Fibroblast membrane vesicles overexpressing fgf-1 and preparation method and application thereof

Abstract

The application discloses a kind of fibroblast membrane vesicles of overexpressing FGF-1 and its preparation method and application, process includes: the plasmid containing the gene of expressing FGF-1 is mixed with E. coli competent cell culture, the plasmid of FGF-1 coding obtained is extracted, FGF-1 coding plasmid is transfected fibroblast, obtain the fibroblast of overexpressing FGF-1, destroy the fibroblast of overexpressing FGF-1 and obtain containing cell membrane supernatant, with liposome extruder to make containing cell membrane supernatant sequentially through different pore size multiple reciprocating extrusion to complete the reconstruction of cell membrane, obtain the nanovesicle of overexpressing FGF-1.The advantages include: the fibroblast membrane vesicle of overexpressing FGF-1 of the application can improve body insulin resistance, improve glucose metabolism level, also can retain good cell proliferation activity, can realize double effect of wound repair and blood sugar regulation simultaneously.

Description

Technical Field

[0001] This invention relates to the field of wound healing drugs, and more particularly to cell membrane vesicle-related drugs that promote wound healing. Background Technology

[0002] Diabetes is a major public health problem. People with diabetes may suffer from chronic, slow-healing wounds. Diabetic wound healing generally involves three processes: inflammation, proliferation, and remodeling. Blood glucose levels, as a crucial parameter in the wound microenvironment, play a key role in regulating each stage of the healing process. Currently, the treatment of infected diabetic wounds primarily relies on adjunctive therapy, including insulin therapy to lower blood glucose and novel pro-healing biological dressings incorporating various growth factors.

[0003] However, insulin, a commonly used hypoglycemic drug in clinical practice, is not very effective in treating diabetic patients with insulin resistance. Thiazolidinediones are currently the only drugs that can treat insulin sensitivity, but their clinical application is limited by adverse side effects such as weight gain, hepatic steatosis, and bone loss. Finding alternative treatments that can improve insulin sensitivity therapy has become an urgent need. In vivo injection of FGF-1 has been shown to exert a sustained hypoglycemic effect without interference from insulin resistance and without producing side effects similar to those of thiazolidinediones. Furthermore, the FGF-1 protein may activate the PI3K signaling pathway in vivo through FGF1-FGFR1 to increase insulin-dependent glucose uptake and inhibit hepatic glucose production without interference from insulin resistance. This suggests that FGF-1 holds promise as a next-generation hypoglycemic drug and an alternative treatment option for insulin sensitivity therapy. However, due to the instability of FGF-1, most current studies on hypoglycemic treatment or related research use central or peripheral injection or increase endogenous secretion. There are still no studies on the dual regulatory effects of FGF-1 on wound repair and systemic blood glucose levels through transdermal administration.

[0004] Building upon systemic regulation of blood glucose levels, a key focus for future research is to accelerate the transition of wound healing from the inflammatory infection phase to the proliferative remodeling phase, achieving more complete coverage of the repair process in diabetic wounds. Fibroblasts, as repair cells encompassing multiple functions in normal wound repair, have emerged as a prominent example. However, in diabetic wounds, the biological functions of fibroblasts are impaired. Although recent studies have shown that allogeneic fibroblast therapy can restore the proliferation and migration of wound fibroblasts, promote angiogenesis, and enhance collagen expression, the short half-life and low yield of bioactive factors secreted by fibroblasts lead to high treatment costs, limiting their widespread clinical application.

[0005] Existing studies have confirmed the feasibility of a simple technique for preparing cell nanovesicles from allogeneic cell-derived membranes, demonstrating superior biocompatibility, drug loading capacity, and the ability to enrich and express bioactive factors. Fibroblast-derived cell membrane vesicles represent a more productive and promising alternative material for diabetic wound treatment. However, natural cell membrane vesicles do not offer advantages in glycemic regulation. Functional improvements to fibroblast-derived cell membrane vesicles, enabling them to address the multi-dimensional needs of glycemic regulation and wound repair in the treatment of diabetic infected wounds, would hold immense application potential. Summary of the Invention

[0006] The purpose of this invention is to provide a fibroblast membrane vesicle overexpressing FGF-1, its preparation method and application, in order to solve the problem of lack of effective drugs for treating refractory wounds.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing fibroblast membrane vesicles overexpressing FGF-1 includes the following steps: co-culturing a plasmid containing the gene expressing FGF-1 with competent Escherichia coli cells; extracting the plasmid encoding FGF-1 from the obtained bacterial solution; transfecting the plasmid encoding FGF-1 into fibroblasts to obtain fibroblasts overexpressing FGF-1; destroying the fibroblasts overexpressing FGF-1 and obtaining a cell membrane supernatant; and using a liposome extruder to repeatedly squeeze the cell membrane supernatant through different pore sizes to reconstruct the cell membrane, thereby obtaining fibroblast membrane vesicles overexpressing FGF-1.

[0009] Further, the specific process for extracting the plasmid encoding FGF-1 from the obtained bacterial culture is as follows: 1 μL of plasmid was added to a centrifuge tube containing 10 μL of thawed DH5α competent cells and mixed well. The mixture was placed on ice for 30 min, then heat-shocked in a 42 ℃ water bath for 45 s. The centrifuge tube was then quickly transferred to ice, and 500 μL of antibiotic-free sterile LB medium was added to the centrifuge tube. After mixing well, the mixture was placed in a 37 ℃ constant temperature shaker and cultured at 200 rpm for 1 h. Subsequently, 150 μL of the bacterial culture was spread onto LB plates and cultured at 37 ℃ for 14–16 h. Single colonies were then selected from the cloning plates and added to 15 mL of LB medium containing 15 μL of ampicillin. The plates were then cultured at 37°C with shaking for 14–16 h.

[0010] Furthermore, the specific process of transfecting fibroblasts with plasmids encoding FGF-1 is as follows: fibroblasts are passaged one day before transfection, so that on the day of transfection, the cells cover 70%-90% of the culture dish. The plasmid encoding FGF-1 is mixed with the transfection-promoting reagent at a ratio of 1:2-5. The mixture is added to the cell culture system and cultured in an incubator for 12-24 h, then the medium is changed, and the culture is continued for 48-96 h.

[0011] Furthermore, the specific process for preparing fibroblast membrane vesicles overexpressing FGF-1 is as follows: The cell suspension obtained after digestion and centrifugation of fibroblasts overexpressing FGF-1, which had undergone plasmid transfection, was added to hypotonic lysis buffer to obtain 1×10⁻⁶ cells / mL. 7 Cells / mL cell suspension was incubated overnight at 4 °C. Subsequently, the cell suspension was repeatedly ground 20 times using a tissue grinder and centrifuged at 3200g for 5 min at 4 °C to obtain cell membrane supernatant. Then, the cell membrane supernatant was extruded sequentially through polycarbonate membranes with pore sizes of 800 nm and 200 nm using a liposome extruder. Each pore size was extruded repeatedly to complete the reconstruction of the cell membrane, resulting in fibroblast membrane vesicles FGF1@EVs overexpressing FGF-1.

[0012] The present invention also provides fibroblast membrane vesicles overexpressing FGF-1 obtained by the preparation method described above.

[0013] The present invention also provides the application of the fibroblast membrane vesicles overexpressing FGF-1 in the preparation of drugs for treating wounds.

[0014] The present invention also provides the application of the fibroblast membrane vesicles overexpressing FGF-1 in the preparation of a drug for treating refractory wounds.

[0015] The present invention also provides the application of the fibroblast membrane vesicles overexpressing FGF-1 in the preparation of a drug for treating diabetic wounds.

[0016] The advantages of this invention are that fibroblast membrane vesicles overexpressing FGF-1 can improve insulin resistance and increase glucose metabolism, while retaining good cell proliferation activity, thus achieving the dual effects of wound repair and blood glucose regulation.

[0017] The FGF-1 overexpressing fibroblast membrane vesicles prepared by this invention have a simple preparation process, low production cost, and wide applicability. They have good clinical treatment value for diabetic wound patients with insulin resistance symptoms and effectively make up for many defects and deficiencies in existing related treatment technologies. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, do not constitute an undue limitation of the invention. In the drawings:

[0019] Figure 1 A map showing the relative protein expression levels of the L929 cell line overexpressing FGF-1.

[0020] Figure 2 The preparation and characterization of EVs and FGF1@EVs are presented, among which, Figure 2 A shows the transmission electron microscope (TEM) images of EVs and FGF1@EVs; Figure 2 B shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of FGF1@EVs and L929 cells overexpressing FGF-1; Figure 2 C represents the protein map of FGF1@EVs and L929 cells overexpressing FGF-1; Figure 2 D is the fluorescence imaging of EVs and FGF1@EVs; Figure 2 E represents the relative protein expression levels of FGF-1 in EVs and FGF1@EVs.

[0021] Figure 3 The NTA and Zeta potential spectra of FGF1@EVs are shown, where, Figure 3 A is the NTA spectrum of FGF1@EVs; Figure 3 B represents the Zeta potential spectrum of FGF1@EVs.

[0022] Figure 4 The in vitro glucose-lowering performance of FGF1@EVs was tested, among which, Figure 4 A is the Oil Red O staining pattern of a diabetic cell model; Figure 4 B represents the in vitro relative glucose metabolism levels of different treatment groups.

[0023] Figure 5 The figure shows the results of the molecular mechanism detection of the in vitro glucose-lowering performance of FGF1@EVs.

[0024] Figure 6 Statistical results for FGF1@EVs promoting cell proliferation.

[0025] Figure 7 The scratch test diagram is for FGF1@EVs.

[0026] Figure 8 This is a diagram of the tube forming test of FGF1@EVs.

[0027] Figure 9 The image shows the repair effect of FGF1@EVs on diabetic wounds in mice.

[0028] Figure 10 The in vivo glucose-lowering performance of FGF1@EVs was tested, among which, Figure 10 A represents the relative regulation of overall blood glucose levels in different treatment groups within the body; Figure 10 B shows the relative blood glucose metabolism levels in mice 4 hours before and after treatment on day 5 in different treatment groups.

[0029] Figure 11 Tissue section image showing the biotoxicity of FGF1@EVs. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0031] Example 1: Preparation of fibroblast membrane vesicles overexpressing FGF-1

[0032] (1) Transformation and extraction of plasmid encoding FGF-1 gene: First, the pRP[Exp]-EGFP / Puro-CAG>mFgf1 plasmid vector was ordered from Yunzhou Biotechnology (Guangzhou) Co., Ltd. 1 μL of plasmid was added to a centrifuge tube containing 10 μL of thawed DH5α competent cells and mixed well. The tube was then placed on ice for 30 min. After heat shock in a 42 ℃ water bath for 45 s, the centrifuge tube was quickly transferred to ice. 500 μL of sterile LB medium (antibiotic-free) was added to the centrifuge tube, mixed well, and incubated at 37 ℃ in a shaker at 200 rpm for 1 h. Subsequently, 150 μL of bacterial culture was spread onto LB plates and incubated at 37 ℃ for 14–16 h. Single colonies were then selected from the cloning plate and added to a 50 ml centrifuge tube containing 15 mL of LB medium (15 μL of ampicillin). The tube was then incubated at 37°C with shaking for 14–16 h. The plasmid encoding FGF-1 was extracted from the obtained bacterial culture. The concentration and purity of the extracted plasmid encoding FGF-1 were measured, requiring OD260 / OD280 > 1.8 and OD260 / OD230 > 2. After testing, the qualified plasmid encoding FGF-1 was stored at -20 ℃.

[0033] (2) Preparation of L929 cell line overexpressing FGF-1: L929 cells were transfected with the plasmid encoding FGF-1 obtained in step (1). L929 cells were passaged one day before transfection, ensuring that the cells covered 70%-90% of the culture dish on the day of transfection. The plasmid encoding FGF-1 was mixed with the transfection-promoting reagent according to the instructions. Experimental groups were formed at ratios of 1:2, 1:3, 1:4, and 1:5, respectively. The mixture of each experimental group was added to the corresponding cell culture system and cultured in an incubator for 12-24 h, followed by medium replacement. After culturing the cells for 48-96 h, the cells were lysed, and protein samples were extracted for Western blot analysis to determine the expression effect of FGF-1 protein, thus obtaining the L929 cell line overexpressing FGF-1. The experimental results of Western blot analysis of FGF-1 protein expression in each experimental group are shown below. Figure 1 As shown.

[0034] (3) Preparation of fibroblast membrane vesicles overexpressing FGF-1: The cell suspension obtained by digesting and centrifuging L929 cells overexpressing FGF-1 after plasmid transfection was added to hypotonic lysis buffer (1×10⁻⁶). 7 Cells were collected at a density of 1000 μg / mL to obtain a cell suspension, which was then incubated overnight at 4 °C. The cell suspension was then repeatedly ground 20 times using a tissue grinder and centrifuged at 3200 g for 5 min at 4 °C to obtain a cell membrane supernatant. The cell membrane supernatant was then extruded sequentially through polycarbonate membranes with pore sizes of 800 nm and 200 nm using a liposome extruder. Each pore size was repeatedly extruded to reconstruct the cell membrane, resulting in a solution of FGF-1-overexpressing nanovesicles (FGF1@EVs).

[0035] Comparative Example 1

[0036] The difference between this comparative example and Example 1 is that the plasmid vector used in step (1) was pRP[Exp]-EGFP / Puro-CAG>ORF_Stuffer purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd., and the control plasmid was obtained in the same way, finally obtaining the control fibroblast cell line. In addition, the control nanovesicle (EV) solution was obtained from untransfected fibroblasts using the same extrusion method.

[0037] Test Example 1

[0038] The FGF1@EVs solution and the EVs solution were characterized by transmission electron microscopy. Figure 2 A represents the characterization of the FGF1@EVs solution and the EVs solution, such as... Figure 2 As shown in Figure A, a typical cup-shaped structure with a size of 100–200 nm can be observed, demonstrating the successful preparation of nanovesicles.

[0039] The proteomic profiles of FGF1@EVs and L929 cells overexpressing FGF-1 from Example 1 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Figure 2 B shows the proteomic profiles of FGF1@EVs in step (3) of Example 1 and L929 cells overexpressing FGF-1 in step (2) of Example 1, such as... Figure 2 As shown in B, the proteomic results show that the proteomic spectrum of FGF1@EVs is similar to that of L929 cells, proving that they have the same origin.

[0040] Western blotting was used to detect the surface marker protein profiles of FGF1@EVs and L929 cells overexpressing FGF-1 from Example 1. Figure 2 C represents the expression of surface marker proteins in FGF1@EVs and L929 cells overexpressing FGF-1 in Example 1, such as... Figure 2 As shown in Figure C, the collected FGF1@EVs are rich in exosome markers CD63, TSG101, CD81 and CD9, but do not express calnexin protein, demonstrating that FGF1@EVs have similar characteristics to L929 cell-derived exosomes, indicating the successful preparation of nanovesicles.

[0041] The relative fluorescence intensity of the FGF1@EVs solution and the EVs solution in Comparative Example 1 was detected using a fluorescence imaging system. Figure 2 D represents the relative fluorescence intensity of the FGF1@EVs solution in Example 1 and the EVs solution in Comparative Example 1, as shown below. Figure 2 As shown in Figure D, the relative fluorescence expression of GFP in FGF1@EVs is more significant, proving the successful preparation of FGF1@EVs.

[0042] The expression level of FGF-1 protein in the FGF1@EVs solution and the EVs solution in Comparative Example 1 was detected by Western blotting. Figure 2 E represents the FGF-1 protein expression levels in the FGF1@EVs solution of Example 1 and the EVs solution of Comparative Example 1, such as... Figure 2 As shown in Figure E, a significant increase in the expression level of FGF-1 protein in FGF1@EVs can be observed, demonstrating the successful preparation of FGF1@EVs.

[0043] Test Example 2

[0044] The FGF1@EVs solution was characterized using the NTA assay, such as... Figure 3As shown in Figure A, the particle size of FGF1@EVs is 187.1 ± 2.5 nm, confirming the uniformity and batch stability of the MT NPs preparation.

[0045] The FGF1@EVs solution was characterized using a zeta potential assay, such as... Figure 3 As shown in Figure B, the Zeta potential of FGF1@EVs is approximately -8 to -10 mV.

[0046] Test Example 3

[0047] The in vitro hypoglycemic performance of FGF1@EVs was detected using the glucose oxidase method, and glucose levels were measured according to the glucose assay kit instructions. Figure 4 A shows the Oil Red O staining results of the 3T3-L1 diabetic cell model, as follows: Figure 4 As shown in Figure A, a large number of lipid droplets clearly stained orange-red were visible in the model group, confirming the construction of the diabetic cell model. The 3T3-L1 diabetic cell model was incubated with EVs, FGF1@EVs, insulin, EVs+insulin, and FGF1@EVs+insulin, respectively. The relative glucose metabolism levels of different treatment groups in vitro were measured as follows: Figure 4 As shown in Figure B, compared to the negative control group, the glucose metabolism levels in the FGF1@EVs group, the insulin-positive control group, and the combination therapy group were significantly increased 4 hours after treatment. Among them, the combined FGF1@EVs and insulin treatment group showed the most significant effect. These results indicate that FGF1@EVs can alleviate insulin resistance and significantly improve the glucose metabolism capacity of 3T3-L1 diabetic cells.

[0048] Test Example 4: Detection of the molecular mechanism of in vitro glucose-lowering performance of fibroblast membrane vesicles overexpressing FGF-1

[0049] Referring to Test Example 3, the 3T3-L1 diabetic cell model was incubated with EVs, FGF1@EVs, insulin, EVs+insulin, and FGF1@EVs+insulin, respectively. Cell lysis samples were collected from each treatment group after 24 h of treatment, and protein samples were extracted. Western blotting was used to detect the PI3K protein expression levels in each treatment group. The results of PI3K protein expression levels in different treatment groups are shown below. Figure 5 As shown, compared to other groups, the phosphorylation level of p-PI3K protein was significantly increased in the FGF1@EVs group and the FGF1@EVs combined with insulin treatment group, with the FGF1@EVs combined with insulin treatment group showing the most significant improvement. These results indicate that FGF1@EVs enhances insulin sensitivity and promotes glucose metabolism by activating PI3K phosphorylation.

[0050] Test Example 5: Cell proliferation, scratch assay, and tube formation assay of fibroblast membrane vesicles overexpressing FGF-1.

[0051] The FGF1@EVs cell proliferation promotion assay was performed as follows: HSF cells were incubated at 3×10⁻⁶ cells / year. 3 Cells were seeded at a density in 96-well plates and cultured for 24 h. They were then treated with FGF1@EVs at different concentrations. CCK-8 reagent was added after 24 h, and the absorbance was measured at 450 nm after incubation at 37 °C for 1 h.

[0052] The migration performance of FGF1@EVs was tested as follows: The wound healing effect of FGF1@EVs in Example 1 was evaluated in vitro using a scratch assay. HSF cells were cultured at 1.0 × 10⁶ cells / mL. 5 Cells were seeded at a density in 6-well plates and cultured to 100% confluence. Parallel scratches were made in each well using the tip of a 200 μL pipette, followed by washing once with PBS and then treatment with FGF1@EVs or PBS. Images were taken using an optical microscope at 0 h, 12 h, and 24 h.

[0053] The FGF1@EVs were subjected to a tube formation assay, specifically as follows: The wound healing effect of FGF1@EVs in Example 1 was evaluated in vitro using a tube formation assay. HUVECs cells were cultured at 5.0 × 10⁶ cells / year. 4 Cells were seeded at a density in 48-well plates pre-coated with matrix gel, then treated with FGF1@EVs or PBS as described in Example 1, and co-cultured at 37 °C for 3 h. After removing the supernatant, 200 μL of Calcein-AM working solution was added for staining. Finally, the formation of vascular-like structures was observed and photographed using an inverted fluorescence microscope.

[0054] Figure 6 Statistical results showing that FGF1@EVs promote cell proliferation. Figure 6 It can be seen that the cell number increases significantly in a dose-dependent manner, and FGF1@EVs in Example 1 have a significant cell proliferation promoting function.

[0055] Figure 7 Scratch test for FGF1@EVs. Figure 7 It can be seen that, compared with the control group, FGF1@EVs in Example 1 significantly promoted cell migration.

[0056] Figure 8 Tube formation test for FGF1@EVs. (By...) Figure 8 It can be seen that, compared with the control group, FGF1@EVs in Example 1 significantly promoted the tube formation ability of cells.

[0057] Test Example 6: Diabetic Wound Treatment Trial Using Fibroblast Membrane Vesicles Overexpressing FGF-1

[0058] The specific steps for constructing a diabetic mouse model of infectious wounds are as follows: Four-week-old male SPF-grade C57BL / 6J mice, purchased from Guangdong Zhiyuan Biomedical Technology Co., Ltd., were selected. A diabetic mouse model was constructed using intraperitoneal injection of STZ. All mice were housed in the same environment under constant temperature and humidity conditions of 25 ℃±2, following a 12-hour light / dark cycle. After one week of adaptation to a standard diet, the mice were switched to a high-fat diet, with fresh food, water, and bedding replaced daily to maintain optimal growth and minimize mortality. After 4-6 weeks of high-fat feeding, STZ solution was injected intraperitoneally for 3 consecutive days to induce diabetes. Mice were fasted for 12 hours before injection, and water was prohibited during this period. Each mouse was injected with 100 mg / kg of 1% STZ solution. After injection, all mice were placed in cages and fasted for another 4 hours before being fed the high-fat diet. Sufficient water was provided to prevent mouse mortality. For one week after STZ injection, tail vein blood was collected daily using a blood glucose meter to monitor and record blood glucose levels, and body weight was also monitored. Diabetes modeling can only be considered successful when serum glucose levels are stable at or above 16.7 mmol / L; otherwise, STZ solution needs to be injected again for induction.

[0059] On day 0, after anesthetizing diabetic mice, the backs of the mice were shaved using a razor and depilatory cream. A full-thickness skin incision of approximately 6 mm in diameter was made on the dorsal side of the mice, and Staphylococcus aureus (10 μL, 1×10⁻⁶) was inoculated onto the wound surface. 6 A diabetic infected wound model was established using CFU / mL. The infected wounds were divided into two groups: a control group treated with PBS only, and an experimental group treated with a solution containing FGF1@EVs prepared in Example 1. Subsequently, the appropriate PBS and FGF1@EVs solutions were instilled every other day, and wound healing was recorded on days 0, 1, 3, 5, 7, 9, and 12. Results are as follows: Figure 9 As shown, the wound healing in the FGF1@EVs solution treatment group was better than that in the control group.

[0060] Test Example 7: Detection of the in vivo glucose-lowering performance of fibroblast membrane vesicles overexpressing FGF-1

[0061] Diabetic infected wounds were divided into two groups: the control group was treated with PBS only, and the experimental group was instilled with a solution containing FGF1@EVs prepared in Example 1. Subsequently, the corresponding PBS and FGF1@EVs solutions were instilled every other day, and the wound recovery status was recorded on days 0, 1, 3, 5, 7, 9, and 12.

[0062] Record random blood glucose levels in mice on day 0 and day 12 of treatment. The overall change in blood glucose levels after treatment is compared to... Figure 10 As shown in Figure A, random blood glucose levels in mice were recorded 4 hours before and after treatment on day 5. Changes in blood glucose metabolism levels were compared to... Figure 10 As shown in B.

[0063] Test Example 8: Biocompatibility assay of fibroblast membrane vesicles overexpressing FGF-1

[0064] Following the grouping and treatment guidelines of Test Example 7, the hearts, livers, spleens, lungs, and kidneys of mice treated with PBS and FGF1@EVs were then stained with hematoxylin and eosin (HE). Figure 11 As shown, no obvious pathological changes or inflammatory infiltration were observed in the visceral organs after treatment with PBS and FGF1@EVs, indicating that the prepared FGF1@EVs have good biocompatibility.

[0065] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing fibroblast membrane vesicles overexpressing FGF-1, characterized in that, The process includes: co-culturing a plasmid containing the gene expressing FGF-1 with competent E. coli cells; extracting the plasmid encoding FGF-1 from the obtained bacterial solution; transfecting the plasmid encoding FGF-1 into fibroblasts to obtain fibroblasts overexpressing FGF-1; destroying the fibroblasts overexpressing FGF-1 and obtaining a supernatant containing the cell membrane; using a liposome extruder to repeatedly squeeze the supernatant containing the cell membrane through different pore sizes to complete the reconstruction of the cell membrane, thereby obtaining fibroblast membrane vesicles overexpressing FGF-1.

2. The method for preparing fibroblast membrane vesicles overexpressing FGF-1 according to claim 1, characterized in that, The specific process for extracting the plasmid encoding FGF-1 from the obtained bacterial culture was as follows: 1 μL of plasmid was added to a centrifuge tube containing 10 μL of thawed DH5α competent cells and mixed well. The mixture was placed on ice for 30 min, then heat-shocked in a 42 ℃ water bath for 45 s. The centrifuge tube was then quickly transferred to ice, and 500 μL of antibiotic-free sterile LB medium was added to the centrifuge tube. After mixing well, the mixture was placed in a 37 ℃ constant temperature shaker and cultured at 200 rpm for 1 h. Subsequently, 150 μL of the bacterial culture was spread onto LB plates and cultured at 37 ℃ for 14–16 h. Single colonies were then selected from the cloning plates and added to 15 mL of LB medium containing 15 μL of ampicillin. The plates were then cultured at 37°C with shaking for 14–16 h.

3. The method for preparing fibroblast membrane vesicles overexpressing FGF-1 according to claim 1, characterized in that, The specific process of transfecting fibroblasts with plasmids encoding FGF-1 is as follows: fibroblasts are passaged one day before transfection, so that on the day of transfection, the cells cover 70%-90% of the culture dish. The plasmid encoding FGF-1 is mixed with the transfection-promoting reagent at a ratio of 1:2-5. The mixture is added to the cell culture system and cultured in an incubator for 12-24 h, then the medium is changed, and the culture is continued for 48-96 h.

4. The method for preparing fibroblast membrane vesicles overexpressing FGF-1 according to claim 1, characterized in that, The specific process for preparing FGF-1 overexpressing fibroblast membrane vesicles is as follows: FGF-1 overexpressing fibroblasts, obtained through plasmid transfection, are digested and centrifuged, and the resulting cell suspension is added to hypotonic lysis buffer to obtain 1×10⁻⁶ cells / mL. 7 Cells / mL cell suspension was incubated overnight at 4 °C. Subsequently, the cell suspension was repeatedly ground 20 times using a tissue grinder and centrifuged at 3200g for 5 min at 4 °C to obtain cell membrane supernatant. Then, the cell membrane supernatant was extruded sequentially through polycarbonate membranes with pore sizes of 800 nm and 200 nm using a liposome extruder. Each pore size was extruded repeatedly to complete the reconstruction of the cell membrane, resulting in fibroblast membrane vesicles FGF1@EVs overexpressing FGF-1.

5. A fibroblast membrane vesicle overexpressing FGF-1 obtained by any of the preparation methods described in claims 1-4.

6. The use of fibroblast membrane vesicles overexpressing FGF-1 as described in claim 5 in the preparation of drugs for treating wounds.

7. The use of fibroblast membrane vesicles overexpressing FGF-1 as described in claim 6 in the preparation of drugs for treating refractory wounds.

8. The use of fibroblast membrane vesicles overexpressing FGF-1 as described in claim 7 in the preparation of a drug for treating diabetic wounds.

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