiment 18 is the method of any one of Embodiments 1-17, wherein the peripheral vascular disease, vascular disorder, or vascular insufficiency comprises atherosclerosis, ischemia, deep vein thrombosis, pulmonary embolism, varicose veins, chromic venous insufficiency, Buerger's disease, Raynaud's phenomenon, thrombophlebitis, or aneurysm.

[0078] Embodiment 19 is the method of any one of embodiments 1 to 18, wherein the subject is a human.

[0079] Embodiment 20 is the method of any one of embodiments 1 to 19, wherein the subject has an ischemic wound and the method enhances ischemic wound closure compared to a method without PEP. [Example]

[0080] cell culture Human umbilical vascular endothelial cells (HUVECs, Lonza Group AG, Basel, Switzerland) and GFP-tagged HUVECs (Essen BioScience, Inc., Ann Arbor, MI) were cultured in EBM-2 endothelial cell growth basal medium (Lonza Group AG, Basel, Switzerland) using the EGM-2 BULLET kit (Lonza Group AG, Basel, Switzerland) in a humidified chamber at 37°C supplemented with 5% CO2.

[0081] Purified Exosome Product (PEP) PEP (Rion LLC, Rochester, MN) was isolated by subjecting pooled platelets to thermal shock activation as previously described (Kamath et al., 2001, Eur Heart 22:1561-1571). The resulting product was then subjected to repeated enucleation, serial filtration, and stepwise centrifugation for the elimination of non-exosomal components. Following the encapsulation step, PEP were isolated from approximately 5 x 10 12The 100% PEP solution was obtained as a dry powder by lyophilization with each lyophilization vial containing 5 x 10 vesicles / mL. 12 The equivalent of vesicles / mL was defined as dissolving one vial of lyophilized PEP in 1 mL of phosphate-buffered saline (PBS). Prior to use, the resuspended PEP solution was filtered using a 0.22 μm filter system (STERIFLIP, MilliporeSigma, Burlington, MA). For cell culture experiments, PEP was reconstituted in the designated culture medium.

[0082] TISSEEL-PEP biogel preparation and scanning electron microscopy (SEM) TISSEEL-PEP biogels were prepared by reconstituting PEP with fibrinolysis inhibitor solution from a TISSEEL fibrin glue preparation kit (Baxter International, Inc., Deerfield, IL). The TISSEEL preparation protocol was then followed according to the manufacturer's instructions.

[0083] For SEM, samples were fixed with Trumps fixative overnight at 4 °C, washed with PBS, rinsed with deionized H2O, drained, and critical point dried before being imaged by a cold field emission scanning electron microscope (S-4700, Hitachi High-Technologies Corp., Tokyo, Japan) at an accelerating voltage of 5 kV.

[0084] Nanoparticle Tracking Analysis (NTA) PEP samples were diluted with PBS. Nanoparticle tracking analysis of exosome size and particle number was performed using a NANOSIGHT NS300 system (Malvern Panalytical, Malvern, United Kingdom) according to the manufacturer's instructions.

[0085] Zeta potential measurement PEP samples were diluted with PBS (Sigma) for zeta potential analysis using a Zetasizer Nanos Dynamic Light Scattering (Malvern Panalytical, Malvern, United Kingdom). All experiments were performed at a constant temperature of 25°C and were carried out at the Matexcel Materials Analysis Laboratory, Bohemia, NY.

[0086] Atomic force microscope (AFM) The diluted PEP samples were plated on freshly cleaved mica substrates (Ted Pella, Inc., Redding, CA) for 20 min, washed three times with deionized HO, and gently dried using a stream of nitrogen gas. 2 μm × 2 μm (width × length) images were collected at room temperature using an atomic force microscope (NANOSCOPE IV PICOFORCE multimode atomic force microscope; Bruker Scientific Instruments, Inc., Billerica, MA) in contact mode and analyzed using NANOSCOPE analysis software (Bruker Scientific Instruments, Inc., Billerica, MA).

[0087] Stiffness measurement Particle stiffness testing was performed as previously described (Zhang et al., 2018, Nat Cell Biol 20(3):332-343). Freshly cleaved mica coverslips were coated with poly-L-lysine (0.1% wt / vol in HO) for 30 minutes and subsequently incubated with the sample for 45 minutes. The samples were then rinsed three times with PBS buffer and submerged in PBS for measurement. All measurements were performed using an atomic force microscope (MFP 3D, Oxford Instruments Asylum Research, Santa Barbara, CA). The cantilever spring constant was calibrated using a thermal method, yielding a constant of 1.2–1.8 N / m. The cantilever radius of curvature was ~10 nm, and the Hertzian model was used to analyze the force curves for stiffness measurements. An array of force curves for each sample was measured with at least 10 data points collected per sample.

[0088] LC-MS / MS cholesterol quantification Bligh and Dyer extraction was performed on the reconstituted PEP samples. Samples containing the test article were injected with the appropriate diluent for LC-MS / MS analysis by reversed-phase LC-MS / MS, which was performed in part in the lipid analysis laboratory at Avanti Polar Lipids, Inc. (Alabaster, AL).

[0089] Moisture content The moisture content of the PEP was measured using a moisture analyzer (MB90, Ohaus Corp., Parsippany, NJ). Samples were weighed, rapidly heated in a halogen oven to evaporate the moisture, and then reweighed to determine the percent moisture content.

[0090] Protein extraction and quantification HUVEC or PEP samples were homogenized in lysis buffer containing the following: 50 mM NaPyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, 10 mM HEPES pH 7.4, 1% Triton X-100, 1% protease inhibitor, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mg / mL leupeptin. Protein quantification was performed using a BCA protein assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA).

[0091] Cell proliferation assay HUVECs were seeded at a density of 5,000 cells / well in 96-well plates (Corning, Inc., Corning, NY) and treated with supplement-free growth medium, regular growth medium, or PEP. Subsequently, they were stained with INCUCYTE NUCLIGHT Rapid Red (1:500, Essen BioScience, Inc., Ann Arbor, MI). The stained cell plates were placed in an INCUCYTE S3 live cell analysis system (Essen BioScience, Inc., Ann Arbor, MI) and scanned every 6 hours. Fluorescence was quantified using INCUCYTE integrated analysis software (Essen BioScience, Inc., Ann Arbor, MI) to calculate proliferation rates.

[0092] Intracellular staining, antibodies, and flow cytometry HUVECs were washed twice with PBS, stained with zombie dye (BioLegend, Inc., San Diego, CA), fixed (PERM FIX, BioLegend, Inc., San Diego, CA), washed twice with permeabilization buffer (eBioscience, San Diego, CA), and stained with anti-CD31 antibody (744361, BD Biosciences, Franklin Lakes, NJ) and anti-Ki-67 antibody (11-5698-82, ThermoFisher Scientific, Inc., Waltham, MA) for at least 30 minutes at room temperature. Cells were washed twice with permeabilization buffer (eBioscience, San Diego, CA) before flow cytometry collection. Staining antibodies were diluted 1:100 before staining. Flow cytometry experiments were performed using an 11-color system (ATTUNE NXT, Life Technologies Corp., ThermoFisher Scientific, Inc., Carlsbad, CA). Data were then analyzed with FlowJo software (BD Biosciences, Franklin Lakes, NJ).

[0093] Cell migration assay HUVECs were seeded in 96-well plates (Corning, Inc., Corning, NY). Cells were cultured until confluence, and then the cell monolayer was scratched using a wound maker (Essen BioScience, Inc., Ann Arbor, MI). Two PBS washes and a PEP (2.5 × 10) smear with serum-free medium were performed. 11 After the addition of 1000 μg / mL of 10 ...

[0094] Matrigel tube formation assay A 96-well plate (Corning, Inc., Corning, NY) was precoated with MATRIGEL (Corning Life Sciences, Corning, NY) and allowed to solidify at 37°C for 1 hour before cell seeding. GFP-tagged HUVECs (1 × 10 4 Cells (0.01% / well) were added to individual wells in the medium designated for treatment. Images were acquired using an inverted microscope (DMI6000B, Leica Microsystems GmbH, Wetzlar, Germany) at 0 h, 1 h, 3 h, and 6 h after treatment. All images were analyzed using Angiotool (Zudaire et al., 2011, PLoS One 6:e27385).

[0095] PKH26 vesicle labeling PEP vesicles were labeled with PKH26 red fluorescent dye (MilliporeSigma, Burlington, MA) according to the manufacturer's protocol. Briefly, PEP were resuspended in 1 mL of Diluent C, mixed with 4 μL of PKH26, and incubated at room temperature for 5 minutes. Labeling was quenched by adding 2 mL of 10% BSA and 8.5 mL of serum-free medium (Lonza Group AG, Basel, Switzerland). Labeled exosomes were ultracentrifuged at 190,000 × g for 2 hours, washed with PBS, and concentrated by centrifugation at 3,000 × g using a 10 kDa filter column (AMICON, Merck KGaA, Darmstadt, Germany).

[0096] Endocytosis inhibition assay HUVECs were cultured in EBM-2 basal medium (Lonza Group AG, Basel, Switzerland) at a density of 150,000 cells / well in two-well chamber slides (NUNC LAB-TEK II, Thermo Fisher Scientific, Inc., Waltham, MA). Cells were pretreated with inhibitors heparin, amiloride, dynasore, Pitstop2, or omeprazole for 30 minutes before adding labeled PEP to the HUVECs, followed by incubation at 37°C for 6 hours. The medium was then discarded, and the cells were washed with PBS to remove excess exosomes. Cells were fixed with 4% (vol / vol) paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, blocked (blocking buffer: 5% normal donkey serum, 0.2% Triton X-100 in PBS), and stained with ALEXA FLUOR (Molecular Probes, Inc., Eugene, OR) 488 Phalloidin (Thermo Fisher Scientific, Inc., Waltham, MA). Fluorescent images were acquired using a confocal microscope (LSM 780, Carl Zeiss AG, Oberkochen, Germany). Microscopic images were exported as tiff files using Zen Blue and analyzed using ImageJ software (Schneider et al., 2012, Nature Methods 9(7):671-675).

[0097] Imaging of cellular uptake in live cells To observe the fate of PEP intracellular uptake, HUVECs were cultured with PKH26-labeled PEP for 18 hours and analyzed using a confocal microscope (LSM 780, Carl Zeiss AG, Oberkochen, Germany) as previously described (Schott et al., 2019, J Cell Biol 218:3320-3335).

[0098] Rat hindlimb ischemia (HLI) model The rat study was conducted using 11 male Sprague-Dawley (SD) rats weighing between 220 and 265 g. The animals were randomly assigned to three groups: Sham group (n = 4), TISSEEL group (n = 3), and TISSEEL-PEP group (n = 4).

[0099] During both surgical procedures and SPY intraoperative laser angiography (SPY, Stryker Corp., Kalamazoo, MI), animals were anesthetized with 1–3% isoflurane, and body temperature was maintained with a circulating warm water pad. Following skin incision, the right femoral nerve, artery, and vein were visualized under a stereoscope. A unilateral ischemic wound in the right limb was achieved by ligation and excision of a segment of the femoral artery, as previously described (Mirabella et al., 2017, Nat Biomed Eng 1:0083). The artery was occluded, the surgical site was sutured, and perfusion of the operated limb was assessed by SPY immediately after surgery. Rats received an intramuscular (IM) injection of 0.3 mL of saline (sham), 0.3 mL of TISSEL (Baxter International, Inc., Deerfield, IL), or 0.3 mL of TISSEL-PEP in the surgical tissue. The surgical site was inspected for infection throughout the study period. In this moderate ischemia model, perfusion assessment was performed immediately before surgery, and 10 minutes and 21 days after surgery, as previously described (Mucke et al., 2020, Sci Rep. 10:939). No animals underwent spontaneous tissue necrosis or self-amputation during the study.

[0100] Rabbit ear ischemic wound model A rabbit study was conducted using 12 female New Zealand White rabbits weighing between 2.0 kg and 3.5 kg and randomly assigned to four groups: Sham group (n = 3), TISSEEL group (n = 3), PEP group (n = 3), and TISSEEL-PEP group (n = 3).

[0101] During surgical procedures and SPY intraoperative laser angiography (SPY, Stryker Corp., Kalamazoo, MI), animals were anesthetized with ketamine (35–40 mg / kg) and xylazine (5 mg / kg) and maintained with 2.5–3.5% isoflurane. Body temperature was maintained with a heating pad. Ischemic ear injury was performed as previously described (Chen, S., 2007, Wound Rep Reg. 15:928–935). The central and cranial arteries were ligated, and the accompanying nerves were severed. A circular subcutaneous tunnel was created through three incisions, and all subcutaneous tissue, muscles, nerves, and small blood vessels were excised. A circular, full-thickness skin wound was created on the ventral side of each ear using an 8-mm stainless steel punch. Perfusion of the operated ear was assessed by angiographic imaging (SPY Elite Fluorescent, Stryker Corp., Kalamazoo, MI) immediately before surgery, and 10 minutes and 21 days after surgery. This wound model did not develop spontaneous tissue necrosis during the study. Wound closure analysis was performed using ImageJ software (Schneider et al., 2012, Nature Methods 9(7):671-675).

[0102] Histology The rats were sacrificed 21 days after surgery. The excised muscles were fixed in 4% (vol / vol) paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 10 μm.

[0103] The rabbits were sacrificed 4 weeks after surgery, and the ear skin, including either healthy or wounded skin, was fixed in 10% neutral formalin, rinsed in 30% sucrose and 0.1% sodium azide, embedded in paraffin, and sectioned at a thickness of 5 μm.

[0104] Hematoxylin and eosin (H&E) staining and Masson's trichrome staining were performed according to standard procedures. To quantify the percentage of vascular area, images were recorded using NIS-ELEMENTS (Nikon Instruments, Inc., Melville, NY) software. ImageJ software (Schneider et al., 2012, Nature Methods 9(7):671-675) was used for image analysis.

[0105] Immunohistological analysis Immunohistochemistry was performed on deparaffinized sections. Antigens were retrieved using an acid-based antigen unmasking solution (R&D Systems, Inc., Minneapolis, MN). Next, tissue sections were permeabilized with 0.5% Triton X-100 in PBS and blocked (5% normal donkey serum, 0.2% Triton X-100 in PBS). Then, they were incubated overnight at 4°C in primary antibodies diluted in blocking buffer: anti-vWF (1:400, ab6994, Abcam, Cambridge, United Kingdom), anti-CD31 (1:400, NB6300-562, Novus Biologicals, LLC, Centennial, CO), and anti-α-SMA (1:400, NB300-978, Novus Biologicals, LLC, Centennial, CO). Samples were then stained with fluorescent secondary antibodies (Thermo Fisher Scientific Inc., Waltham, MA) for 1 hour at room temperature. For EdU staining, the EdU Cell Proliferation Kit (CLICK IT Plus, Thermo Fisher Scientific Inc., Waltham, MA) was used according to the manufacturer's protocol. Slides were mounted with Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Burlingame, CA) to visualize nuclei and captured using a confocal microscope (LSM 780, Carl Zeiss AG, Oberkochen, Germany). Microscopic images were exported as tiff files using Zen Blue and analyzed using ImageJ software (Schneider et al., 2012, Nature Methods 9(7):671-675).

[0106] Proteome profile array To measure PEP-induced angiogenic factors in vitro, HUVEC lysates with or without PEP treatment were analyzed using a Human Angiogenesis Array Kit (ARY007, R&D Systems Inc., Minneapolis, MN) according to the manufacturer's instructions. Blots were analyzed using HLImage. ++ Analysis was performed using the Quick Spots tool in Western Vision Software (Salt Lake City, Utah). Heatmap visualization was performed using pheatmap (V1.0.12; Luo et al., 2013, Bioinformatics 29:1830-1831).

[0107] Angiogenesis RT 2 PCR array Total RNA was extracted from muscle samples using the RNEASY Fibrous Tissue Kit (Qiagen, Hilden, Germany). A total of 0.5 μg of RNA was extracted using RT 2 The first strand kit (Qiagen, Hilden, Germany) was used for cDNA synthesis. 2 A Profile PCR array (Qiagen, Hilden, Germany) was designed to detect the expression of angiogenesis-related genes. The procedure was performed according to the manufacturer's instructions. Data were analyzed by the online Gene Globe Data Analysis Center (Qiagen, Hilden, Germany). For consensus clustering, analysis and heat map generation were performed using Morpheus software (Broad Institute, Cambridge, MA).

[0108] statistical analysis Unless otherwise stated, data are presented as mean ± SD, and p values less than 0.05 were considered significant. For in vitro studies, Student's t-test, one-way ANOVA followed by Dunnett's post hoc test, or Mann-Whitney test were used as appropriate. For in vivo studies, data were analyzed in an investigator-blinded manner. Two-way ANOVA, the nonparametric Mann-Whitney U test, or Fisher's exact test were used as appropriate.

[0109] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (e.g., including nucleotide sequence deposits in GenBank and RefSeq, and amino acid sequence deposits in SwissProt, PIR, PRF, and PDB, and translations from annotated coding regions in GenBank and RefSeq), are incorporated by reference in their entirety. In the event of any inconsistency between the disclosure of this application and the disclosure of any document incorporated herein by reference, the disclosure of this application shall control. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be construed therefrom. The invention is not limited to the exact details shown and described, for variations obvious to those skilled in the art are within the invention defined by the claims.

[0110] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by ordinary rounding techniques.

[0111] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible, however, all numerical values inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0112] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

1. Purified exosome products (PEP); and A carrier that is acceptable as a pharmaceutical; A pharmaceutical composition for treating a target peripheral vascular disease, vascular disorder, or vascular dysfunction, comprising the above.

2. The pharmaceutical composition according to claim 1, wherein the PEP comprises spherical or spheroidal exosomes having a diameter of 300 nm or less.

3. The pharmaceutical composition according to claim 1, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm ± 90 nm.

4. The pharmaceutical composition according to claim 3, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm ± 50 nm.

5. The pharmaceutical composition according to claim 4, wherein the PEP comprises spherical or spheroidal exosomes having an average diameter of 110 nm ± 30 nm.

6. The aforementioned PEP, 1% to 20% CD63 － exosomes; and 80% to 99% CD63 ＋ exosomes, A pharmaceutical composition according to any one of claims 1 to 5, comprising:

7. The aforementioned PEP contains at least 50% CD63 － A pharmaceutical composition according to any one of claims 1 to 5, comprising exosomes.

8. The aforementioned PEP is 1 × 10 11 PEP exosomes ~ 1 x 10⁻⁶ 13 A pharmaceutical composition according to any one of claims 1 to 5, comprising PEP exosomes.

9. The aforementioned PEP is 1 × 10 12 PEP exosomes ~ 1 x 10⁻⁶ 13 The pharmaceutical composition according to claim 8, comprising PEP exosomes.

10. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition further comprises a support matrix.

11. The pharmaceutical composition according to claim 10, wherein the support matrix comprises a collagen scaffold.

12. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition further comprises a tissue sealant, a fibrin adhesive, or a hydrogel.

13. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is applied in an amount effective in enhancing angiogenesis-promoting activity compared to peripheral vascular disease treated without the pharmaceutical composition.

14. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is applied in an amount effective in enhancing post-ischemia in vivo perfusion compared to peripheral vascular disease treated without the pharmaceutical composition.

15. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is applied in an amount effective in enhancing the drive of the MAPK pathway or AKT pathway compared to peripheral vascular disease treated without the pharmaceutical composition.

16. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition is delivered by intramuscular injection.

17. The pharmaceutical composition according to any one of claims 1 to 5, wherein the aforementioned peripheral vascular disease, vascular disorder, or vascular dysfunction includes peripheral artery disease.

18. The pharmaceutical composition according to any one of claims 1 to 5, wherein the aforementioned peripheral vascular disease, vascular disorder, or vascular dysfunction includes atherosclerosis, ischemia, deep vein thrombosis, pulmonary embolism, varicose veins, chronic (chromic) venous insufficiency, Buerger's disease, Raynaud's phenomenon, thrombophlebitis, or aneurysm.

19. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject is a human.

20. The pharmaceutical composition according to any one of claims 1 to 5, wherein the subject suffers from an ischemic wound, and the pharmaceutical composition enhances ischemic wound closure compared to the case in which the pharmaceutical composition is not used.

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