Preparation method of pericyte-like phenotype mesenchymal stem cell exosome and application thereof in diabetic wound surface
By preparing P-MSC-Exo rich in PDGFR-β, the problems of insufficient targeting and repair efficacy of conventional exosomes in diabetic wounds were solved, achieving precise repair of endothelial cells and functional vascular reconstruction, significantly accelerating wound healing, and exhibiting high safety and high efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE SEVENTH MEDICAL CENTER OF PLA GENERAL HOSPITAL
- Filing Date
- 2026-02-05
- Publication Date
- 2026-06-05
AI Technical Summary
Existing conventional exosomes have limited targeting ability in the treatment of diabetic wounds, and their repair and angiogenesis effects are limited, failing to effectively repair endothelial damage and rebuild functional vascular networks.
By inducing pericyte-like phenotypes in mesenchymal stem cells, platelet-derived growth factor receptor β (PDGFR-β)-rich exosomes (P-MSC-Exo) were prepared and purified by differential centrifugation and ultracentrifugation to achieve targeted repair of endothelial cell damage, activate the eNOS/NO signaling pathway, and promote vascular remodeling.
P-MSC-Exo can precisely target damaged vascular endothelium, repair mitochondrial damage in endothelial cells, activate the eNOS/NO signaling pathway, promote functional vascular reconstruction, significantly accelerate wound healing, avoid the risks of cell transplantation, and has high safety and high efficiency.
Smart Images
Figure CN122146592A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of biotechnology, nanomedicine and regenerative medicine, and specifically relates to a method for preparing functionally enhanced exosomes from phenotypic engineered mesenchymal stem cells, and the application of these exosomes in promoting diabetic wound healing and functional angiogenesis, specifically a method for preparing pericyte-like phenotype mesenchymal stem cell exosomes and their application in diabetic wounds. Background Technology
[0002] Diabetic foot ulcers (DFUs) and other refractory wounds are among the most serious complications of diabetes, characterized by high infection rates, high disability rates, and high mortality rates, imposing a heavy medical and economic burden globally. The core pathology lies in the dysfunction of the vascular microenvironment, leading to persistent local ischemia and tissue repair failure. Although hyperglycemia and chronic inflammation are known to be major contributing factors, the direct attacking factors initiating vascular structural collapse remain unclear. Furthermore, in the pathological state of diabetes, key signaling communication between vascular endothelial cells and their supporting cells—pericytes—is severely disrupted, particularly the endothelial nitric oxide (NO) signaling pathway mediated by endothelial nitric oxide synthase (eNOS). As a core regulator of vascular homeostasis and pericyte recruitment, impaired function of this pathway leads to vascular instability and the inability to form a mature functional vascular network.
[0003] Cell therapy, such as the application of mesenchymal stem cells (MSCs), has shown some potential, but it faces challenges such as low cell survival rates, unstable efficacy, and potential safety risks. Therefore, cell-free exosome therapy has emerged as a more promising alternative strategy. Exosomes are nanoscale vesicles secreted by cells that can deliver bioactive substances (such as proteins and miRNAs) from their parent cells and mediate intercellular communication. However, exosomes derived from conventional MSCs have relatively mild functions, and there is an urgent need to develop more powerful engineered exosomes to specifically address the complex pathology of diabetic wounds. Summary of the Invention
[0004] This invention aims to address the problems of poor targeting, limited repair and angiogenesis efficacy of conventional exosomes in the treatment of diabetic wounds. Specifically, this invention is committed to providing a novel method for preparing functionally enhanced exosomes that can effectively repair initial endothelial damage and reactivate the endothelial-pericylic cell signaling axis, thereby driving the reconstruction of functional blood vessels and ultimately achieving highly effective treatment of diabetic wounds.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] A method for preparing pericyte-like mesenchymal stem cell exosomes (P-MSC-Exo) includes the following steps:
[0007] Step 1: Providing pericyte-like mesenchymal stem cells (P-MSCs): Mesenchymal stem cells (MSCs) are induced and cultured in a medium containing an effective concentration of nitric oxide (NO) donor to obtain pericyte-like mesenchymal stem cells (P-MSCs).
[0008] Step 2: Collect conditioned medium: The P-MSCs were cultured in an exosome-free medium, and the cell culture supernatant containing exosomes was collected;
[0009] Step 3: Separation and purification of exosomes: The cell culture supernatant is separated and purified to obtain the P-MSC-Exo.
[0010] Furthermore, the nitric oxide (NO) donor in step 1 is sodium nitroprusside (SNP), and the final concentration of sodium nitroprusside in the culture medium is 10 µM to 1000 µM, preferably 200 µM.
[0011] Further, the separation and purification process in step 3 includes differential centrifugation and ultracentrifugation. The centrifugal force of the ultracentrifugation is 100,000g to 150,000g. Specifically, the cell supernatant is centrifuged at 300g for 30 minutes at 4°C to remove dead cells, and then centrifuged at 2000g for 20 minutes to remove cell debris and microvesicles. The supernatant is then transferred to a 15ml ultrafiltration tube and centrifuged at 3500g for 20 minutes at 4°C to obtain a concentrated supernatant. After filtering the concentrated supernatant through a 0.22μm filter membrane, it is ultracentrifuged at 120,000g for 90 minutes at 4°C. The collected precipitate is P-MSC-Exo.
[0012] A pericyte-like phenotype mesenchymal stem cell exosome prepared by the above-described method, wherein the pericyte-like phenotype mesenchymal stem cell exosome (P-MSC-Exo) has a surface rich in platelet-derived growth factor receptor β (PDGFR-β), and the exosome has one or more of the following characteristics:
[0013] Morphologically, it exhibits a typical cup-shaped bilayer film structure with a diameter of approximately 120 nm under transmission electron microscopy.
[0014] In terms of molecular markers, it still expresses exosome marker proteins CD63, CD81 and TSG101, and is rich in pericyte-associated marker platelet-derived growth factor receptor β (PDGFR-β).
[0015] Functionally, it can be efficiently internalized by vascular endothelial cells and can repair mitochondrial damage in endothelial cells induced by high glucose or neutrophil extracellular traps (NETs), restoring mitochondrial membrane potential.
[0016] A pharmaceutical composition or extracellular vesicle formulation comprising a therapeutically effective amount of P-MSC-Exo and one or more pharmaceutically acceptable carriers, excipients, or delivery systems.
[0017] Furthermore, its application in the preparation of drugs for promoting angiogenesis, enhancing vascular barrier function, or promoting vascular maturation.
[0018] Furthermore, its application in the preparation of drugs for the prevention or treatment of ischemic diseases.
[0019] Furthermore, ischemic diseases include diabetic wounds, diabetic foot, lower extremity venous ulcers, or pressure sores.
[0020] This invention provides a method for preparing pericyte-like mesenchymal stem cell exosomes and their application in diabetic wounds. It has the following beneficial effects:
[0021] 1. This invention provides a method for preparing pericyte-like mesenchymal stem cell exosomes and their application in diabetic wounds. Compared with existing technologies, this invention has the advantages of strong targeting and precise repair. The P-MSC-Exo prepared by this invention is rich in PDGFR-β on its surface, enabling it to specifically target and accumulate in damaged vascular endothelium expressing its ligand PDGF-BB, achieving precise delivery. Its contents can directly repair mitochondrial damage in endothelial cells, restoring cell function from the root cause.
[0022] 2. This invention provides a method for preparing pericyte-like phenotype mesenchymal stem cell exosomes and their application in diabetic wounds. Compared with existing technologies, the angiogenesis mechanism is innovative and efficient. P-MSC-Exo not only repairs the endothelium, but more importantly, it can activate the eNOS / NO signaling pathway of endothelial cells, transforming endothelial cells into signaling stations, releasing NO in situ and continuously to recruit the host's own pericytes, achieving a leap from repair to reconstruction, and constructing a complete, mature and stable functional blood vessel.
[0023] 3. This invention provides a method for preparing pericyte-like phenotype mesenchymal stem cell exosomes and their application in diabetic wounds, achieving "cell-free" treatment with high safety: As a cell-free nanoscale biological product, P-MSC-Exo avoids the risks of immune rejection and tumorigenicity that may be caused by cell transplantation, has higher biosafety, lower immunogenicity, and is easy to standardize production, storage and use.
[0024] 4. This invention provides a method for preparing pericyte-like phenotype mesenchymal stem cell exosomes and their application in diabetic wounds. The in vivo therapeutic effect is significant. In diabetic animal wound models, the exosomes of this invention exhibit a strong healing-promoting effect, significantly accelerating wound closure and promoting the formation of high-quality neovascular networks, demonstrating its great clinical translational potential. Attached Figure Description
[0025] Figure 1 Morphological images of P-MSC-Exo captured by transmission electron microscopy;
[0026] Figure 2 To detect the exosome size distribution using an NTA instrument;
[0027] Figure 3 Western blot was used to detect the expression of CD63, CD81 and TSG101 proteins in P-MSC-Exo.
[0028] Figure 4 The expression of PDGFR-β in P-MSC-Exo was detected by ELISA.
[0029] Figure 5 The internalization of P-MSC-Exo in vascular endothelial cells was captured by an inverted fluorescence microscope;
[0030] Figure 6 To detect the co-localization of P-MSC-Exo with neovascularization using immunofluorescence staining.
[0031] Figure 7 Images of mitochondrial morphology in vascular endothelial cells after different treatments, captured by transmission electron microscopy;
[0032] Figure 8 Results of succinate dehydrogenase (SDH) activity assay in vascular endothelial cells that have undergone different treatments;
[0033] Figure 9 Mitochondrial membrane potential of vascular endothelial cells treated with different methods, captured by an inverted fluorescence microscope;
[0034] Figure 10To detect eNOS expression in vascular endothelial cells treated with different methods using qRT-PCR;
[0035] Figure 11 To detect NO release from vascular endothelial cells treated with different methods using the Griess method;
[0036] Figure 12 To detect pericytosis of vascular endothelial cells treated with different methods using the Transwell assay;
[0037] Figure 13 To detect angiogenesis in damaged vascular endothelial cells treated with different methods in an angiogenesis assay;
[0038] Figure 14 To detect the VE-cadherin and ZO-1 protein linkages between damaged vascular endothelial cells treated with different methods using cellular immunofluorescence.
[0039] Figure 15 Transendothelial resistance (TEER) and FITC-dextran permeability of damaged vascular endothelial cells subjected to different treatments;
[0040] Figure 16 To detect the contractile capacity of damaged vascular endothelial cells subjected to different treatments using a collagen gel contraction assay;
[0041] Figure 17 The wound healing status of diabetic mice that received different treatments, captured by dermoscopy;
[0042] Figure 18 To detect the co-localization of neovascularization and eNOS in the wounds of diabetic mice treated with different methods using immunofluorescence staining.
[0043] Figure 19 To detect neovascularization and ZO-1 localization in wounds of diabetic mice treated with different methods using immunofluorescence staining.
[0044] Figure 20 To detect the localization of neovascularization and α-SMA in the wounds of diabetic mice that received different treatments using immunofluorescence staining.
[0045] Figure 21 The results of calculating the diameter of neovascularization in wounds of diabetic mice that received different treatments were obtained by immunofluorescence staining scans. Detailed Implementation
[0046] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0047] This invention provides a method for preparing pericyte-like mesenchymal stem cell exosomes and their application in diabetic wounds, including...
[0048] Example 1: As Figure 1-6 Preparation and identification of P-MSC-Exo as shown
[0049] Step 1: Preparation of P-MSC-Exo: P-MSCs obtained by induction with 200 μM SNP for 3 days were cultured to 80%-90% confluence, then replaced with culture medium containing exosome-free serum and cultured for another 48 hours. The cell culture supernatant was collected and centrifuged at 4°C using the following differential speeds: 300g for 30 minutes to remove dead cells; 2000g for 20 minutes to remove cell debris; the supernatant was transferred to a 15 mL ultrafiltration tube (10 KD molecular weight cutoff) and concentrated by centrifugation at 3500g for 20 minutes at 4°C. The concentrated supernatant was filtered through a 0.22 µm filter membrane and then centrifuged at 120,000g for 90 minutes at 4°C. The supernatant was discarded, and the resulting precipitate was P-MSC-Exo. The precipitate was resuspended in sterile PBS, and the protein concentration was determined by the BCA method. After aliquoting, the precipitate was stored at -80°C for later use.
[0050] Step 2: Morphological and physicochemical property identification:
[0051] Transmission electron microscopy (TEM) observation: A suitable amount of exosome suspension was dropped onto a copper grid, fixed with 1% glutaraldehyde, and stained with uranyl oxalate. The results showed that P-MSC-Exo exhibited a uniform, cup-shaped vesicle structure with a double membrane, such as... Figure 1 ;
[0052] Nanoparticle tracking analysis (NTA): Exosome suspensions were analyzed using an NTA instrument. Results showed that the peak particle size distribution of P-MSC-Exo was around 120 nm, consistent with the typical size range of exosomes. Figure 2 ;
[0053] Western Blot analysis: Total exosome protein was extracted, and exosome markers were detected. Results showed that CD63, CD81, and TSG101 proteins were all positively expressed in the P-MSC-Exo sample, confirming its exosome identity. Figure 3 ;
[0054] Targeting and internalization capability verification:
[0055] In vitro internalization: P-MSC-Exo was labeled with PKH67 green fluorescent dye and co-incubated with SVEC4-10 endothelial cells for 4 hours. Fluorescence microscopy revealed a large amount of green fluorescent signal entering the cytoplasm, indicating that P-MSC-Exo can be efficiently internalized by endothelial cells. Figure 5 ;
[0056] In vivo homing: PKH67-labeled P-MSC-Exo was injected subcutaneously into the area surrounding the wound in db / db mice. Forty-eight hours later, frozen sections of the wound tissue were collected, and blood vessels were labeled with anti-CD31 antibody (red fluorescence). Microscopic observation revealed abundant green exosome signals co-localizing with red vascular structures, demonstrating that P-MSC-Exo actively targets and accumulates in damaged vascular areas, such as... Figure 6 ;
[0057] PDGFR-β enrichment: ELISA detection revealed that the PDGFR-β content in P-MSC-Exo was significantly higher than that in conventional MSC-Exo, such as... Figure 4 .
[0058] Example 2: As Figure 7-9 The P-MSC-Exo method is shown to repair mitochondrial function in endothelial cells.
[0059] SVEC4-10 cells were exposed to a 30 mM high-glucose environment or NETs conditioned medium for 24 hours to induce a damage model. Then, they were treated with PBS, MSC-Exo (100 μg / mL), P-MSC-Exo (100 μg / mL), or SNP (200 μM) for 24 hours, respectively.
[0060] Mitochondrial ultrastructural observation (TEM): Results showed that mitochondria in the high glucose / NETs treatment group exhibited severe swelling, vacuolation, and cristae breakage and disappearance. However, after P-MSC-Exo treatment, mitochondrial morphology returned to normal, with intact structure and clearly defined cristae. Figure 7 ;
[0061] Mitochondrial function assay (SDH activity): The activity was detected using a succinate dehydrogenase (SDH) activity assay kit. Results showed that P-MSC-Exo significantly improved SDH activity repair in damaged endothelial cells compared to the MSC-Exo treatment group. Figure 8 ;
[0062] Mitochondrial membrane potential (MMP) detection: Staining was performed using the JC-1 probe. Results showed that the damaged group cells mainly emitted green fluorescence (decreased MMP), while the P-MSC-Exo treatment group recovered strong red fluorescence (restored MMP), indicating that it effectively reversed mitochondrial depolarization. Figure 9 .
[0063] Example 3: As Figure 10-12 The P-MSC-Exo shown promotes pericyte recruitment via the eNOS / NO pathway;
[0064] Activate the eNOS / NO signaling pathway:
[0065] SVEC4-10 cells were treated with PBS, MSC-Exo (100 μg / mL), P-MSC-Exo (100 μg / mL), or SNP (200 μM), respectively.
[0066] qRT-PCR detection of eNOS mRNA: Results showed that after 24 hours of P-MSC-Exo treatment, the relative upregulation level of eNOS mRNA expression was significantly higher than that in the MSC-Exo group, such as... Figure 10 ;
[0067] NO release was detected using the Griess method: Supernatants were collected at 1 and 4 hours after treatment. Results showed that after 4 hours, the cumulative NO release in the P-MSC-Exo group was significantly higher than that in the MSC-Exo group. Figure 11
[0068] Pericytokinesis assay:
[0069] Migration experiments were performed using Transwell chambers. SVEC4-10 cells pretreated with PBS, MSC-Exo (100 μg / mL), or p-MSC-Exo (100 μg / mL), or untreated SVEC4-10 cells were cultured in the lower chamber with SNP (200 μM). Primary pericytes treated with serum starvation were added to the upper chamber. After 24 hours of culture, pericytes that had migrated below the membrane were stained with crystal violet and counted. The results showed that endothelial cells pretreated with p-MSC-Exo had the strongest recruitment ability for pericytes, with a significantly higher number of migrating cells than the MSC-Exo group and the SNP group (as a positive control). Figure 12 .
[0070] Example 4: Figure 13-16 The P-MSC-Exo reconstructed functional vascular network is shown.
[0071] Functional evaluation of the co-culture system of SVEC4-10 and pericytes under simulated high glucose damage conditions;
[0072] Vascular network stability: In the Matrigel tube formation experiment, the vascular network formed in the MSC-Exo group disintegrated after 18 hours, while the vascular network formed in the P-MSC-Exo group remained stable and mature. The total number of branch points and total length in the P-MSC-Exo group were significantly higher than those in all other groups. Figure 13 ;
[0073] Vascular barrier function: After P-MSC-Exo treatment, the broken VE-cadherin (adhesion junction) and ZO-1 (tight junction) proteins between endothelial cells were re-arranged continuously and linearly at the cell junctions, such as... Figure 14 .
[0074] Transendothelial resistance (TEER) values recovered after injury, and FITC-dextran permeability was also significantly reduced, such as Figure 15 ;
[0075] Vasomotor Function: In the collagen gel contraction assay, only the cell-collagen complex treated with P-MSC-Exo showed significant contraction after 24 hours, indicating the formation of vasomotor units with contractile function, such as... Figure 16 .
[0076] Example 5: Figure 17-21 The P-MSC-Exo shown accelerates the healing of diabetic wounds in vivo;
[0077] Animal Model and Treatment: An 8 mm full-thickness skin defect was created on the back of db / db diabetic mice. They were randomly divided into four groups and injected subcutaneously at multiple points around the wound margin with PBS, MSC-Exo (100 μg / 100 μL), P-MSC-Exo (100 μg / 100 μL), or SNP, respectively. Administration was repeated every 3 days.
[0078] Wound healing assessment: Wound size was recorded regularly by photographing. Results showed that the P-MSC-Exo group healed the fastest, significantly faster than the MSC-Exo group, the SNP-positive control group, and the PBS blank control group. Figure 17 ;
[0079] Angiogenesis and Maturity Assessment:
[0080] On day 14, wound tissue was taken for immunofluorescence staining to examine angiogenesis and function: In the P-MSC-Exo group, numerous CD34+ neovascularizations (red) were observed, and the expression level of eNOS (green) within the vessels was significantly upregulated. Figure 18 ;
[0081] Vascular integrity and maturity: Neovascularization (CD31+, red) in the P-MSC-Exo group showed continuous and intact ZO-1 tight junctions (green), such as... Figure 19 Furthermore, it is tightly wrapped by α-SMA-positive pericytes (green), exhibiting high vascular maturity, such as... Figure 20 Quantitative analysis showed that the average vessel diameter in the P-MSC-Exo group was significantly larger than that in other groups, such as... Figure 21 .
[0082] The following points should be noted in this article:
[0083] 1. The accompanying drawings of the embodiments disclosed herein only relate to the structures involved in the embodiments disclosed herein; other structures can be referred to in general design.
[0084] 2. Where there is no conflict, the embodiments of this disclosure and the features in the embodiments can be combined with each other to obtain new embodiments.
[0085] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A method for preparing pericyte-like mesenchymal stem cell exosomes, characterized in that: The method for preparing pericyte-like mesenchymal stem cell exosomes (P-MSC-Exo) includes the following steps: Step 1: Providing pericyte-like mesenchymal stem cells (P-MSCs): Mesenchymal stem cells (MSCs) are induced and cultured in a medium containing an effective concentration of nitric oxide (NO) donor to obtain pericyte-like mesenchymal stem cells (P-MSCs). Step 2: Collect conditioned medium: The P-MSCs were cultured in an exosome-free medium, and the cell culture supernatant containing exosomes was collected; Step 3: Separation and purification of exosomes: The cell culture supernatant is separated and purified to obtain the P-MSC-Exo.
2. The method for preparing pericyte-like mesenchymal stem cell exosomes according to claim 1, characterized in that: The nitric oxide (NO) donor in step 1 is sodium nitroprusside (SNP), and the final concentration of sodium nitroprusside in the culture medium is 10 µM to 1000 µM, preferably 200 µM.
3. The method for preparing pericyte-like mesenchymal stem cell exosomes according to claim 1 and its application in diabetic wounds, characterized in that: The separation and purification process in step 3 includes differential centrifugation and ultracentrifugation. The centrifugal force of the ultracentrifugation is 100,000g to 150,000g. Specifically, the cell supernatant is centrifuged at 300g for 30 minutes at 4°C to remove dead cells, and then centrifuged at 2000g for 20 minutes to remove cell debris and microvesicles. The supernatant is then transferred to a 15ml ultrafiltration tube and centrifuged at 3500g for 20 minutes at 4°C to obtain a concentrated supernatant. After filtering the concentrated supernatant through a 0.22μm filter membrane, it is ultracentrifuged at 120,000g for 90 minutes at 4°C. The collected precipitate is P-MSC-Exo.
4. A pericyte-like mesenchymal stem cell exosome prepared by the method described in any one of claims 1-3, characterized in that: The pericyte-like phenotype mesenchymal stem cell exosomes (P-MSC-Exo) have a surface rich in platelet-derived growth factor receptor β (PDGFR-β), and these exosomes possess one or more of the following characteristics: Morphologically, it exhibits a typical cup-shaped bilayer film structure with a diameter of approximately 120 nm under transmission electron microscopy. In terms of molecular markers, it still expresses exosome marker proteins CD63, CD81 and TSG101, and is rich in pericyte-associated marker platelet-derived growth factor receptor β (PDGFR-β). Functionally, it can be efficiently internalized by vascular endothelial cells and can repair mitochondrial damage in endothelial cells induced by high glucose or neutrophil extracellular traps (NETs), restoring mitochondrial membrane potential.
5. A pharmaceutical composition or extracellular vesicle formulation, characterized in that: Includes a therapeutically effective amount of the P-MSC-Exo of claim 4, and one or more pharmaceutically acceptable carriers, excipients, or delivery systems.
6. The use of the pericyte-like phenotype mesenchymal stem cell exosomes (P-MSC-Exo) of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for promoting angiogenesis, enhancing vascular barrier function, or promoting vascular maturation.
7. The use of the pericyte-like phenotype mesenchymal stem cell exosomes (P-MSC-Exo) of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for the prevention or treatment of ischemic diseases.
8. The application according to claim 7, characterized in that: The ischemic diseases mentioned are diabetic wounds, diabetic foot, lower extremity venous ulcers, or pressure sores.