Compositions and methods for wound healing

Abstract

Generally, a method of promoting wound healing comprises administering to a wound an amount of a PEP exosome preparation effective to promote wound healing. In one or more embodiments, the PEP exosome preparation comprises a tissue sealant or surgical adhesive. In one or more embodiments, the wound is an ischemic wound, a puncture wound, a laceration, an abrasion, a surgical wound, a skin graft, or a traumatic wound.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 271,486, filed October 25, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a sequence listing that has been electronically submitted to the U.S. Patent and Trademark Office via the Patent Center as an XML file entitled "0560-000014 WO01", created on October 25, 2022, and having a size of 31.8 kilobytes. With the electronic filing of the sequence listing, the electronically submitted sequence listing serves as both the paper copy required by 37 CFR § 1.821(c) and the CRF required by § 1.821(e). The information contained in the sequence listing is incorporated herein by reference. Summary of the Invention

[0003] overview In one aspect, this disclosure describes a method of promoting wound healing. In general, the methods involve administering to the wound a PEP preparation in an amount effective to promote healing of the wound. In one or more embodiments, the PEP preparation comprises a hydrogel that includes a basement membrane protein. In one or more embodiments, the PEP preparation comprises a hydrogel that includes a thrombin sealant or a fibrin sealant. In one or more embodiments, the wound is an ischemic wound, a puncture wound, a laceration, an abrasion, a surgical wound, a skin graft, or a traumatic wound.

[0004] In one or more embodiments, the amount of the PEP preparation administered to the wound is effective to enhance angiogenesis, enhance fibroblast migration to the wound, or enhance keratinocyte migration to the wound compared to a comparable untreated wound. In one or more embodiments, the amount of the PEP preparation administered to the wound is effective to provide TGF-β to enhance expression of COL1A or COL3A compared to a comparable untreated wound.

[0005] In one or more embodiments, the amount of the PEP preparation administered to the wound is effective to reduce the Wagner Ulcer Classification grade of the wound compared to a comparable untreated wound. In one or more embodiments, the amount of the PEP preparation administered to the wound is effective to reduce reaction force change (Rc) or increase resistance to tensile force compared to a comparable untreated wound.

[0006] In one or more embodiments, the amount of the PEP preparation administered to the wound is effective to enhance expression of SMAD2, RAS, MKK3, RHOA, P38, or periostin in keratinocytes compared to untreated keratinocytes. In one or more embodiments, the amount of the PEP preparation administered to the wound is effective to enhance expression of SMAD2, RAS, MKK3, ERK1, or TAK1 in fibroblasts compared to untreated fibroblasts.

[0007] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The following description more particularly illustrates exemplary embodiments. In several places throughout the 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 description of the drawings]

[0008] [Figure 1]PEP extracellular vesicles display exosomal characteristics. (A) Representative transmission electron microscopy images of PEP exosomes. Scale bar, 200 nm. (B) Representative Western blotting of CD63, CD9, and Alix in PEP exosomes. GAPDH was used as a loading control. (C) Size distribution of PEP exosomes measured by Nanoparticle Tracking Analysis (NTA) with a peak at 105.4 nm in diameter. (D) Size distribution of PEP exosomes measured by nanoflow cytometry (NanoFCM) with a mean of 123.49 nm. [Diagram 2] PEP extracellular vesicles display exosome surface markers. (A) Representative simplified Western blot (Jess, ProteinSimple) shows the presence of CD41 (platelet marker), CD9, CD63, and Flotillin-1 (EV marker) on PEP. (B) Representative plot from nanoflow cytometry of PEP demonstrates the presence of CD41a (platelet marker) on PEP extracellular vesicles (MemGlow488+, lipid bilayer staining). (C) Pie chart showing affinity-based capture of CD41a+ vesicles followed by immunofluorescence staining for CD9, CD63, or CD81 surface markers (NanoView). (D) Bar graph showing affinity-based capture of CD41a+ vesicles followed by immunofluorescence staining for CD9, CD63, or CD81 surface markers (NanoView). [Diagram 3]PEP microvesicles promote angiogenesis in vitro. (A) In vitro angiogenesis assay using co-culture of GFP-tagged human umbilical vascular endothelial cells (HUVEC) and human dermal fibroblasts (HDFB) in the presence of VEGF, PEP, or suramin (angiogenesis inhibitor). Scale bar, 200 μm. (B) Graph showing quantification of tube formation at 6-hour increments. (C) 3D organoid differentiation assay of human keratinocytes treated with PEP or serum-free medium. Organoid harvesting was performed on day 24, and sections were prepared for microscopic observation. Hematoxylin and eosin (H&E) staining was performed for each group. Arrows in upper panel: organized differentiated keratinocytes with multiple epidermis-like layers. Arrows in lower panel: non-organized keratinocyte differentiation. (D) Brightfield microscopy images showing in vitro angiogenesis assay culture of HUVECs on extracellular matrix-coated plates under serum-free or PEP conditions. (E) Quantification of tube network formation in an in vitro angiogenesis assay of HUVECs cultured on extracellular matrix-coated plates under serum-free, FBS, or PEP conditions. [Figure 4] PEP microvesicles promote wound healing in vitro. (A) Scratch assay assessing migration of primary fibroblasts treated with FBS, PEP, or serum-free medium. Representative photographs of wound closure for FBS vs. PEP vs. serum-free medium at 0 and 32 hours. (B) Graph showing quantification of fibroblast wound closure measured by microscopic imaging performed every 2 hours. (C) Scratch assay examining migration of human keratinocytes treated with FBS, PEP, or serum-free medium. Representative photographs of wound closure for FBS vs. PEP vs. serum-free at 0 and 72 hours. (D) Graph showing quantification of keratinocyte wound closure measured by microscopic imaging performed every 3 hours. ***p<0.001, ****p<0.0001. [Diagram 5]PEP microvesicles stimulate TGF-β-mediated wound healing in vitro. (A) Schematic diagram of the mechanism of PEP-induced wound healing in vitro. (B) Representative Western blot analysis of TGF-β in PEP exosomes. GAPDH was used as a loading control. (C) ELISA-based analysis of TGF-β concentration in four different lots of PEP (Ella, ProteinSimple). (D) Procollagen I protein concentration in PEP-treated fibroblasts (ELISA). (E) Procollagen III protein concentration in PEP-treated fibroblasts (ELISA). **p<0.01. [Figure 6] PEP microvesicles stimulate TGF-β-mediated wound healing in vitro. (A) Quantification of Smad2, Ras, MKK3, RhoA, P38, and periostin mRNA expression in PEP-treated keratinocytes. (B) Quantification of Smad2, Ras, MKK3, Erk1, and TAK1 mRNA expression in PEP-treated fibroblasts. Unpaired two-tailed Student's t-test was used for each group compared to the untreated control group. *p<0.05, **p<0.01. [Figure 7] PEP is eluted from PEP-TISSEEL biogels over time. (A) Representative scanning electron microscopy (SEM) images of TISSEEL alone vs. TISSEEL+PEP showing that PEP binds to fibrils in TISSEEL. (B) PEP extracellular vesicle concentration eluted from TISSEEL over 7 days in an in vitro elution assay quantified by Nanoparticle Tracking Analysis (NTA, Nanosight NS300). (C) Average PEP extracellular vesicle size eluted from TISSEEL over 7 days quantified by NTA (Nanosight). [Figure 8]TISSEEL-PEP biogel promotes cell migration in a scratch assay. (A) Representative brightfield microscopy images of MSC migration towards PEP-TISSEEL biogel at time=0. (B) Representative brightfield microscopy images of MSC migration towards PEP-TISSEEL biogel at 4 hours. (C) Representative brightfield microscopy images of MSC migration towards PEP-TISSEEL biogel at 21 hours. (D) Representative brightfield microscopy images of MSC migration towards PEP-TISSEEL biogel at 48 hours. (E) Representative brightfield microscopy images of MSC migration towards PEP-TISSEEL biogel at 144 hours. (F) Scratch area quantified from brightfield microscopy images. [Figure 9] TISSEEL-PEP biogel promotes ischemic wound healing in vivo. (A) Schematic of the rabbit ischemic ear punch biopsy wound model. Arterial ligation created an ischemic wound environment. Animals were divided into four groups. Skin problems were left untreated or treated with PEP only, TISSEEL (Baxter International, Inc., Deerfield, IL) only, or PEP-TISSEEL biogel. (B) Photographs of representative wounds from each of the four groups. [Figure 10] TISSEEL-PEP biogel promotes ischemic wound healing in vivo. (A) Bar graphs showing quantification of wound healing (Figure 9). Each bar evaluates the average wound size of each group at day 28 as a percentage of the original wound. (B) Sebum levels in skin tissue 4 weeks after injury. (C) Hydration of skin tissue 4 weeks after injury. Skin hydration and sebum levels of the different treatment groups were measured and compared to the untreated group. Normal skin served as the baseline. (D) Clinical assessment of wound closure. All groups were evaluated weekly after injury by a certified physician using the Wagner ulcer classification system. Each individual data point was plotted on a graph with the generated smoothed spline curve. ***p<0.001. [Figure 11]PEP contributes to structural reorganization in injured tissue. (A) Hematoxylin and eosin (H&E) staining analysis was performed on untreated, TISSEEL only control, PEP only, TISSEEL-PEP, and normal control. Tissue samples were taken at sacrifice 28 days after injury for analysis. Representative pictures from each group shown at two magnifications. Scale bar in normal skin column represents 100 μm. ▲: non-healed area. Yellow arrow: hair follicle. Red arrow: neovessel. [Figure 12] PEP contributes to structural reorganization in injured tissue. H&E stained tissue sections taken from wound sites (FIG. 11) were analyzed using Image J software. (A) Quantification of epithelial layer thickness 28 days after treatment typically performed in 10 separate locations per slide. N=4. (B) Quantification of epithelial layer thickness 28 days after treatment typically performed in 5 separate locations per slide. N=4. ***p<0.001. [Figure 13] PEP contributes to structural reorganization in injured tissue. Representative 3D electron microscopy of reconstructions of injured tissues (n=3) taken at sacrifice 28 days after treatment. Yellow arrows: fibroblasts. Red arrows: disorganized collagen deposition. Colored areas: new capillaries with red blood cells. Reference bar for normal skin is 1 μm. [Figure 14] PEP biogel activated TGF-β signaling and promoted collagen organization. Masson Trichrome staining analysis (row 1), TGF-β immunofluorescence staining (row 2), Col1A immunofluorescence staining (row 3), Col3A staining (row 4), and Col1A / Col3A combined staining of untreated, TISSEEL only control, PEP only, TISSEEL-PEP, and normal skin. Skin tissues were obtained 28 days after surgery. Scale bar, 200 μm. [Figure 15]PEP biogel activated TGF-β signaling and promoted collagen organization. (A) Quantification of immunofluorescent staining of TGF-β. (B) Quantification of immunofluorescent staining of Col1A, Col3A, and calculated ratio of Col3A:Col1A. (C) Repeated tensile tests for all groups. Untreated and TISSEEL-only treated groups were stiffer and less like normal skin. Rc=variation of reaction force. (D) Maximum tensile test for all groups. The PEP-TISSEEL group had skin that could resist the highest tensile force. [Figure 16] TISSEEL-PEP treatment mediates transcriptional changes in genes that promote wound healing events. mRNA was isolated from tissue biopsies of the wound site taken 28 days after treatment and analyzed using RNA sequencing (RNA-seq). A third analysis of the resulting gene expression data demonstrated genes that were differentially expressed in injured tissue taken from TISSEEL alone compared to the TISSEEL-PEP (three columns on the right) treated group (N=3, genes were sorted using a normalized count of 100, and sorted genes met the criteria of |log2FC|>0.5 and p<0.05 were considered significantly changed). Heatmaps demonstrate differentially upregulated (red) and downregulated (blue) genes, expressed as fold change relative to untreated controls. [Figure 17]TISSEEL-PEP treatment mediates transcriptional changes in genes associated with pre-wound healing events. Gene ontology and pathway analysis of significantly differentially expressed genes determined by RNA sequencing analysis. The top 10 up- and down-regulated pathways are shown (p<0.05). The most significant and non-redundant biological processes or pathways with their respective gene numbers and p-values ​​are shown. (A) Heatmap of differentially regulated genes involved in organization of extracellular structures. (B) Heatmap of differentially regulated genes involved in regulating angiogenesis. (C) Heatmap of differentially regulated genes involved in skin development. (D) Heatmap of differentially regulated genes involved in VEGF signaling. (E) Heatmap of differentially regulated genes involved in response to wounding. (F) Heatmap of differentially regulated genes involved in collagen metabolic processes. (G) Heatmap of differentially regulated genes involved in positive regulation of cell cycle. (H) Heatmap of differentially regulated genes involved in the regulation of NIK / NF-κB signaling. The heatmap demonstrates differentially upregulated (red) and downregulated (blue) genes expressed as fold change relative to untreated controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS This disclosure describes the use of clinical grade platelet-derived exosome products (PEP) supplemented with TGF-β (transforming growth factor beta) formulated as a lyophilized, pre-formed regenerative platform for wound treatment. While described below in exemplary context with respect to the treatment of ischemic wounds, the compositions and methods described herein may include the treatment of any type of wound, including, but not limited to, ischemic wounds (e.g., ischemic ulcers), puncture wounds, lacerations, abrasions, surgical wounds, skin grafts, burn wounds, radiation-induced wounds, or traumatic wounds.

[0010] Topical treatment of ischemic wounds with PEP loaded into fibrin sealant (TISSEEL, Baxter International, Inc., Deerfield, Ill.) promoted full-thickness healing with reacquisition of hair follicles and sebaceous glands. Over untreated and TISSEEL-only treated controls, TISSEEL-PEP drove skin healing associated with collagen synthesis and restoration of skin architecture. Furthermore, PEP promoted epithelial and vascular cell activity and enhanced angiogenesis to restore blood flow and mature skin function. Transcriptomic analysis of wounds treated with TISSEEL-PEP versus TISSEEL alone prioritized regenerative pathways including neovascularization, matrix remodeling, and tissue growth. The composition is stable at room temperature when lyophilized, and therefore may provide bioactive growth factors to drive regenerative events.

[0011] Ischemic wounds affect millions of patients worldwide, resulting in life-threatening amputations and severe morbidity. For example, chronic ischemic wounds can progress to limb amputation with an associated 57% 5-year mortality rate. Wound development is believed to be the result of defective cell proliferation, reduced vascularization, and limited epithelialization. Current management includes wound dressings, topical medications, and surgery. However, no treatment to date has achieved restoration of normal skin architecture. To improve outcomes, cell-based treatments are being considered as an adjunct to standard care. Lack of ease of use and hindered by high cost, their utility remains limited, supporting the development of feasible and widely available regenerative alternatives.

[0012] Extracellular vesicles (EVs) and their exosomal subset offer a next-generation, scalable option for wound healing. Exosomes have been shown to promote healing through angiogenesis, cell proliferation and migration, and eventual re-epithelialization. Transmissible through cell membranes to mediate cell-cell communication, exosomes are highly consistent cell-secreted vesicles ranging from 30 to 150 nm in diameter that can transport lipid-encapsulated signaling proteins and nucleotides between cells.

[0013] PEP has been fully characterized and methods for preparing PEP are described in International Patent Application No. PCT / US2018 / 065627 (published as International Publication No. WO2019 / 118817), which is incorporated herein by reference in its entirety. Briefly, for example, PEP generally has a spherical or spheroidal structure rather than a crystalline structure. Spherical or spheroidal exosome structures generally have a diameter of 300 nm or less. Typically, PEP preparations contain spherical or spheroidal exosome structures with a relatively narrow size distribution. In some preparations, PEP contains spherical or spheroidal exosome structures with a mean diameter of about 110 nm ± 90 nm, with most of the exosome structures having a mean diameter of 110 nm ± 50 nm, such as 110 nm ± 30 nm.

[0014] This disclosure describes the use of a PEP preparation prepared from CD63+, CD9+, Alix-positive, activated platelets. The PEP preparation accelerated wound healing by releasing bioactive TGF-β into the wound bed. Sustained delivery using fibrin sealant resulted in significant regenerative benefits with full-thickness ischemic wound healing. The results herein provide the first evidence of the ability to retain TGF-β bioactivity in a lyophilized exosome product applied to accelerate wound healing.

[0015] Extracellular vesicles contained within PEP exhibit exosome characteristics To ensure the homogeneity of cGMP-repaired exosomes, PEP vesicles were analyzed and evaluated for vesicle morphology, exosomal surface markers using nanoparticle tracking analysis (NTA) and nanoflow cytometry (NanoFCM) (Figure 1A-D). Transmission electron microscopy (TEM) documented the intact bilayer nanostructure of PEP vesicles (Figure 1A). PEP identity was confirmed in different cGMP production lots with expression of exosomal markers, CD63, CD9, and Alix (Figure 1B), in parallel with additional release criteria. The hydrodynamic diameter of PEP had a mean value of 129.7 nm (Figure 1C). Further analysis of PEP by NanoFCM showed a similar mean diameter of 123.49 nm (Figure 1D). This multiparametric quality control assessment helped to validate the homogeneous exosomal content of PEP.

[0016] Additional surface marker characterization was performed using automated Western blotting (JESS, Protein Simple), nanoflow cytometry (Flow Nanoanalyzer, NanoFCM), and affinity capture-based probes (ExoView R200, Nanoview Biosciences) (Figure 2). Western blot analysis demonstrates the presence of exosomal surface markers CD9, CD63, and Flotillin-1, as well as the platelet-specific marker CD41 (Figure 2A). Nanoflow cytometry analysis shows that the lipid membrane-bound vesicle (MemGlow488+) population was also positive for the CD41 platelet-specific surface marker, indicating that the vesicles were of platelet origin (Figure 2B). Affinity-based capture of vesicles and subsequent fluorescent antibody staining show that both CD63 and CD81 are detected at low levels, along with enrichment of the CD9 surface marker, respectively, in the CD41+ captured population of vesicles (Figure 2C-D).

[0017] Next, PEP stimulation of skin cell healing was evaluated in vitro by its effect on neovascularization and cell proliferation (Figures 3-4). Human umbilical vascular endothelial cells (HUVECs) were cultured on fibroblast monolayers with PEP, VEGF (vascular endothelial growth factor) or suramin (VEGF inhibitor). It was noted that PEP stimulated angiogenesis of HUVECs more effectively than VEGF, as shown by a significant increase in endothelial tube formation (Figures 3A-B). Human keratinocytes (hKCs) cultured in 3D with PEP showed differentiation of hKCs in air-liquid interface culture and regenerated normal epithelial architecture within 21 days (Figure 3C). The angiogenic potential of PEP was further evaluated by culturing HUVECs in extracellular matrix and treating them with PEP. PEP significantly enhanced the ability of HUVECs to form ductal networks (meshes) over FBS and serum-free controls, indicating that PEP promotes the formation of new vasculature (Figure 3D-E). Furthermore, PEP promoted the migration of primary rabbit dermal fibroblasts and hKCs as recorded in wound scratch assays (Figure 4A-D).

[0018] Further studies pinpointed PEP-encapsulated TGF-β as a driver of wound healing events (Figure 5A). The presence of TGF-β was confirmed by Western blot (Figure 5B) and ELISA-based assay (Figure 5C). Treatment of human fibroblasts (hFBs) with PEP significantly increased the secretion of collagen types I and III versus controls (Figure 5D-E). To confirm the activity of TGF-β, downstream targets were probed in both hFBs and hKCs. Compared to controls, PEP-treated hKCs upregulated TGF-β targets including Smad2, Ras, MKK3 (mitogen-activated protein kinase kinase 3), RhoA (Ras homolog family member), P38, and periostin, facilitating epithelial transdifferentiation (Figure 6A). Upregulation was also observed in PEP-treated hFBs (Figure 6B). Increased expression of Smad2, Ras, MKK3, Erk1 (extracellular signal-regulated kinase), and TAK1 (transforming growth factor beta-activated kinase 1) confirmed the ability of PEP to provide TGF-β and promote fibroblast activation, proliferation, and collagen deposition in the wound area.

[0019] TISSEEL-PEP biogel promotes ischemic wound healing in vivo Fibrin sealant (TISSEEL, Baxter International, Inc., Deerfield, IL) was evaluated as a delivery vehicle for PEP to administer extracellular vesicles to the wound bed (Figure 7). PEP extracellular vesicles were bound to the fibrin fibrils of the fibrin sealant (Figure 7A) and provided sustained release of the vesicles over a 7-day period (Figure 7B). There was no significant change in vesicle size after mixing with and elution from the fibrin sealant (Figure 7C), indicating that the vesicles maintained their integrity and no significant aggregation occurred. To further evaluate the biocompatibility of the PEP-TISSEEL combination, an in vitro scratch assay was performed with the PEP-TISSEL mixture administered to the scratch site (Figures 8A-F). There was rapid progression of cells toward the bioreinforced fibrin scaffold and closure of the scratch (Figure 8F).

[0020] In vitro translation, we were able to demonstrate that PEP stimulates skin regeneration events and investigated the therapeutic potential of PEP in relation to a clinically established surgical sealant for wounds (TISSEEL, Baxter International, Inc., Deerfield, IL). Wound healing in an ischemic rabbit ear model was used to evaluate the efficacy of PEP-bioenhanced TISSEEL biogel (TISSEEL-PEP) compared to control groups including untreated, TISSEEL-only, and PEP-only treated animals (Figure 9A). At day 28, all groups except the TISSEEL-PEP group showed persistent wounds (Figure 9B). TISSEEL-PEP resulted in the fastest wound closure rate (Figure 10A).

[0021] Furthermore, skin hydration and sebum levels are markers of healed skin. Both sebum concentration (Figure 10B) and hydration (Figure 10C) were significantly higher in TISSEEL-PEP-treated animals, suggesting restoration of skin homeostasis. The results were further supported by Wagner ulcer classification analysis, which showed the accelerated wound healing of TISSEEL-PEP biogel (Figure 10D). Collectively, these findings suggest that TISSEEL-PEP facilitates ischemic wound healing.

[0022] PEP contributes to wound tissue reorganization To evaluate the quality of the regenerated skin, the physiology of the healed skin was investigated. Over a follow-up period of up to 4 weeks, wounds treated with TISSEEL-PEP restored normal skin architecture comparable to that of normal skin, whereas the control group demonstrated abnormal architecture (Figure 11). One-third of untreated animals exposed cartilage and minimal collagen deposition, confirming the severity of the model applied. TISSEEL-PEP wounds also redeveloped hair follicles and sebaceous glands, which were absent in the other groups. Evaluation of the skin architecture showed that TISSEEL-PEP-treated wounds displayed a predominance of organized epidermal architecture with a normalized epithelial layer (Figures 12A-B). The accelerated re-epithelialization, which minimized scar formation and prevented transepithelial water loss, may be attributed to PEP-stimulated keratinocyte migration and differentiation. For further comparison of the histology, three-dimensional electron microscopy (3D EM) was used to visualize the full thickness of the wound architecture (Figure 13). Control tissues showed disorganized and sparse collagen fibers, and PEP-only animals showed aligned collagen fibers typical of new scar formation. In contrast, TISSEEL-PEP animals showed a basket weave collagen structure (similar to the mature extracellular matrix of normal skin) (Figure 13).

[0023] PEP biogel drives TGF-β signaling to promote collagen organization Due to the differences in collagen organization and expression among the treatment groups, collagen distribution and TGF-β expression were investigated in vivo and examined for re-epithelialization, collagen synthesis, and deposition (Figure 14). Consistent with the enhanced cell migration in vitro (Figures 4, 8) and induction of TGF-β downstream gene expression (Figure 6), TISSEEL-PEP stimulated higher tissue expression of TGF-β (Figure 15A) and drove expression of collagen type I (COL1A) and collagen type III (COL3A) (Figure 15B). While having similar collagen density, the TISSEEL-PEP group had a higher COL3A / COL1A ratio compared to the normal skin group, which may suggest healing with less scar formation mediated by COL3A. In comparison, the control group showed delayed healing with scar-type cell alignment and abnormal collagen distribution (Figure 9B).

[0024] Collagen content may affect the biomechanical properties of skin, as there are different collagen concentrations and organization among the groups. When wounded skin was subjected to repeated stretching, PEP-only and TISSEEL-PEP tissues responded most closely to intact skin and showed functional collagen content relative to the other treatment groups (Figure 15C). In contrast, the untreated and TISSEEL-only groups showed the poorest elasticity. Tensile strength tests demonstrated that TISSEEL-PEP tissues had strength similar to intact skin (Figure 15D). These findings suggest that in ischemic situations, TISSEEL-PEP can regenerate skin and regain biomechanical integrity similar to normal skin. TISSEEL-PEP treatment induces transcriptional remodeling that underlies pre-wound healing events.

[0025] The molecular events underlying the TISSEEL-PEP-driven regenerative outcomes were probed by transcriptome profiling evaluating over 700 gene targets (Figure 16). Compared to the TISSEEL group, 213 genes were significantly upregulated and 523 genes were downregulated in the PEP-TISSEEL group. Gene ontology enrichment analysis and KEGG pathway analysis showed that PEP-enhanced biogel regulated genes related to pre-wound healing (Table 1).

[0026] [Table 1]

[0027] Specifically, genes related to extracellular organization, angiogenesis, skin development, and VEGF signaling were upregulated (Figure 17A-D). Genes related to TGF-β pathway and NIK / NF-κB signaling were downregulated (Figure 17E-H). Further analysis identified the PEP effect with modulation of TGF-β pathway and NIK / NF-κB signaling. These findings are consistent with the in vivo results that PEP modulated downstream mediators of TGF-β, including enhanced RhoA, Smad2, TAK1, and Ras pathways (Figure 6), underlying enhanced epithelialization, fibroblast activation, and collagen production. Interestingly, suppressed collagen metabolism was observed in TISSEEL-PEP-treated wounds, suggesting an earlier transition to the remodeling phase of wound healing. This was further supported by the transcriptional and phenotypic development of skin development and maturation.

[0028] The results provided herein document the therapeutic potential of PEP bioenhanced biogels as a cell-independent, preformed, regenerative platform for ischemic wound healing. PEP drives mitogenic events in dermal progenitor cells that initiate rapid healing of ischemic wounds, characterized by epithelial transdifferentiation and enhanced collagen deposition and organization, through the provision of bioactive TGF-β. Furthermore, TISSEEL-PEP-treated wounds demonstrated restored architecture and gene expression patterns favoring a physiological healing process. The concertation of biological events driven by TISSEEL-PEP resulted in regenerated tissue with similar properties to normal skin in histological, biomechanical, and functional assessments.

[0029] Global transcriptomic fingerprints of PEP-depleted versus PEP-enriched treatments highlighted normalization of molecular events toward a healthy state. Taken together, the data presented herein demonstrate that PEP serves as a pre-exosomal product of regeneration that generates a TGF-β-centric program to promote wound healing within an ischemic wound bed.

[0030] Evidence of favorable effects on multiple biological activities in the healing process was observed in this study. In particular, vascular events were observed in the histology of the TISSEEL-PEP group, suggesting that PEP may also target endothelial cell activity. This was further supported in vivo as transcriptome profiling revealed higher expression of genes related to angiogenesis, including VEGF signaling. Furthermore, tissue transcriptome profiling suggested downregulation of inflammatory and NIK / NF-κB-related events, suggesting that PEP may have polyvalent effects in tissues to drive regenerative events.

[0031] In summary, the data presented herein show that specialized PEP bioenhanced hydrogels promote ischemic wound healing by orchestrating epithelial transdifferentiation, collagen reorganization, and overall induction of skin tissue development via the TGF-β pathway. Thus, PEP offers a promising acellular regenerative treatment for patients suffering from chronic ischemic wounds. Therefore, PEP preparations may serve as a technological platform to provide an off-the-shelf, cell-independent regenerative treatment.

[0032] In one aspect, this disclosure describes compositions and methods for treating a wound in a subject. In various embodiments, the wound may be an ischemic wound (e.g., an ischemic ulcer), a puncture wound, a laceration, an abrasion, a surgical wound, a skin graft, or a traumatic wound. Generally, the composition includes a PEP preparation and a medicamentous acceptable carrier. In one or more embodiments, the medicamentous acceptable carrier may include, for example, a surgical adhesive or a tissue adhesive material.

[0033] 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, companion animals, or laboratory animals. Exemplary non-human animal subjects include, but are not limited to, members of the Hominidae family (including, for example, a chimpanzee, gorilla, or orangutan), bovine (including, for example, a cow), caprine (including, for example, a goat), ovine (such as a sheep), porcine (such as a pig), equine (such as a horse), members of the Cervidae family (such as a deer, elk, moose, caribou, reindeer, etc.), members of the Bisonidae family (including, for example, a bison), feline (including, for example, a domestic cat, a tiger, a lion, etc.), canine (including, for example, a domestic dog, a wolf, etc.), avian (including, for example, a turkey, a chicken, a duck, a goose, etc.), rodent (including, for example, a mouse, a rat, etc.), members of the Leporidae family (including, for example, a rabbit or a hare), members of the Mustelidae family (including, for example, a ferret), or members of the Chiroptera order (including, for example, a bat).

[0034] Thus, the method includes administering an effective amount of the composition to a wound in need of repair. An "effective amount" is an amount effective to reduce the time to wound closure compared to a suitable comparable wound that is either untreated or has received a different wound closure treatment. In one or more embodiments, an "effective amount" is an amount effective to enhance angiogenesis, enhance fibroblast migration to the wound, or enhance keratinocyte migration to the wound compared to a comparable untreated wound. In one or more other embodiments, an "effective amount" is an amount effective to enhance TGF-β, COL1A, or COL3A compared to a comparable untreated wound. In one or more other embodiments, an "effective amount" is an amount effective to reduce the Wagner Ulcer Classification grade of the wound compared to a comparable untreated wound. In one or more other embodiments, an "effective amount" is an amount effective to reduce reaction force change (Rc) or enhance resistance to tensile force compared to a comparable untreated wound. In one or more other embodiments, an "effective amount" is an amount effective to enhance expression of SMAD2, RAS, MKK3, RHOA, P38, or periostin in keratinocytes compared to untreated keratinocytes. In one or more other embodiments, an "effective amount" is an amount effective to enhance expression of SMAD2, RAS, MKK3, ERK1, or TAK1 in fibroblasts compared to untreated fibroblasts. In certain embodiments, the control may be a wound, keratinocyte, or fibroblast treated with a tissue sealant or surgical adhesive that does not include PEP. In certain embodiments, the control may be a wound, keratinocyte, or fibroblast treated with PEP in the absence of a tissue sealant or surgical adhesive.

[0035] PEP may be formulated with a pharma- ceutically acceptable carrier to form a pharmaceutical composition. As used herein, a "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antibacterial and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. In one or more embodiments, the pharma- ceutically acceptable carrier may include a hydrogel. In certain embodiments, the pharma- ceutically acceptable carrier may include a fibrin sealant (e.g., TISSEEL, Baxter International, Inc., Deerfield, IL; VISTASEAL, Johnson & Johnson Corp., New Brunswick, NJ; EVICEL, Johnson & Johnson Corp., New Brunswick, NJ; ARTISS, Baxter International, Inc., Deerfield, IL; TACHOSIL, Corza Health, Inc., Del Mar, CA; RECOTHROM, Baxter International, Inc., Deerfield, IL), tissue sealant, or surgical adhesive. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients can also be incorporated into the composition. Thus, for example, the pharma- ceutical acceptable carrier can include hydrogels including basement membrane proteins (e.g., collagen). Furthermore, multiple pharma- ceutical acceptable carriers can be combined. Thus, in certain embodiments, the pharma- ceutical acceptable carrier can include hydrogels including, for example, thrombin sealant, fibrin sealant, tissue sealant, or surgical adhesive.As used herein, "pharmacologically acceptable" refers to a material that is biologically or otherwise undesirable, i.e., that may be administered to an individual together with PEP without causing any undesirable biological effects or interacting in a deleterious way with any of the other components of the pharmaceutical composition in which it is included.

[0036] Pharmaceutical compositions containing PEP may be formulated in various forms adapted to the preferred route of administration. Thus, pharmaceutical compositions may 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.). Pharmaceutical compositions may be administered to mucosal surfaces, such as, for example, by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). Pharmaceutical compositions may also be administered via sustained or delayed release.

[0037] Thus, 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 form of mixture.The pharmaceutical composition may be delivered in a formulation with any pharma- ceutically acceptable excipient, carrier, or vehicle.For example, the formulation may be delivered in a conventional topical dosage form, such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, etc.The formulation may further include one or more additives, including, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a moisturizer, a thickener, etc.

[0038] The formulations may conveniently be present in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing compositions with 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.

[0039] The amount of PEP administered may vary depending on various factors, including but not limited to the content and / or source of the PEP administered, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of PEP contained in a given unit dosage form may vary widely and depends on factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Therefore, it is not practical to generally prescribe an amount that constitutes an amount of PEP that is effective for all possible uses. However, a person skilled in the art can easily determine an appropriate amount after due consideration of such factors.

[0040] In one or more embodiments, the dose of PEP can also be measured in terms of a dose of PEP exosomes delivered. Thus, in one or more embodiments, the method can include, for example, administering about 1×10 6 Approximately 1 × 10 15 This may include administering a sufficient amount of 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. Thus, in one or more embodiments the method includes administering at least 1×10 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 11PEP 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, or at least 5 × 10 12 PEP exosomes, at least 1 × 10 13 PEP exosomes, or at least 1 × 10 14 This may include administering a sufficient amount of PEP to provide a minimal dose of PEP exosomes.

[0041] In one or more embodiments, the method comprises: 15 PEP exosomes: 1×10 14 PEP exosomes: 1×10 13 PEP exosomes: 1×10 12 PEP exosomes: 1×10 11 PEP exosomes or less, or 1×10 10 This may include administering a sufficient amount of PEP to provide a maximum dose of no more than the PEP exosomes.

[0042] In one or more embodiments, the method may include administering a sufficient amount of 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 selected minimum dose. For example, in ... 11 ~5×10 12 Dose of PEP-exosomes: 1 x 10 12 ~1×10 13 PEP-exosome dose, or 5 x 10 12 ~1×10 13 Dose of PEP exosomes e.g. 1 x 10 11 ~1×10 13In certain embodiments, the method may include administering a sufficient amount of PEP to provide a dose of PEP-exosomes equal to any minimum dose or any maximum dose as described above. Thus, for example, the method may include administering a sufficient amount of PEP to provide a dose of PEP-exosomes equal to 1×10 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, or 1 × 10 14 Optionally, the method includes administering a dose of the PEP exosome.

[0043] Alternatively, in one or more embodiments, the method can include administering sufficient PEP to provide a subject with a dose of, for example, from about a 0.01% solution to a 100% solution, although in one or more embodiments, the method can be practiced by administering a dose of PEP outside this range. As used herein, a 100% solution of PEP refers to about 75 mg of PEP dissolved in 1 ml of a liquid or gel carrier (e.g., water, phosphate buffered saline, serum-free medium, surgical glue, tissue adhesive, etc.). For comparison, a dose of 0.01% PEP is roughly equivalent to a standard dose of exosomes prepared using conventional exosome acquisition methods, such as isolating exosomes from cells in vitro using standard cell conditioned media.

[0044] Thus, in one or more embodiments, the method may include administering a sufficient amount of 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%.

[0045] In one or more embodiments, the method may include administering a sufficient amount of 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.

[0046] In one or more embodiments, the method may include administering a sufficient amount of 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 selected minimum dose. For example, in one or more embodiments, the method may include administering a sufficient amount of PEP to provide a dose of 1% to 50%, such as a dose of 5% to 20%. In certain embodiments, the method may include administering a sufficient amount of PEP to provide a dose equal to any minimum dose or any maximum dose described above. Thus, for example, the method may include administering a dose of 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100%.

[0047] A single dose may be administered at once, continuously for a certain period of time, or in multiple doses. When multiple doses are used, the amount of each dose may be the same or different. For example, a daily amount may be administered continuously over 24 hours as a single dose, or may be administered as two or more doses, which may be equal or unequal. When multiple doses are used to deliver a single dose, the interval between doses may be the same or different. In certain embodiments, PEP may be administered from a single dose, for example, during surgery.

[0048] In an exemplary embodiment, the PEP composition may be administered as soon as the subject presents with a wound in need of repair. The subject may receive a single dose of the PEP composition, or multiple doses of the PEP composition. In certain embodiments in which multiple doses of the PEP composition are administered to the subject, the PEP composition may be administered as needed until the wound is sufficiently healed. Alternatively, the PEP composition may be administered 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times. The interval between doses can be at least a minimum of 1 day, such as, for example, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, or at least 21 days. The interval between doses can be up to 6 months, such as, for example, 3 months, 2 months, 1 month, 21 days, or 14 days.

[0049] In one or more embodiments, the method may include multiple administrations of PEP at intervals (for 2 administrations) or intervals (for more than 2 administrations) characterized by a range with endpoints defined by any minimum interval identified above and any maximum interval greater than the selected minimum interval. For example, in one or more embodiments, the method may include multiple administrations of PEP at intervals of 1 day to 6 months, such as, for example, 3 days to 10 days. In certain embodiments, the method may include multiple administrations of PEP at intervals equal to any minimum interval or any maximum interval listed above. Thus, for example, the method may 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.

[0050] In one or more embodiments, the method may include administering a cocktail of PEPs prepared from a variety of cell types, each cell type having a unique wound healing profile - e.g., protein composition and / or gene expression. In this manner, the PEP composition may provide a broader range of wound healing activity than if the PEP composition was prepared from a single cell type.

[0051] 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 "comprise", "comprising" and variations thereof are to be interpreted as open ended, i.e., additional elements or steps are optional and may or may not be present. Unless otherwise specified, "a", "an", "the", and "at least one" are used interchangeably and mean one or more than one. The recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 means 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0052] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "one or more embodiments," or "some embodiments" mean that a particular feature, configuration, composition, or characteristic described in connection with this embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in various places in this specification does not necessarily refer to the same embodiment of the present disclosure. Moreover, particular embodiments may be described separately for clarity. Thus, unless expressly specified that a feature of a particular embodiment is incompatible with a feature of another embodiment, a particular feature, configuration, composition, or characteristic may be combined in any suitable manner in one or more embodiments. Thus, a feature described in the context of one embodiment may be combined with a feature described in the context of a different embodiment, unless the features are mutually exclusive.

[0053] The words "preferred" and "preferably" refer to embodiments of the invention that may offer certain advantages, under particular circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0054] For any method disclosed herein that includes discontinuous steps, the steps may be performed in any practicable order, and, where appropriate, any combination of two or more steps may be performed simultaneously.

[0055] 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. EXAMPLES

[0056] PEP preparation Clinical grade purified exosome product (Rion LLC, Rochester, MN) vials were obtained from the Advanced Product Incubator biomanufacturing facility at Mayo Clinic. Briefly, PEP represents an advanced regenerated exosome fraction isolated from apheresis platelets by a series of filtration, enucleation, and centrifugation steps. A final encapsulation step allows for lyophilization of the exosome population while maintaining the integrity of the exosome lipid bilayer. Additional process details are provided in U.S. Pat. No. 10,596,123 and International Publication No. WO2019 / 118817A1. Sealed PEP (5 × 10 12 A vial of PEP (representing exosomes) was dissolved in 1 mL of phosphate-buffered saline (PBS) and defined as 100% (w / v). Prior to use, the PEP solution was filtered through a STERIFLIP-GP sterile 0.22 μm filtration system (MilliporeSigma, Burlington, MA). The 100% PEP solution was diluted with PBS for characterization or dissolved in culture medium for cell culture.

[0057] Electron microscope The suspended PEP was dropped onto formvar carbon-coated nickel grids for electron microscopy examination. After staining with 2% uranyl acetate, the grids were air-dried and visualized by transmission electron microscopy (TEM, H-7650 Hitachi High-Technology Science Corp., Tokyo, Japan).

[0058] Exosome characterization: NANOSIGHT, NanoView, and NanoAnalyzer A NANOSIGHT NS300 (Malvern Panalytical Ltd., Malvern, UK) was used for real-time characterization of PEP vesicle size and concentration. A NanoAnalyzer (NanoFCM) was used for nanoflow cytometry real-time characterization of PEP vesicle size and concentration. Additionally, PEP was fluorescently labeled with MEMGLOW488 lipid membrane stain and CD41a APC antibody (a platelet-specific surface marker). NanoView: Two lots of PEP were reconstituted and further diluted 1000-fold. Fifty microliters of sample was incubated on CD41a capture chips for 16 hours. For fluorescent labeling of PEP expressing the aforementioned surface markers, the chips were washed and incubated with antibodies to CD9, CD63, and / or CD81. Data were collected using an R100 reader and analyzed with EXOVIEW Scanner 3.0 software (NanoView Biosciences, Inc., Brighton, MA).

[0059] Western blot analysis PEP vesicles were reconstituted in RIPA-based lysis buffer and homogenized with an ultrasonic homogenizer (Branson Ultrasonics, Brookfield, CT). Protein concentrations were quantified using a BCA protein analysis kit (Thermo Fisher Scientific, Inc., Waltham, MA). Equal amounts of protein were resolved on SDS-PAGE gels and probe-bound on ODYSSEY nitrocellulose membranes (LI-COR Biosciences, Inc., Lincoln, NE). Overnight incubation was performed with diluted antibodies against CD63 (1:1000, Abcam ab59479, Abcam plc, Cambridge, UK), CD9 (1:1000, Cell Signaling 13174s, Cell Signaling Technology, Inc., Danvers, MA), Alix (1:1000, Cell Signaling 2171s, Cell Signaling Technology, Inc., Danvers, MA), GAPDH (1:1000, Cell Signaling 2118s, Cell Signaling Technology, Inc., Danvers, MA), followed by incubation with appropriate diluted secondary antibodies (Invitrogen, Carlsbad, CA). Bound antibodies were detected using the Odyssey System (LI-COR Biosciences, Inc., Lincoln, NE).

[0060] Simple Western blot analysis PEP vesicles were concentrated using the EXOEASY Maxi kit (QIAGEN, Hilden Germany). Total protein concentration was quantified by BCA protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA). The JESS automated Western blot system (ProteinSimple, Santa Clara, CA) was used according to the manufacturer's protocol. Proteins were loaded at 1 mg / mL to detect CD9 and Flotillin-1, 0.5 mg / mL to detect CD63, and 0.02 mg / mL to detect CD41. Primary antibodies used included rabbit anti-human CD9 (1:30, Cell Signaling 13403S, Cell Signaling Technology, Inc., Danvers, MA), rabbit anti-human Flotillin-1 (1:50, Abcam ab133497, Abcam plc, Cambridge, UK), rabbit anti-human CD63 (100 μg / mL, MAB50482, R&D Systems, Inc., Minneapolis, MN), and rabbit anti-human CD41 (1:30, NBP1-84581, Novus Biologicals, LLC, Centennial, CO). Data were analyzed with COMPASS software (ProteinSimple, Santa Clara, CA).

[0061] Ella, Automated ELISA with ProteinSimple PEP vesicles were reconstituted in 1× RIPA lysis buffer, vortexed, and incubated at room temperature for 5 min. Samples were centrifuged at 14,000 rpm for 10 min, and then the supernatant was filtered through a 0.22 μm detergent-free cellulose acetate (SFCA) filter syringe. Latent TGF-β was activated to an immunoreactive form with 1N HCl and then neutralized with 1.2N NaOH / 0.5M HEPES. Samples were diluted with sample diluent. Samples were loaded onto a TGF-β cartridge and run on an automated ELISA instrument (ELLA, ProteinSimple, Santa Clara, CA).

[0062] Quantitative real-time polymerase chain reaction (qRT-PCR) PEP-stimulated gene expression in human keratinocytes (ABC-TC536S, AcceGen, Fairfield, NJ) and fibroblasts (C0135C, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA) was examined by qRT-PCR. Keratinocytes and fibroblasts were treated with 5% PEP for 3, 12, or 24 h, while blank DMEM served as control. Total RNA was isolated from keratinocytes and fibroblasts using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's standard protocol. Complementary DNA (cDNA) was reverse transcribed from equal amounts of RNA (1 μg) by iSCRIPT cDNA Synthesis Kit (Bio-Rad laboratories, Inc., Hercules, CA). All runs were performed using SYBR Green PCR Master Mix (Quantabio, Beverly, MA) on a thermocycler (C1000 TOUCH, Bio-Rad Laboratories, Inc., Hercules, CA) for the following genes: Smad2, Ras, MKK3, Erk1, Periostin, P38, RhoA, and TAK1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as an internal control. Each cDNA sample was run in duplicate to control for pipetting errors. Primers used for amplification are listed in Table 2. Data from target genes were normalized to GAPDH and then 2 -ΔCt was calculated using the method.

[0063] [Table 2]

[0064] Collagen type I / III ELISA The concentrations of collagen types I and III were measured separately by ELISA (R&D Systems, Inc., Minneapolis, MN). The absorbance at 450 nm was measured using a microplate spectrophotometer (FLUOstar Omega, BMG Labtech, Ortenberg, Germany).

[0065] Co-culture HUVEC angiogenesis assay The INCUCYTE 96-well angiogenesis assay (Essen BioScience, Inc., Ann Arbor, MI) was performed according to the manufacturer's protocol. Briefly, lentivirus-infected green fluorescent protein (GFP)-expressing HUVECs (Essen BioScience, Inc., Ann Arbor, MI) were co-cultured with normal human dermal fibroblasts (Essen BioScience, Inc., Ann Arbor, MI) in 96-well microplates. Plates were placed in an INCUCYTE imager (Essen Bioscience, inc., Ann Arbor, MI) and images were automatically acquired in both phase and fluorescence every 6 hours for 10 days. On day 4, small molecule inhibitors were added to the endothelial tube network and maintained throughout the experiment. Tube length and branch points were quantified using the Angiogenesis Analysis Module (Essen BioScience, Inc., Ann Arbor, MI). Eight biological replicates were included for each condition.

[0066] HUVEC angiogenesis assay in extracellular matrix While keeping the plate and reagents on ice, each well of a 96-well plate was coated with 50 μl of extracellular matrix solution using an angiogenesis kit (Abcam plc, Cambridge, UK). A control well was included for each sample tested. The plate was transferred to a 37° C. incubator for 1 hour to allow the extracellular matrix solution to form a gel. PEP samples were diluted in serum-free medium containing heparin (1 U / mL) at a final concentration of 20% (v / v). HUVECs were added to each well at a density of 3,200 cells per well in 100 μl of serum-free tissue culture medium containing heparin at a concentration of 1 U / mL. The plate was placed in a 37° C. incubator for 18 hours, with pictures taken every 3 hours at 10× magnification using an INCUCYTE scanner (Essen Bioscience, inc., Ann Arbor, MI). After 18 hours, the cells were stained according to the manufacturer's protocol of the angiogenesis assay kit. Briefly, the stain provided in the kit was diluted in washing buffer. Bright field and fluorescent microscopy images were acquired after cell staining. Images were uploaded to ImageJ software and analyzed using the Angiogenesis Analyzer tool (Carpentier, et al., Sci Rep 10(1):11568, 2020).

[0067] Cell migration assay Primary rabbit dermal fibroblasts (FB) were isolated from the ear skin of healthy rabbits (approximately 6 months old, weighing 2.0-3.5 kg) and maintained in DMEM containing 10% fetal bovine serum. Primary human keratinocytes (KC) were purchased from Gibco (C0055C, Thermo Fisher Scientific, Inc. Waltham, MA) and maintained. Cell migration was analyzed by scratch wound assay (measured by a live cell imaging system (INCUCYTE S3, Essen BioScience Inc., Ann Arbor, MI)). FB or KC were seeded in 96-well plates (Corning, Inc., Corning, NY). Cells were grown under standard tissue culture conditions until confluent, and then scratched onto the cell monolayer using a 96-pin WOUNDMAKER device (Essen BiosScience Inc., Ann Arbor, MI). After two phosphate-buffered saline washes and the addition of 10% PEP with blank medium, the plates were placed in an INCUCYTE imager (Essen BiosScience Inc., Ann Arbor, MI) for timed imaging.

[0068] Adipose-derived MSCs were plated and grown to 80% confluence. Migration was measured by scratch wound assay. Wounds were created by scratching the cell monolayer using a pipette tip. Cells were washed with PBS and the effect of PEP in TISSEEL Biogel on cell migration was assessed by applying a thin thread of biogel to the center of a scratch made in the MSC monolayer. Cells were allowed to migrate for 7 days during which continuous imaging was performed by INCUCYTE and brightfield microscopy. Scratch area was quantified using ImageJ.

[0069] Skin tissue organoid assay Skin tissue organoid assays (Thermo Fisher Scientific, Inc., Waltham, MA) were performed according to the manufacturer's protocol. Briefly, human adult cortical cells (C0055C, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA) were plated on pre-coated cell culture inserts at 750,000 cells / cm. 2 The cells were seeded at 37°C and 5% CO in 50 μL of EPILIFE growth medium with supplements (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA). 2 After incubation at 4°C for 2 days, the inserts were repositioned to the desired hanging height in a 24-well plate and the medium was replaced while leaving the top compartment inside the cell culture insert empty. The skin tissue inserts were grown for 28 days after seeding and then fixed using overnight incubation in 4% paraform at 4°C. The inserts were paraffin embedded, sectioned, and subsequently processed for hematoxylin and eosin (H&E) staining. The tissue sections were photographed using an Olympus BH-2 microscope (Olympus Life Science, PA) at 400x magnification to examine the stratification of the cell layer.

[0070] Preparation of PEP biogels and SEM To prepare PEP in TISSEEL (Baxter International, Inc., Deerfield, IL), 2 ml of a standard kit was used. PEP was dissolved in fibrinolysis inhibitor solution (human fibrinogen, aprotinin, human albumin, L-histidine, nicotinamide, sodium citrate dihydrate, polysorbate, water) from the TISSEEL fibrin glue preparation kit. The solution was then prepared for topical administration according to the manufacturer's standard TISSEEL preparation protocol, and diluted with thrombin solution (500 IE / ml) / CaCl 2 (40 μmol / ml).

[0071] For SEM, samples were fixed in Trump's fixative overnight at 4°C, washed in PBS, then water, and dried. Samples were imaged with a cold-field emission scanning electron microscope (S-4700, Hitachi High-Tech Global, Tokyo, Japan).

[0072] In vivo wound healing animal model Twelve female New Zealand White rabbits (weighing 2.0-3.5 kg and approximately 6 months of age) were used in this study. The rabbits were assigned to each treatment group in turn. Ischemia was induced in both ears by ligating two of the three vascular bundles of the ear. A circular full-thickness skin defect measuring 2 cm in diameter was created in each ear. Postoperative ear ischemia was confirmed using indocyanine green angiography with a SPY Elite fluorescent imaging system (Stryker Corp., Kalamazoo, MI). The rabbits were then randomly assigned to three groups. In each group, one ear was left untreated whereas the other side was treated with 0.6 mL of TISSEEL (Baxter healthcare Corp., Deerfield, IL), 0.6 mL of 20% PEP, or 0.6 mL of TISSEEL-PEP (20%) combination. Wound healing was observed and recorded daily for the first week and weekly thereafter until the rabbits were sacrificed after 4 weeks.

[0073] Clinical assessment of wound closure To grade wound healing based on clinical perspective, all ischemic wounds were evaluated by a board-certified plastic surgeon at the Mayo Clinic once a week according to the Wagner Ulcer Classification System.

[0074] Measuring moisture and sebum levels in wounds Skin moisture and sebum levels were objectively assessed using a digital skin moisture / sebum detection device (Zinnor, Korea). All measurements were taken under standard climatic conditions (temperature, 25±1°C; relative humidity, 50±5 percent).

[0075] Biomechanical Testing Full-thickness wound specimens were cut into strips 2 mm wide. The specimens were then attached to a tensile testing device using strong adhesive (Gorilla Glue Co., Sharonville, OH). The adhesive was applied to the bare skin at the extremities to prevent slippage at the sites where the specimens were secured in the grips. The grips had a serrated inner surface to reduce slippage. Cyclic tensile testing of the specimens was performed using a custom tensile testing machine equipped with a 25-lb load cell (MLP-25, Transducer Techniques LLC, Temecula, CA). The specimens were tested at a constant strain rate of 0.1 mm / s with a peak displacement of 1 mm for 20 cycles. A preload of 1 N was applied before the start of each test. After the 20th cycle, the specimens were tested for failure using a strain rate of 0.1 mm / s. Motion control and data acquisition were operated via a custom NI LabVIEW 2018 application (National Instruments Corp., Austin, TX) with load and displacement data sampling at a rate of 50 Hz.

[0076] Histology Rabbits were sacrificed up to 4 weeks after surgery. Ear skin from the original injury site was removed and fixed in 10% neutral formalin overnight at 4°C, then rinsed in PBS containing 30% sucrose and 0.1% sodium azide for 24 hours at 4°C, with PBS replaced after approximately 12 hours, to remove all formalin residues. Specimens were then embedded in paraffin wax (Thermo Fisher Scientific, Inc., Waltham, MA) by a tissue processor (EXCELSIOR AS, Thermo Fisher Scientific, Inc., Waltham, MA). Specimens were then sliced ​​into longitudinal sections (5 μm) and prepared onto slides (SUPERFROST PLUS, New Erie Scientific LLC, Fremont, CA). After embedding, specimens were sectioned transversely at 5 μm for further use. Hematoxylin and eosin (H&E) staining and Masson's trichrome staining were performed according to standard procedures.

[0077] Immunohistochemical IHC analysis (TGF-β, Col I and III, α-smooth muscle actin, CD31) Skin sections were deparaffinized and permeabilized with 0.5% Triton® X-100 in PBS for 5 min. They were then incubated with blocking buffer (5% normal donkey serum, 0.2% Triton®-X in PBS) followed by primary antibody incubation overnight at 4°C with the following antibodies diluted in blocking buffer: anti-TGF-β (1:400, MAB240-SP, R&D Systems, Inc., Minneapolis, MN), anti-type I collagen (1:200, ab24821, Abcam, Cambridge, UK), and anti-type III collagen (1:400, ab6310, Abcam, Cambridge, UK). After three 30-min washes with PBS + 0.05% Triton®-X, samples were stained with fluorescent secondary antibodies (Thermo Fisher Scientific, Inc., Waltham, MA) for 1 h at room temperature, followed by two washes with PBS. Slides were mounted with anti-fade mounting medium containing DAPI (h-1200, Vector Laboratories, Inc., Burlingame, CA) and examined under a spinning disk confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

[0078] 3D-EM reconstruction of injured tissue Full-thickness wound samples were fixed, stained, and prepared for serial block-face microscopy using a protocol adapted from one previously described (Hua et al., 2015, Nat Commun 6(1):7923). Briefly, tissue samples were fixed by immersion in 2% glutaraldehyde + 2% paraform in 0.15 M cacodylate buffer containing 2 mM calcium chloride until further processing (minimum 24 h). Fixed specimens were washed in 0.15 M cacodylate buffer and incubated in 2% osmium tetroxide in 0.15 M cacodylate for 1.5 h at room temperature. Without rinsing, samples were incubated in 2% osmium tetroxide + 2.5% ferric cyano in 0.15 M cacodylate for an additional 1.5 h at room temperature. Deionized water (dH2O4) was used to wash the tissue. 2 Following a rinse with H2O, the samples were 2 Incubated in 1% thiocarbohydrazide in dHO for 45 min at 50°C. 2 After another rinse with dHO, the samples were washed with dHO between each reagent. 2 Successive H2O rinses were performed with several 2 They were incubated in 2% osmium tetroxide in O for 1.5 h at room temperature, in 1% aqueous uranyl acetate overnight at 4°C, and in 7% lead aspartate solution at 50°C for 1 h.

[0079] Following dehydration through a series of ethanol and acetone, the samples were infiltrated with polyepoxide resin (DURCUPAN, MilliporeSigma, St. Louis, MO), finally mounted, and polymerized in an oven at 60°C for a minimum of 24 h. To prepare the mounted samples for placement in a scanning electron microscope and subsequent imaging, 1 mm 3The pieces were roughly trimmed of any excess resin and embedded in 8 mm aluminum stubs using silver epoxy EPO-TEK (Epoxy Technology, Inc., Billerica, MA). The encapsulated samples were then carefully trimmed into 0.5 mm x 0.5 mm x 1 mm tall towers using a diamond trimming knife (trimtool 45, DiATOME, Hatfield, PA). The entire trimmed sample and stub were coated with gold palladium to aid in charge dissipation. The coated samples were then inserted into a serial block face scanning electron microscope (VOLUMESCOPE Thermo Fisher Scientific, Inc., Waltham, MA) and conditioned to high vacuum for 12 hours before imaging began.

[0080] High-resolution block-face images were acquired in a low vacuum environment using a beam energy of 3.0 kV with a current of 100 pA, a scan dwell time of 2 μs, and a pixel size of 10 nm. A stack of approximately 500 block-face images was acquired while cutting the block at 50 nm increments. The image stacks were then aligned and filtered using Amira software (Thermo Fisher Scientific, Inc., Waltham, MA) with further analysis performed using reconstruction software (Fiala, JC, 2005, J Microsc 218(1):52-61).

[0081] Drug release assay 0.3 mL of TISSEEL (Baxter Healthcare Corp., Deerfield, IL) and TISSEEL-PEP were prepared and incubated in 2 mL of PBS. The mixture was stored in an incubator at 37° C. The vesicle concentration in the supernatant was measured on days 1, 3, 7, and 14 using a NANOSIGHT NS300 (Malvern Instruments, Malvern, UK).

[0082] RNA-Seq and data analysis Injured tissues were harvested 28 days after operation. RNA was extracted using the TRIZOL PLUS RNA Purification Kit (Thermo Fisher Scientific, Inc., Waltham, MA) according to the protocol. RNA library generation and sequencing reactions were performed at GENEWIZ, LLC. (South Plainfield, NJ). Data analysis was performed following a standard mRNA analysis pipeline. mRNA expression levels were calculated as normalized counts with Poisson Distribution-based statistical analysis (Fang et al., 2012, Cell Biosci 2(1):26). Significantly changed genes (|log 2 FC|>0.5 and p<0.05) were used for heatmap visualization and further analysis. Gene ontology (GO) and pathway enrichment analysis were performed by clusterProfiler (V3.12.0; Yu et al., 2012, OMICS 16(5):284-287), and heatmap visualization was performed by pheatmap (V1.0.12; Luo, W and Brouwer, C., 2013, Bioinformatics 29(14):1830-1831).

[0083] statistical analysis Quantitative results were expressed as mean ± standard error. Statistical analysis was performed using two-way ANOVA and unpaired two-tailed Student's t-test (SPSS Statistics 13.0, SPSS Inc., Chicago, IL; Prism 8.0, GraphPad Software, San Diego, CA). A value of p<0.05 was considered statistically significant.

[0084] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (including, for example, nucleotide sequence submissions such as GenBank and RefSeq, and amino acid sequence submissions such as SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. In the event of a discrepancy between the disclosure of this application and the disclosure(s) of a document incorporated herein by reference, the disclosure of this application shall govern. The foregoing detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, as variations obvious to one skilled in the art are within the invention as defined by the claims.

[0085] Unless otherwise noted, all numerical values ​​expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood in all instances as modified by the term "about." Accordingly, unless specifically indicated to the contrary, 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 without attempting 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 applying ordinary skill in the art.

[0086] 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 in their respective testing measurements.

[0087] 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 specifically stated.

Claims

1. A pharmaceutical composition for promoting wound healing, comprising a PEP preparation in an amount effective to promote wound healing.

2. 10. The pharmaceutical composition of claim 1, wherein the PEP preparation comprises a hydrogel comprising a basement membrane protein.

3. 10. The pharmaceutical composition of claim 1, wherein the PEP preparation comprises a hydrogel comprising a thrombin sealant or a fibrin sealant.

4. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the wound comprises an ischemic wound, a puncture wound, a laceration, an abrasion, a surgical wound, a skin graft, or a traumatic wound.

5. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the amount of the PEP preparation is effective to enhance angiogenesis, enhance fibroblast migration into the wound, or enhance keratinocyte migration into the wound compared to a comparable untreated wound.

6. 4. The pharmaceutical composition of claim 1, wherein the amount of the PEP preparation is effective to provide TGF-β to enhance expression of COL1A or COL3A compared to a comparable untreated wound.

7. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the amount of the PEP preparation is effective to reduce the Wagner Ulcer Classification grade of the wound compared to a comparable untreated wound.

8. 4. The pharmaceutical composition of any one of claims 1 to 3, wherein the amount of the PEP preparation is effective to reduce reaction force change (Rc) or increase resistance to tensile force compared to a comparable untreated wound.

9. 4. The pharmaceutical composition of claim 1, wherein the amount of the PEP preparation is effective to enhance expression of SMAD2, RAS, MKK3, RHOA, p38, or periostin in keratinocytes compared to untreated keratinocytes.

10. 4. The pharmaceutical composition of claim 1, wherein the amount of the PEP preparation is effective to enhance expression of SMAD2, RAS, MKK3, ERK1, or TAK1 in fibroblasts compared to untreated fibroblasts.

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