Preparation method of endothelial-targeting peptide modified extracellular vesicles and application thereof

Abstract

The present application relates to a preparation method and application of endothelial targeting peptide modified extracellular vesicles, to solve the problems of poor targeting, unstable therapeutic molecules and limited efficacy in the prior art, realize enrichment of diabetic wounds, efficient promotion of angiogenesis and sustained release, and significantly accelerate the healing of diabetic wounds. Specifically, the preparation method comprises the steps of culturing mesenchymal stem cells, isolating and purifying extracellular vesicles of mesenchymal stem cells, loading drugs on the extracellular vesicles, modifying the extracellular vesicles with VHP peptides, and compounding with hydrogel carriers, to finally obtain endothelial targeting peptide modified extracellular vesicles. It can be applied in wound dressings or drugs for treating chronic wounds caused by diabetes.

Description

Technical Field

[0001] This invention relates to the field of bionanomaterials technology, and in particular to a method for preparing extracellular vesicles modified with endothelial-targeting peptides and its application. Background Technology

[0002] In recent years, with the increasing aging of the population and changes in people's dietary structure, the incidence of diabetes has risen rapidly. Chronic wounds caused by diabetes, especially diabetic foot ulcers, have always been a challenging problem in clinical treatment. In severe cases, they can lead to amputation or even endanger life, thus diabetic foot ulcers are attracting increasing attention. Currently, effective treatments for diabetic foot ulcers remain limited. Traditional drug treatments often struggle to precisely target damaged cells and suffer from poor drug stability and difficulty in achieving long-term release. Therefore, there is an urgent need for a new therapy that can precisely target and repair cells, provide long-term stable drug release, and significantly promote wound healing.

[0003] Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have become highly attractive therapeutic carriers and drug delivery systems in regenerative medicine due to their natural biocompatibility, low immunogenicity, and ability to deliver bioactive molecules. However, natural EVs have insufficient tissue targeting, and in specific sites, due to rapid clearance by body fluids and circulation, it is difficult to achieve sustained and slow release of EVs, which limits their therapeutic efficacy.

[0004] Histone deacetylase 7 (HDAC7)-derived 7-amino acid peptide (7A) can promote stem cell migration and plays an important role in maintaining endothelial integrity and promoting angiogenesis. However, existing natural endothelial cells (EVs) and active pharmaceutical ingredients (7A) are simply used in combination, and 7A is not effectively loaded into EVs, which not only reduces their stability but also further limits their therapeutic effects.

[0005] Therefore, developing a composite nano-formulation with active targeting capability to solve the problems of poor targeting ability, unstable drug active ingredients, and poor therapeutic effect in existing technologies is of great research significance and practical application value. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a method for preparing endothelial-targeting peptide-modified extracellular vesicles and its application, so as to solve the problems of poor targeting, unstable therapeutic molecules, and limited efficacy in the prior art, and to achieve enrichment of diabetic wounds, efficient angiogenesis promotion, sustained release, and significantly accelerate the healing of diabetic wounds.

[0007] First aspect:

[0008] A method for preparing extracellular vesicles modified with endothelial-targeting peptides includes the following steps: Culture of mesenchymal stem cells: Human platelet lysate (HPL) is mixed with DMEM / F12 medium to obtain a mixed medium, which creates a 3D culture environment for culturing mesenchymal stem cells; Isolation and purification of extracellular vesicles from mesenchymal stem cells: The supernatant of the mixed culture medium was collected and the extracellular vesicles (EVs) were isolated and purified by ultracentrifugation. Drug loading on extracellular vesicles: Extracellular vesicles are mixed with drugs, and the drugs are loaded into the extracellular vesicles by ultrasound-mediated loading to obtain drug-loaded extracellular vesicles, wherein the drugs include a 7-amino acid peptide (7A) derived from histone deacetylase 7 (HDAC7). VHP peptide-modified extracellular vesicles: VHP-CP05, a fusion peptide of VHP and CP05, was prepared with the sequence number VHPKQHR-CRHSQMTVTSRL. The drug-loaded extracellular vesicles and VHP-CP05 peptide were incubated at 4°C with shaking for 6 hours at a mass ratio of 1:1-2 to obtain VHP peptide-modified extracellular vesicles. Composite hydrogel carrier: VHP peptide-modified extracellular vesicles, hydrogel carrier and CaCl2 solution are mixed evenly and allowed to stand to form hydrogel, thus obtaining the endothelial-targeting peptide-modified extracellular vesicles.

[0009] This invention uses human platelet lysate (HPL) instead of traditional fractionated platelets (FBS) for cell culture. HPL contains a large amount of growth factors, sufficient to support the in vitro growth and expansion of mesenchymal stem cells (MSCs). Furthermore, MSCs cultured in HPL have been clinically proven to be safe and do not induce immune rejection. In addition, HPL contains a large amount of fibrinogen, which can be hydrolyzed into fibrin during cell culture to form a hydrogel, creating a 3D culture environment. This invention utilizes this convenient 3D matrix to culture MSCs, thereby increasing the secretion of extracellular vesicles and the expression levels of bioactive molecules.

[0010] Extracellular vesicles derived from mesenchymal stem cells possess a closed lipid membrane structure and specific surface composition, which endows them with good modifiability. Flexible modification can achieve targeted drug delivery, significantly improving therapeutic efficacy. The VHPKQHR peptide (VHP) can target vascular cell adhesion molecule-1 (VCAM-1) expressed on activated endothelial cells in inflamed blood vessels. Compared to non-VCAM-1-targeting nanoparticles, VHP modification significantly enhances the binding of nanoparticles to endothelial VCAM-1, thereby improving the diagnostic sensitivity for early atherosclerotic plaques and enhancing its ability to target diseased vascular endothelial cells, significantly improving drug accumulation efficiency at the lesion site and overcoming the insufficient targeting of existing therapies. This invention utilizes the EV membrane protein CD63-specific binding peptide CP05 as an anchoring peptide to construct the fusion peptide VHP-CP05, achieving highly efficient targeted modification of EVs, enhancing their ability to target diseased vascular endothelial cells, and improving therapeutic potential.

[0011] Histone deacetylase 7 (HDAC7)-derived 7-amino acid peptide (7A) can promote stem cell migration and plays an important role in maintaining endothelial integrity and promoting angiogenesis. This invention utilizes the natural vesicle structure of extracellular vesicles to effectively protect their biological activity, avoid rapid degradation by proteases in the wound microenvironment, and achieve long-term delivery.

[0012] As a preferred embodiment, the mesenchymal stem cells are derived from at least one of umbilical cord, bone marrow, or adipose tissue. Among these, mesenchymal stem cells derived from human umbilical cord have good biocompatibility and low immunogenicity, making them the optimal choice.

[0013] As a preferred embodiment, in the mesenchymal stem cell culture step, 3D culture is performed using a mixed culture medium. When the cell density reaches 60-70%, the culture medium is replaced with DMEM / F12 ordinary culture medium and cultured for 1-2 days. In the separation and purification step of the extracellular vesicles of the mesenchymal stem cells, the supernatant of the DMEM / F12 ordinary culture medium is collected and the extracellular vesicles are separated and purified by ultracentrifugation.

[0014] As a preferred embodiment, the hydrogel carrier comprises at least one of sodium alginate, chitosan, gelatin, hyaluronic acid, and polyethylene glycol.

[0015] The choice of hydrogel carrier can further optimize the therapeutic effect. Sodium alginate hydrogel not only mimics the extracellular matrix structure, providing a suitable microenvironment for wound repair, but also avoids the rapid clearance of bioactive molecules. In this embodiment of the invention, sodium alginate is selected as the hydrogel carrier because it has the best sustained-release effect, prolonging the duration of drug action to achieve better therapeutic results.

[0016] As a preferred embodiment, the mass ratio of the VHP peptide-modified extracellular vesicles, sodium alginate, and CaCl2 is 1:(1-2):(0.04-0.1), and the concentration of the CaCl2 solution is 1-2 wt%.

[0017] Sodium alginate determines the gel strength and skeletal structure; too high a content results in a too-hard gel, while too low a content results in a too-soft gel. CaCl2 determines the cross-linking rate and density of the hydrogel carrier; excessive use leads to agglomeration, while insufficient use makes it difficult to form a hydrogel system.

[0018] As a preferred embodiment, the extracellular vesicle drug loading step includes, in addition to ultrasound-mediated drug loading, at least one of transfection, high-pressure loading, electroporation, freeze-thaw, extrusion, and transmembrane chemical reagent methods.

[0019] As a preferred embodiment, the isolation and purification of extracellular vesicles of the mesenchymal stem cells can also be achieved by at least one of PEGbase precipitation, exosome marker protein immunoaffinity chromatography, and size exclusion chromatography.

[0020] The second aspect: Application of an endothelial-targeting peptide-modified extracellular vesicle prepared in the first aspect in wound dressings.

[0021] Third aspect: A drug for treating chronic wounds caused by diabetes, comprising extracellular vesicles modified with endothelial-targeting peptides prepared in the first aspect. Attached Figure Description

[0022] Figure 1 These are 2D and 3D culture morphology diagrams of MSCs.

[0023] Figure 2 The CCK-8 assay was used to detect the effect of HPL-mediated 3D culture on MSC proliferation.

[0024] Figure 3 This is a characterization of 3D cultured extracellular vesicles derived from MSCs.

[0025] Figure 4 This refers to the intake of EV and VHP@EV by HUVECs under high glucose inflammatory conditions.

[0026] Figure 5 This study investigated the effects of VHP@EV-7A on the proliferation and angiogenesis of HUVECs under hyperglycemic inflammatory conditions.

[0027] Figure 6 This is a graph showing the effects of different groups of drugs on promoting wound healing in diabetic patients.

[0028] Figure 2-6In this study, all data are expressed as mean ± standard deviation (n=3). p<0.05, p<0.01, p<0.001, one-way ANOVA and Bonferroni multiple comparison post-hoc test were used. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] A method for preparing extracellular vesicles modified with endothelial-targeting peptides includes the following steps: (1) 3D culture of mesenchymal stem cells Extracellular vesicles released from cells cultured in fetal bovine serum (FBS) medium contain heterologous serum proteins and are unsuitable for clinical use. In this embodiment of the invention, human platelet lysate (HPL) is used instead of FBS for the production of extracellular vesicles. HPL contains a large number of growth factors, sufficient to support the in vitro growth and expansion of mesenchymal stem cells (MSCs).

[0034] HPL was mixed with DMEM / F12 basal medium to prepare a medium containing 5-10 wt% HPL for MSC culture. Under the catalysis of enzymes secreted by MSCs, fibrinogen in HPL was hydrolyzed into fibrin monomers, which aggregated and combined with water in the medium to form a hydrogel, creating a 3D culture environment. When the cell density reached 60-70%, the medium was replaced with ordinary DMEM / F12 medium, and the culture was continued for 1-2 days before collecting the supernatant.

[0035] (2) Isolation and purification of extracellular vesicles of MSCs Collect the supernatant obtained in step (1), centrifuge at 300×g and 2000×g for 10 min at 4-6℃, and then centrifuge at 10000×g for 30 min to remove cells and debris. Then filter through a 0.22μm filter (Merck Millipore) to remove large extracellular vesicles and collect extracellular vesicles smaller than 200nm. Then concentrate the supernatant by centrifugation with a 100kDa ultrafiltration column by 10 times. Finally, use an Optima L-80 XP ultracentrifuge (Beckman Coulter) and Ti-70 rotor to precipitate extracellular vesicles by ultracentrifugation at 100000×g for 2 h at 4℃. The obtained extracellular vesicles precipitated on the tube wall and bottom, appearing translucent and almost invisible. They were washed twice with PBS filtered through a 0.22 μm pore size membrane, and then resuspended in PBS. The vesicles were repeatedly eluted by pipetting several times to ensure maximum dissolution and recovery of extracellular vesicles (EVs). After aliquoting, they were stored at -80°C for later use.

[0036] (3) Extracellular vesicles loaded with drug 7A peptide The EVs obtained in step (2) were mixed with the 7-amino acid peptide (7A) at a mass ratio of 1:1-2, and then subjected to sonication with parameters set as follows: amplitude 30%, temperature 25℃, sonication for 30s, ice bath for 30s, for a total of 10 cycles. Then, the extracellular vesicles (EV-7A) loaded with 7A peptide were recovered by centrifugation at 100000×g for 2h at 4℃ to remove free 7A peptide from the supernatant. The precipitate was resuspended in PBS, aliquoted, and stored at -80℃ for later use.

[0037] (4) Preparation of composite nano-formulation VHP@EV-7A Key technologies for enhancing the vascular targeting of EVs through engineered targeted modification. Peptides, as active targeting agents, possess advantages such as ease of synthesis and coupling, and low immunogenicity, making them suitable for the functional modification of drug delivery systems to prepare smart nanocarriers. Vascular cell adhesion molecule-1 (VCAM-1) exhibits high expression levels in atherosclerotic and occluded arteries of diabetic patients, while the VHP peptide (amino acid sequence VHPKQHR) has a high affinity for VCAM-1; therefore, modifying EVs with VHP peptides can enhance their targeted migration and accumulation into diseased blood vessels of diabetic patients. VHP peptide modification of EVs can be conveniently performed through the EV membrane anchoring peptide CP05.

[0038] The specific steps are as follows: First, prepare the VHP-CP05 fusion peptide of VHP and CP05, with the sequence VHPKQHR-CRHSQMTVTSRL and a purity ≥95%; In order to perform surface modification of extracellular vesicles, the EV-7A obtained in step (3) and the VHP-CP05 peptide are incubated at 4°C with shaking for 6 hours at a mass ratio of 1:1-2 to promote the binding of the peptide with EV-7A. The VHP-modified extracellular vesicle preparation is named VHP@EV-7A; Then, centrifuge at 120000×g at 4°C for 2 hours to remove unbound peptides. The purified VHP@EV-7A precipitate is finally resuspended in PBS, aliquoted and stored at -80°C for later use.

[0039] (5) Preparation of VHP@EV-7A and sodium alginate composite gel First, sodium alginate powder was dissolved in PBS to prepare a 3wt% solution. Then, VHP@EV-7A formulation, sodium alginate solution and 1-2wt% CaCl2 solution were mixed evenly in a ratio of 1:(1-2):(0.04-0.1). After standing for 5 minutes, a hydrogel was formed, and endothelial-targeting peptide-modified extracellular vesicles were obtained.

[0040] Specifically, the MSCs can be derived from at least one of umbilical cord, bone marrow, or adipose tissue. Since umbilical cord-derived MSCs have the best biocompatibility and low immunogenicity, this embodiment of the invention takes umbilical cord-derived MSCs as an example.

[0041] In step (2), in addition to ultracentrifugation to separate EVs, EVs can also be separated and purified by exosome precipitation reagent, PEGbase precipitation, exosome marker protein immunoaffinity chromatography, and size exclusion chromatography. Since ultracentrifugation is highly efficient and low-cost in separating EVs, this embodiment of the invention uses ultracentrifugation as an example.

[0042] In step (3), in addition to loading the 7A peptide, other bioactive molecules with functions such as promoting angiogenesis, anti-inflammation, antibacterial or cell regeneration can also be loaded, such as other functional peptides, small molecule drugs, siRNA, etc.

[0043] In step (3), in addition to ultrasound-mediated drug loading, drugs can also be loaded by transfection, high-pressure loading, electroporation, freeze-thaw, extrusion, and transmembrane chemical reagent methods. Since ultrasound can effectively promote the entry of 7A into the EV through transient cavitation, and has high encapsulation efficiency and is easy to operate, ultrasound-mediated drug loading is selected in this embodiment of the invention.

[0044] In step (4), in addition to VHP, other targeted peptides that can specifically bind to VCAM-1 or other inflammatory vascular markers, such as ICAM-1, E-selectin, and integrin α4β1, can also be modified onto the EV surface through CP05 or other anchoring strategies (such as DMPE-PEG, DMPE-PEG, Pal, etc.) to achieve different targeting purposes. Simultaneously, dual-target modification strategies can be explored, and through multi-receptor synergistic recognition, it is hoped that their enrichment efficiency at lesion sites can be further improved and off-target effects reduced. Since VHP peptides can specifically and with high affinity recognize VCAM-1, which is significantly highly expressed on the surface of inflamed and diseased vascular endothelial cells, VHP is used as an example in this embodiment of the invention.

[0045] In step (5), in addition to using sodium alginate as a hydrogel, chitosan, gelatin, hyaluronic acid, and polyethylene glycol (PEG) can also be used as hydrogel materials.

[0046] The endothelial-targeting peptide-modified extracellular vesicles prepared in this invention can be used in wound dressings or in drugs for treating chronic wounds caused by diabetes.

[0047] The targeted delivery strategy and compound formulation of this invention can not only be used to treat diabetic chronic wounds, but can also be extended to other diseases characterized by inflammatory vascular damage and tissue ischemia, such as myocardial infarction, lower limb ischemia, rheumatoid arthritis, and chronic non-healing wounds.

[0048] Example 1 3D culture of mesenchymal stem cells In this example, DMEM / F12 medium containing 10 wt% FBS was used as a control, and compared with DMEM / F12 medium containing 5 wt% HPL.

[0049] HPL was mixed with DMEM / F12 basal medium to prepare a medium containing 5 wt% HPL for MSC culture. Under the catalysis of enzymes secreted by MSCs, fibrinogen in HPL was hydrolyzed into fibrin monomers, which then aggregated and combined with water in the medium to form a hydrogel, creating a 3D culture environment.

[0050] The results are as follows Figure 1 As shown, MSC cells are cultured in 2D medium containing 10 wt% FBS in DMEM / F12 medium, where the cells grow in a monolayer. In 3D medium containing 5 wt% HPL in DMEM / F12 medium, the cells grow in a 3D spherical shape. It can be seen that 3D culture has a better effect.

[0051] Example 2 Detecting the effect of HPL-mediated 3D culture on MSC cell proliferation The proliferation of MSCs in DMEM / F12 medium containing 5 wt% HPL was detected using a CCK-8 cell proliferation assay. Conventional culture in DMEM / F12 medium containing 10% FBS served as a control. The specific steps are as follows: (1) With 1×10 3 -5×10 3 Cells were seeded at a density of 100 μL per well in 96-well plates. The plates were divided into an experimental group containing 5 wt% HPL in DMEM / F12 medium and a control group containing 10 wt% FBS in DMEM / F12 medium. Each group had 6 replicates and blank wells containing only the medium. (2) The 96-well plate was placed in a 37℃, 5% CO2 incubator and incubated for 1, 2, 3, 4, 5 and 6 days respectively; (3) At each detection time point, discard the culture medium, gently rinse the cells with PBS solution, add 100 μL of culture medium containing 10% CCK-8 reagent, continue incubation for 1-4 h, and then use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0052] The results are as follows Figure 2 As shown, MSCs exhibited a continuous and stable growth trend in a 3D culture system containing 5 wt% HPL.

[0053] Example 3 Isolation and purification of extracellular vesicles from 3D cultured MSCs Collect the supernatant obtained in step (1), centrifuge at 300×g and 2000×g for 10 min at 4-6℃, and then centrifuge at 10000×g for 30 min to remove cells and debris. Then filter through a 0.22μm filter (Merck Millipore) to remove large extracellular vesicles. Then concentrate the supernatant by centrifugation with a 100kDa ultrafiltration column 10 times. Finally, use an Optima L-80 XP ultracentrifuge (Beckman Coulter) with a Ti-70 rotor to precipitate extracellular vesicles at 100000×g for 2 h at 4℃. The obtained extracellular vesicles precipitate on the tube wall and bottom, are translucent and almost invisible. Wash twice with PBS filtered through a 0.22μm pore size membrane, and then resuspend in PBS. Repeat the elution process three times to ensure maximum dissolution and recovery of extracellular vesicles (EVs). Aliquot and store at -80℃ for later use.

[0054] Example 4 Characterization of 3D cultured MSC-derived EVs The extracellular vesicles (EVs) obtained in Example 3 were characterized and analyzed as follows: (1) The morphology of EVs was observed using transmission electron microscopy (TEM). The results are as follows: Figure 3 As shown in Figure A, 3D cultured MSC-derived EVs exhibit a typical membrane vesicle structure.

[0055] (2) The particle size distribution of extracellular vesicles was detected by nanoflow cytometry. The results are as follows: Figure 3 As shown in B, the particle size of fine EV is mainly distributed in the range of 50-120 nm, with an average particle size of 70.2 nm.

[0056] (3) Under the 2D and 3D culture conditions in Example 1, the particle concentration of EVs was directly determined by nanoflow cytometry. The results are as follows: Figure 3 As shown in Figure C, under the same cell number conditions, the concentration of EV particles derived from 3D culture is 2.2 times that of 2D culture.

[0057] (4) The expression of VEGF-A, CD47, WNT4, and CD9 in extracellular vesicles under 2D and 3D culture conditions was detected by Western blotting. The results are as follows: Figure 3 As shown in Figure D, extracellular vesicles extracted under both 2D and 3D culture conditions stably expressed the extracellular vesicle marker protein CD9. However, compared with 2D culture, extracellular vesicles cultured under 3D conditions highly expressed VEGF-A, CD47, and WNT4.

[0058] Example 5 Extracellular vesicles loaded with drug 7A peptide The EVs obtained in Example 3 were mixed with the 7-amino acid peptide (7A) at a 1:1 mass ratio and then subjected to sonication. The parameters were set as follows: amplitude 30%, temperature 25°C, sonication for 30 seconds, ice bath for 30 seconds, for a total of 10 cycles. Then, the extracellular vesicles (EV-7A) loaded with the 7A peptide were recovered by centrifugation at 100,000 × g for 2 hours at 4°C to remove free 7A peptide from the supernatant. The precipitate was resuspended in PBS, aliquoted, and stored at -80°C for later use.

[0059] Example 6 VHP peptide-modified extracellular vesicles First, a fusion peptide of VHP and CP05, VHP-CP05, with the sequence VHPKQHR-CRHSQMTVTSRL and a purity ≥95%, was prepared. To perform surface modification of extracellular vesicles, the EV obtained in Example 3 and the VHP-CP05 peptide were incubated at 4°C with shaking for 6 hours at a 1:1 mass ratio to promote the binding of the peptide to EV-7A. The prepared VHP-modified extracellular vesicle formulation was named VHP@EV. Then, the EV was ultracentrifuged at 120,000 × g for 2 hours at 4°C to remove unbound peptides. The purified VHP@EV precipitate was finally resuspended in PBS, aliquoted, and stored at -80°C for later use.

[0060] Example 7 VHP@EV Targeting Capability Assessment This embodiment uses human umbilical vein endothelial cells (HUVECs) to construct a high-glucose inflammation model to examine the targeting ability of VHP@EVs. The specific method is as follows: (1) Constructing an in vitro HUVEC cell hyperglycemic inflammation model To simulate the diabetic microenvironment, HUVECs were treated with 30 mmol / L glucose solution and 100 ng / mL lipopolysaccharide (LPS) for 24 h to induce a hyperglycemic inflammation model in HUVEC cells.

[0061] (2) Uptake of EV and VHP@EV by high-glucose inflammatory vascular endothelial cells EVs and VHP@EVs were labeled with the membrane-bound red fluorescent dye DiI and then co-incubated with HUVEC cells for 6 h. The uptake of EVs and VHP@EVs by the cells was then observed using laser confocal microscopy.

[0062] Experimental results are as follows Figure 4 As shown, VHP@EV uptake was significantly increased in HUVEC cells compared to unmodified EV.

[0063] Example 8 Preparation of composite nanoformulation VHP@EV-7A First, a fusion peptide of VHP and CP05, VHP-CP05, with the sequence VHPKQHR-CRHSQMTVTSRL and a purity ≥95%, was prepared. To perform surface modification of extracellular vesicles, the EV-7A obtained in Example 5 and the VHP-CP05 peptide were incubated at 4°C with shaking for 6 hours at a 1:1 mass ratio to promote the binding of the peptide to EV-7A. The prepared VHP-modified extracellular vesicle formulation was named VHP@EV-7A. Then, the peptide was ultracentrifuged at 120,000 × g for 2 hours at 4°C to remove unbound peptides. The purified VHP@EV-7A precipitate was finally resuspended in PBS, aliquoted, and stored at -80°C for later use.

[0064] Example 9 Effects of VHP@EV-7A on HUVEC proliferation and angiogenesis under hyperglycemic inflammatory conditions A cellular hyperglycemic inflammation model was established using the method described in Example 7 to investigate the effects of VHP@EV-7A on cell proliferation and angiogenesis.

[0065] The experiment included the Ctrl group, Model group, 7A treatment group, EV treatment group, VHP@EV treatment group, EV-7A treatment group, and VHP@EV-7A treatment group.

[0066] In the Ctrl group, normal HUVECs were cultured in conventional endothelial cell culture medium without LPS, and an equal volume of PBS was added.

[0067] Model group: High glucose inflammatory model cells (30 mmol / L glucose + 100 ng / mL LPS), with an equal volume of PBS added.

[0068] 7A treatment group: high glucose inflammatory model cells + 7A.

[0069] EV treatment group: high glucose inflammatory model cells + untargeted EVs.

[0070] VHP@EV treatment group: high glucose inflammatory model cells + VHP@EV.

[0071] EV-7A treatment group: high glucose inflammatory model cells + EV-7A.

[0072] VHP@EV-7A treatment group: high glucose inflammatory model cells + VHP@EV-7A.

[0073] The specific method is as follows: (1) EdU method to detect HUVEC cell proliferation capacity.

[0074] After modeling, the corresponding reagents were administered to the experimental groups for 24 hours, followed by EdU reagent for further incubation. After fixation, permeabilization and staining, the proportion of EdU-positive cells was observed and counted under a fluorescence microscope to quantitatively assess the effect of VHP@EV-7A on the proliferation of HUVECs under high glucose inflammatory conditions.

[0075] (2) Detection of HUVEC angiogenesis capacity by matrix gel lumen formation assay HUVEC cells from each treatment group were seeded into culture plates pre-coated with matrix gel and incubated in a cell culture incubator. The formation of luminal structures was observed periodically under an inverted microscope, and images of typical fields of view were acquired. The number of luminal branch points and the total luminal length were quantitatively analyzed to evaluate the effect of VHP@EV-7A on the angiogenic capacity of HUVECs with high glucose inflammation.

[0076] Experimental results are as follows Figure 5 As shown, compared with the Ctrl group, the Model group had a significantly reduced number of green fluorescent positive cells, and cell proliferation activity was significantly inhibited; at the same time, the number of tubular structures and nodes also decreased significantly, indicating that the angiogenesis capacity was significantly weakened.

[0077] Among the treatment groups, the 7A group showed no significant improvement in cell proliferation; while the number of positive cells increased progressively in the EV group, VHP@EV group, and EV-7A group, indicating a gradual recovery in cell proliferation activity. Furthermore, all treatment groups effectively promoted the formation of cellular tubular networks, with the VHP@EV-7A group showing the most significant effect. It had the highest number of positive cells, significantly higher number of tubular nodes, and significantly longer total length than the other groups, demonstrating the best in vitro cell proliferation and angiogenesis-promoting capabilities. This indicates that the VHP@EV-7A group had the most significant targeted therapeutic effect.

[0078] Example 10 Preparation of VHP@EV-7A and sodium alginate composite gel First, sodium alginate powder was dissolved in PBS to prepare a 3wt% solution. Then, the VHP@EV-7A formulation prepared in Example 8, the sodium alginate solution, and 2wt% CaCl2 solution were mixed evenly at a mass ratio of 1:2:0.1 and allowed to stand for 5 minutes to form a hydrogel, thus obtaining endothelial-targeting peptide-modified extracellular vesicles.

[0079] Example 11 Endothelial-targeting peptides modify extracellular vesicles to promote diabetic wound healing (1) Constructing a mouse model of diabetic wounds Six-week-old male Balb / c mice were used for animal experiments. After one week of acclimatization, streptozotocin (STZ) was administered intraperitoneally for 5 consecutive days to establish the diabetes model. Mice were continuously monitored for one week to confirm stable hyperglycemia. Mice with blood glucose levels consistently exceeding 16.7 mmol / L were selected as successful diabetes models. The hair on the backs of the mice was shaved with a power shaver, and the surgical area was then treated with depilatory cream. After anesthetizing the mice with isoflurane, a 1 cm diameter full-thickness skin wound was created on the backs of the mice using a sterile skin biopsy punch.

[0080] (2) Drug treatment and efficacy evaluation The model mice obtained in step (1) were randomly divided into four groups: PBS control group, 7A treatment group, VHP@EV treatment group, and VHP@EV-7A treatment group. Composite hydrogel was applied to the wounds of the mice for treatment, and the wound healing was dynamically monitored and photographed on days 0, 3, 7, 10, 14, and 21.

[0081] The results are as follows Figure 6 As shown, from day 3, the wounds in all groups showed varying degrees of contraction. The wound area in the VHP@EV-7A group was significantly smaller than that in the Ctrl, 7A, and VHP@EV groups, with a wound healing rate as high as 20%. From day 7, the wound healing rate of the VHP@EV group began to exceed that of the 7A group, but the difference was not significant. At this point, the wound healing rate of the VHP@EV-7A group exceeded 40%, while the healing rates of the other groups were less than 30%. On day 14, a significant difference began to appear between the Ctrl and VHP@EV groups. At this time, the healing rate of VHP@EV reached 65%, while the healing rate of the VHP@EV-7A group was as high as 80%. By day 21, the wound in the VHP@EV-7A group was almost completely closed; while the Ctrl, 7A, and VHP@EV groups still had obvious unhealed areas, and the wound contraction was slow.

[0082] The above embodiments are merely illustrative of several implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the invention patent. For those skilled in the art, any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A method for preparing extracellular vesicles modified with endothelial-targeting peptides, characterized in that, Includes the following steps: Culture of mesenchymal stem cells: Human platelet lysate is mixed with DMEM / F12 medium to obtain a mixed medium, which creates a 3D culture environment for culturing mesenchymal stem cells; Isolation and purification of extracellular vesicles from mesenchymal stem cells: The supernatant of the mixed culture medium was collected, and the extracellular vesicles were isolated and purified by ultracentrifugation. Drug-loaded extracellular vesicles: Extracellular vesicles are mixed with drugs, and the drugs are loaded into the extracellular vesicles by ultrasound-mediated loading to obtain drug-loaded extracellular vesicles, wherein the drugs include a 7-amino acid peptide derived from histone deacetylase 7; VHP peptide-modified extracellular vesicles: VHP-CP05, a fusion peptide of VHP and CP05, was prepared with the sequence number VHPKQHR-CRHSQMTVTSRL. The drug-loaded extracellular vesicles and VHP-CP05 peptide were incubated at 4°C with shaking for 6 hours at a mass ratio of 1:1-2 to obtain VHP peptide-modified extracellular vesicles. Composite hydrogel carrier: VHP peptide-modified extracellular vesicles, hydrogel carrier and CaCl2 solution are mixed evenly and allowed to stand to form hydrogel, thus obtaining the endothelial-targeting peptide-modified extracellular vesicles.

2. The method for preparing extracellular vesicles modified with endothelial-targeting peptides according to claim 1, characterized in that, The mesenchymal stem cells are derived from at least one of the following: umbilical cord, bone marrow, or adipose tissue.

3. The method for preparing extracellular vesicles modified with endothelial-targeting peptides according to claim 1, characterized in that, In the mesenchymal stem cell culture step, 3D culture is carried out using a mixed culture medium. When the cell density reaches 60-70%, the culture medium is replaced with DMEM / F12 ordinary culture medium and cultured for 1-2 days. In the separation and purification step of the extracellular vesicles of the mesenchymal stem cells, the supernatant of the DMEM / F12 ordinary culture medium is collected and the extracellular vesicles are separated and purified by ultracentrifugation.

4. The method for preparing extracellular vesicles modified with endothelial-targeting peptides according to claim 1, characterized in that, The hydrogel carrier includes at least one of sodium alginate, chitosan, gelatin, hyaluronic acid, and polyethylene glycol.

5. The method for preparing extracellular vesicles modified with endothelial-targeting peptides according to claim 4, characterized in that, The mass ratio of the VHP peptide-modified extracellular vesicles, sodium alginate, and CaCl2 is 1:(1-2):(0.04-0.1), and the concentration of the CaCl2 solution is 1-2 wt%.

6. The method for preparing extracellular vesicles modified with endothelial-targeting peptides according to claim 1, characterized in that, The extracellular vesicle drug loading step includes, in addition to ultrasound-mediated drug loading, at least one of transfection, high-pressure loading, electroporation, freeze-thaw, extrusion, and transmembrane chemical reagent methods.

7. The method for preparing extracellular vesicles modified with endothelial-targeting peptides according to claim 1, characterized in that, The isolation and purification of extracellular vesicles of mesenchymal stem cells can also be achieved by at least one of the following methods: PEGbase precipitation, exosome marker protein immunoaffinity chromatography, and size exclusion chromatography.

8. A wound dressing, characterized in that, Including the extracellular vesicles modified with endothelial-targeting peptides prepared according to any one of claims 1 to 7.

9. A medicine for treating chronic wounds caused by diabetes, characterized in that, Including the extracellular vesicles modified with endothelial-targeting peptides prepared according to any one of claims 1 to 7.

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