Preparation method and application of GS-loaded mesenchymal stem cell exosome

Abstract

The invention discloses a preparation method and application of a GS-loaded mesenchymal stem cell exosome, and belongs to the technical field of biology. In order to solve the problems of slow onset and long recovery period of a diabetic skin tissue defect healing method in the prior art, the invention provides a preparation method of a GS-loaded mesenchymal stem cell exosome, the yield of the MSCs exosome is induced to be greatly increased through high-frequency ultrasonic stimulation and a microfluidic circulation technology, and the GS-loaded mesenchymal stem cell exosome is prepared. An original cell membrane structure is broken by using an acoustic fluid nano directional coupling technology, phospholipid bimolecules are driven to aggregate and self-assemble to form exosomes which are wrapped in GS, and effective drug delivery and synergistic tissue repair are realized; according to the method, nucleic acid, protein, saccharide, polypeptide and various small molecule compounds can be loaded in the exosome, and certain universality is achieved. The GS-loaded mesenchymal stem cell exosome provided by the invention can be applied to preparation of drugs for promoting healing of skin tissue defects and promoting regeneration of diabetic skin tissues.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of GS-loaded mesenchymal stem cell exosomes. Background Art

[0002] Diabetes has become an increasingly prevalent global health issue. It is a serious and widespread chronic metabolic disease, with a rapidly increasing incidence in recent years and a steady increase in the number of people with diabetes. The skin, the largest organ in the human body, serves as a physical barrier that protects the body and defends against external pathogens. However, due to its fragile nature, it is particularly susceptible to various forms of damage. Depending on the severity of skin damage, the time required to stop bleeding and fight infection varies. The greater the severity of the damage, the longer the hemostatic and anti-infection phases required to repair the wound, thus delaying tissue regeneration and making wound healing difficult.

[0003] Diabetes-induced wounds (skin tissue defects) and the increased risk of complications can make wound healing more difficult, resulting in prolonged healing times and increased susceptibility to further complications. Diabetic ulcers, one of the most serious complications of diabetes, are slow or difficult to heal, with high rates of recurrence and amputation, placing a heavy burden on diabetic patients and their families. Current traditional treatments for diabetic ulcers suffer from slow onset and a long recovery period, causing considerable pain and suffering for patients.

[0004] Ginseng is a traditional medicinal herb characterized by its sweet and slightly bitter flavor, warm and flat aroma, and a history of over 2,000 years. It possesses a range of biological and pharmacological effects, including anti-tumor, anti-inflammatory, antibacterial, immune-enhancing, and blood sugar-regulating properties. Ginseng saponins (GS) are the most important active compounds in ginseng, and studies have demonstrated their excellent antibacterial properties. However, their water solubility and bioavailability are poor, and their antibacterial properties are unable to effectively penetrate the skin barrier. These active compounds remain confined to the skin's surface, limiting their potential for skin repair and therapeutic efficacy.

[0005] MSC has a significant therapeutic effect on diseases such as inflammation. Research on MSC in recent years has confirmed that its exosomes can achieve targeted delivery of functional neurotransmitters and have higher biosafety. Therefore, the use of exosomes as carriers in treatment has received widespread attention in recent years and has high clinical application value.

[0006] Those skilled in the art are eager to develop a technical method that can shorten the course of the disease, promote the healing of skin tissue defects, and thus promote the healing of diabetic wounds. Summary of the Invention

[0007] The present invention aims to solve the technical problems of slow onset and long recovery period in the existing treatment methods for healing diabetic skin tissue defects, and provides a preparation method and application of GS-loaded mesenchymal stem cell exosomes.

[0008] One of the objectives of the present invention is to provide a method for preparing GS-loaded mesenchymal stem cell exosomes, the preparation method comprising the following steps: S1: Mesenchymal stem cells were digested using acoustofluidic nano-directional coupling technology, and the mesenchymal stem cells were transferred to serum-free OPTI-MEM medium for culture. The culture medium was allowed to stand, and the supernatant was centrifuged three times. The supernatant was filtered through a 0.2 μm syringe, concentrated using a 100 kDa MWCO ultrafiltration, and centrifuged at 4000 × g for 30 min to obtain mesenchymal stem cell exosomes. S2: GS was mixed with the mesenchymal stem cell exosomes obtained in S1 through acoustofluidic nano-directional coupling technology to obtain GS-loaded mesenchymal stem cell exosomes.

[0009] In a preferred embodiment of the present invention, the processing steps of the acoustofluidic nano-directional coupling technology described in S1 and S2 are as follows: centrifugation at 1000×g for 10 min, 5 The cells were resuspended in serum-free OPTI-MEM medium at a density of 10 cells / mL and treated with an ultrasonic spray generator.

[0010] In a preferred embodiment of the present invention, the flow rate of the ultrasonic spray generator is 1.4 L / h.

[0011] In a preferred embodiment of the present invention, the frequency processed by the ultrasonic spray generator is 15 kHz.

[0012] In a preferred embodiment of the present invention, the standing time in S1 is 48 h.

[0013] In a preferred embodiment of the present invention, the conditions of the three centrifugal treatments in S1 are: the first centrifugal treatment condition is centrifugation at 500 rpm for 30 min, the second centrifugal treatment condition is centrifugation at 2000 rpm for 30 min, and the third centrifugal treatment condition is centrifugation at 10000 rpm for 60 min.

[0014] In a preferred embodiment of the present invention, the mixing volume ratio of GS to mesenchymal stem cell exosomes in S2 is 1:60.

[0015] A second object of the present invention is to provide GS-loaded mesenchymal stem cell exosomes, wherein the GS-loaded mesenchymal stem cell exosomes are obtained by the above-mentioned preparation method.

[0016] A third object of the present invention is to provide the use of the above-mentioned GS-loaded mesenchymal stem cell exosomes in the preparation of a drug for promoting the healing of skin tissue defects.

[0017] In a preferred embodiment of the present invention, the skin tissue defect is a skin tissue defect caused by diabetes and its complications.

[0018] The present invention has the following beneficial effects: MSC-exo, a double-membrane extracellular vesicle (EV) produced by stem cells through paracrine signaling, has a smaller particle size and improved skin permeability while retaining many of the beneficial properties of stem cells, including broad differentiation potential, which can be used to promote tissue regeneration. Furthermore, considering the biosafety risks of stem cell proliferation in vivo, exosomes have a higher safety profile compared to stem cells. Therefore, leveraging the tissue repair properties of MSC-exo EVs and their ability to safely deliver drugs to wound sites, the present invention provides a method for preparing GS-loaded MSC exosomes. GS is embedded in MSC-exo (abbreviated as GS / exo), ultrasonic spraying is used to stimulate MSCs to produce a large number of exosomes, and then the exosomes are encapsulated in GS using acoustofluidic nano-directional coupling technology. This method achieves effective drug delivery and synergistic tissue repair, resulting in the efficient production of GS-loaded EVs.

[0019] The present invention conducted effect evaluation in Hacat and diabetic mouse models through relevant experiments, demonstrating that the GS-loaded mesenchymal stem cell exosomes provided by the present invention utilize the natural extracellular vesicles MSC-exo secreted by MSCs, combined with GS with antibacterial properties, so that they work synergistically, have the effect of promoting the elimination of bacterial infection and inflammation, promoting the regeneration of diabetic skin tissue, and establishing an optimal microenvironment for the healing of diabetic skin defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In the following figures, GS represents ginsenoside, MSC-exo represents extracellular vesicles, and GS / exo represents exosomes loaded with ginsenoside.

[0021] Figure 1 This is a flow chart for preparing mesenchymal stem cell exosomes MSC-exo in Example 1; Figure 2 This is a statistical diagram of the number of MSC-exo in Example 1; Figure 3 Figure 1 is a graph showing the effect of cell concentration and flow rate on MSC-exo production in Example 1; A is a graph showing the effect of cell concentration on the number of MSC-exo, B is a graph showing the effect of flow rate on the number of MSC-exo, C is a graph showing the effect of cell concentration on cell viability, and D is a graph showing the effect of flow rate on cell viability; Figure 4This is the MSC-exo particle size detection diagram in Example 1; the vertical axis Particles is the particle concentration, and the horizontal axis Diameter is the particle size; Figure 5 This is the MSC-exo characterization test diagram in Example 1;. Figure 6 The figure is the result of Western blot analysis in Example 1; GAPDH is glyceraldehyde-3-phosphate dehydrogenase; CD63 is a tetraspanin; CD9 is a key marker protein of exosomes; CD81 is a transmembrane protein on the surface of exosomes; Calnexin is a calcium-binding protein; Figure 7 This is the characterization detection diagram of GS / exo in Example 2; Figure 8 High performance liquid chromatography-mass spectrometry detection diagram; A is the HPLC-MS chromatogram of the blank sample, B is the LC-MS spectrum of the standard, and C is the LC-MS spectrum of the test sample; Figure 9 Figure 1 is the encapsulation efficiency analysis result; Figure 10 Figure 2 shows the relative viability of HaCaT cells after different treatments; A is the HG treatment group, B is the GS treatment group, C is the MSC-exo treatment group, and D is the GS / exo treatment group. Figure 11 Figure 2 is the Hoechst staining result of different treatment groups; PKH26 is a red cell membrane fluorescent dye; Hoechst is a blue cell nucleus fluorescent dye; Merge is an overlay image; Figure 12 The results of cell scratch test in different treatment groups are shown in Figure 2. Figure 13 Figure 2 shows the staining results of transwell chambers in different treatment groups; Figure 14 Statistical graph of the number of cells migrating into the upper chamber of the transwell chamber in different treatment groups; Figure 15 The results of flow cytometry detection of reactive oxygen species (ROS) levels in different treatment groups; A is the result of reactive oxygen species (ROS) detection, and B is the quantitative diagram of reactive oxygen species (ROS) detection; Figure 16 Figures 2 and 3 show the results of HaCaT cell apoptosis detection in different treatment groups; A shows the results of HaCaT cell apoptosis detection, and B shows the quantitative diagram of HaCaT cell apoptosis detection; HaCaT cells are human immortalized keratinocytes; Figure 17 Figure 2 shows the results of mitochondrial membrane potential changes in different treatment groups; A is the mitochondrial membrane potential diagram, B is the relative fluorescence intensity diagram; HaCaT is human immortalized keratinocytes; Figure 18Figures 2 and 3 show the results of inflammatory factor expression level detection in different treatment groups; A is the detection graph of TGF-β factor level; B is the detection graph of IL-10 factor level; C is the detection graph of IL-β factor level; D is the detection graph of TGF-α factor level; E is the detection graph of IL-6 factor level; Figure 19 Observation diagram of wound changes on skin defect models in different treatment groups; Figure 20 The results of H&E staining and Masson staining of different treatment groups after 7 days of treatment; A is the result of H&E staining, and B is the result of Masson staining; Figure 21 These are the results of H&E staining and Masson staining of different treatment groups after 14 days of treatment; A is the result of H&E staining, and B is the result of Masson staining. DETAILED DESCRIPTION

[0022] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0024] The experimental group treated with the acoustofluidic nano-directional coupling technology described in the following examples is referred to as the ANDC group.

[0025] The ICR female experimental mice (SPF grade) described in the following examples were purchased from Liaoning Changsheng Experimental Animal Technology Co., Ltd.

[0026] Example 1: Preparation of mesenchymal stem cell exosomes In this embodiment, mesenchymal stem cells were digested by using the acoustofluidic nano-directional coupling technology. The processing steps of the acoustofluidic nano-directional coupling technology method were as follows: centrifugation at 1000×g for 10 min, 5The cells were resuspended in serum-free OPTI-MEM medium at a density of 10 cells / mL and cultured, and then treated with an ultrasonic spray generator at a flow rate of 1.4 L / h and a frequency of 15 kHz. The mesenchymal stem cells were transferred to serum-free OPTI-MEM medium and cultured for 48 hours. The supernatant was centrifuged three times, and the conditions for the three centrifugation treatments were as follows: the first centrifugation condition was 500 rpm for 30 minutes, the second centrifugation condition was 2000 rpm for 30 minutes, and the third centrifugation condition was 10000 rpm for 60 minutes. The exosomes were filtered through a 0.2 μm syringe, concentrated by 100 kDa MWCO ultrafiltration, and centrifuged at 4000×g for 30 minutes to obtain mesenchymal stem cell exosomes MSC-exo.

[0027] The flow chart of obtaining mesenchymal stem cell exosomes (MSC-exo) in this example is as follows Figure 1 As shown, in this example, mesenchymal stem cells (MSCs) were resuspended and quickly passed through an ultrasonic spray generator. During this process, MSCs were dispersed into droplets under ultrasonic stimulation. After 48 hours of culture, the supernatant was collected and subjected to gradient centrifugation, filtration, and ultrafiltration to obtain MSC-exo.

[0028] Effect experiment: (1) In this example, the number of exosomes produced by the control group, the starvation group, and the electroporation group without the acoustofluidic nano-directional coupling technology and the experimental group treated with the acoustofluidic nano-directional coupling technology (ANDC) in this example were counted. The results are as follows: Figure 2 As shown in the figure, the starvation group employed the following specific procedures: mesenchymal stem cells were transferred to serum-free OPTI-MEM medium and cultured for 48 hours. The supernatant was then centrifuged three times to obtain mesenchymal stem cell exosomes (MSC-exo). The electroporation group employed the following specific procedures: mesenchymal stem cell exosomes were placed in an electroporation cuvette and electroporated using the GenePulser Xcell™ system (Bio-Rad). The parameters were set as follows: 160 V, 3 pulses, each lasting 10 ms. As can be seen, the experimental group treated with the acoustofluidic nano-directional coupling technology (ANDC) provided by the present invention increased exosome production by approximately 30-fold compared to the control, starvation, and electroporation groups, demonstrating a significant improvement in exosome production.

[0029] In order to achieve the maximum exosome yield with the smallest cell image, this embodiment optimizes the flow rate of the ultrasonic spray generator in the acoustofluidic nano-directional coupling technology.

[0030] like Figure 3As shown in Figures AD, when the flow rate through the ultrasonic spray generator is 1.4 L / h and the cell concentration is 3×10 5 The preparation method provided in this embodiment maintains a high cell viability while maximizing the production of mesenchymal stem cell exosomes MSC-exo; moreover, there is no regular change between the flow rate through the ultrasonic spray generator and the exosome yield, and there is no obvious positive and negative relationship between the cell concentration through the ultrasonic spray generator and the cell viability.

[0031] (2) Characterization and detection of MSC-exo In this example, nanoparticle tracking analysis (NTA) was used to quantitatively analyze the particle size and concentration of MSC-exo. The morphology of the mesenchymal stem cell exosomes MSC-exo obtained above was observed using transmission electron microscopy (TEM) at 100 kV. Western blot analysis was then used to compare the expression levels of exosome markers CD9, CD63, and CD81 with the intracellular protein (calnexin) to further confirm the prepared MSC-exo.

[0032] After NTA characterization, it was found that the particle size of MSC-exo was in the range of 100-150 nm (e.g. Figure 4 MSC-exo was characterized by transmission electron microscopy, and it was found that MSC-exo was a typical exosome structure with a distinct cup-shaped double-layer membrane structure and clear rounded edges, indicating that the preparation method of mesenchymal stem cell exosomes provided by the present invention can generate well-structured MSC-exo with a good structure (such as Figure 5 As shown in Figure 2 ). Western blot analysis results showed that the extracts were positive and enriched for classic exosome markers CD9, CD63, and CD81, but negative for the intracellular protein calnexin, which is a protein not usually found in exosomes. In summary, the method for preparing mesenchymal stem cell exosomes provided by the present invention successfully prepared MSC-exo, (as shown in Figure 2 ). Figure 6 shown).

[0033] Example 2: Preparation of GS-loaded mesenchymal stem cell exosomes In this example, GS was mixed with the mesenchymal stem cell exosomes obtained in Example 1 by using the acoustofluidic nano-directional coupling technology (ANDC treatment). The treatment steps of the acoustofluidic nano-directional coupling technology were as follows: centrifugation at 1000×g for 10 min, and 3×10 5The mesenchymal stem cells were resuspended in serum-free OPTI-MEM medium at a density of 10 cells / mL and cultured. The mesenchymal stem cells and GS were treated by an ultrasonic spray generator; the flow rate of the ultrasonic spray generator treatment was 1.4 L / h, and the frequency of the ultrasonic spray generator treatment was 15 kHz, to obtain GS-loaded mesenchymal stem cell exosomes GS / exo.

[0034] In this example, the GS / exo exosomes of mesenchymal stem cells loaded with GS obtained above were characterized by transmission electron microscopy. Figure 7 As shown, the membrane structure of GS / exo remained intact.

[0035] Comparative Example 1: In this example, the mesenchymal stem cell exosomes obtained in Example 1 were mixed with GS using an interval sonication method. The sonication was performed for 5 min, followed by a 5-min recovery period. The sonication step was repeated 5 times. The exosomes were concentrated using 100 kDa MWCO ultrafiltration and centrifuged at 4000 × g for 30 min to obtain GS-loaded mesenchymal stem cell exosomes.

[0036] Comparative Example 2: In this example, the mesenchymal stem cell exosomes obtained in Example 1 were mixed with GS by a co-incubation method. The mixture was stirred continuously, and the exosomes were concentrated by 100 kDa MWCO ultrafiltration. The exosomes were centrifuged at 4000 × g for 30 min to obtain GS-loaded mesenchymal stem cell exosomes.

[0037] Effect experiment: (1) High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to quantitatively analyze the GS-loaded mesenchymal stem cell exosomes prepared in Example 2 and Comparative Examples 1-2.

[0038] The results are as follows Figure 8 As shown, the HPLC-MS spectrum of the test sample showed consistent peak times with that of the standard.

[0039] (2) In Example 2 and Comparative Examples 1-2 of the present invention, five different mixing ratios of GS and mesenchymal stem cell exosomes (1:20, 1:40, 1:60, 1:80, and 1:100) were set, and the encapsulation efficiency was analyzed.

[0040] The results are as follows Figure 9As shown, the encapsulation efficiency of GS-loaded mesenchymal stem cell exosomes (GS / exo) prepared using acoustofluidic nano-directional coupling technology (ANDC treatment) in Example 2 was significantly higher than the encapsulation efficiency of GS-loaded mesenchymal stem cell exosomes prepared using the interval ultrasound method and the co-incubation method in Comparative Example 1. Furthermore, when the volume ratio of GS to mesenchymal stem cell exosomes was 1:60, the GS / exo encapsulation efficiency reached as high as 22.5%. This indicates that the acoustofluidic nano-directional coupling technology provided by the present invention enables the loading of GS onto MSC-exo and achieves excellent preparation results.

[0041] (3) Cell viability assay HaCaT cells were cultured at 1×10 4 The cells were seeded in a 96-well plate at a density of 8 × 10³ cells / well. After 24 hours, when the cell confluence reached 70%, the model was induced by treating with high glucose solution for 24 hours. The modeled cells were mixed with different concentrations of HG (0, 30, 35, 40, 45, 50, 55, 60, 65 mM), GS (0, 5, 10, 20, 40, 60, 80, 100, 200 μg / mL), MSC-exo prepared in Example 1 (0, 5, 25, 100, 500, 800, 1000, 1500, 2000 μg / mL) and GS / exo prepared in Example 2 (0, 5, 25, 100, 500, 800, 1000, 1500, 2000 μg / mL) and incubated for 24 hours. Then, 20 μL of 2% HG was added to each well. CCK-8 reagent (APExBIO) was used for incubation for 30 minutes, and the absorbance at 450 nm was measured. HaCaT cell viability was expressed as a percentage of the untreated control group (HG).

[0042] The present invention uses CCK-8 method to measure HaCaT cell viability under high glucose conditions. It is observed that the lowest HaCaT cell viability is observed when the glucose concentration is 60 mM. The 60 mM concentration is selected as the concentration for modeling. The HaCaT cell model is established under this condition 24 hours before administration. The results are as follows Figure 10 As shown in the results, 40 μg / mL GS, 800 μg / mL MSC-exo, and 100 μg / mL GS / exo all had significant repair effects on the damage of HaCaT cells.

[0043] (4) Cell uptake experiment HaCaT cells were cultured at 8×10 5The cells were seeded at a high density in a 6-well plate. When the cells reached 70% confluency, they were allowed to stand in serum-free medium for 12 hours. The cells were then incubated with PKH26 (Umibio)-stained MSC-exo prepared in Example 1 for 15 minutes and 6 hours, respectively. After incubation, the cells were washed twice with PBS and fixed with PBS containing 4% formaldehyde for 15 minutes. The cell nuclei were stained with Hoechst (Solarbio), and fluorescence was observed and recorded using a laser scanning confocal microscope (Zeiss 980).

[0044] In order to study the internalization of MSC-exo, the present invention selected two time points of incubation for evaluation, 15 min and 6 h. Figure 11 As shown, the MSC-exo prepared in Example 1 accumulated around the cell nucleus within 15 minutes of incubation, and the uptake of MSC-exo gradually increased over time. By the 6th hour of incubation, some MSC-exo had been internalized into the cell nucleus. This shows that both cell types can effectively take up the MSC-exo prepared in Example 1, and even reach the cell nucleus.

[0045] (5) Cell scratch test HaCaT cells were cultured at 5×10 5 The cells were seeded at a high density in a 6-well plate. When the cells reached 100% confluence, the monolayer of cells in each well was scraped using a 200 μL pipette tip and washed three times with PBS. The cells were then treated with GS, MSC-exo prepared in Example 1, and GS / exo prepared in Example 2 for 48 hours, respectively. Cell migration was assessed using an inverted optical microscope (Olympus, Tokyo, Japan) at 0, 12, 24, and 48 hours, and the wound area was quantified using ImageJ software.

[0046] The cell scratch test effectively evaluates the migration ability of cells, which is crucial for skin healing; therefore, the present invention monitors the changes in the scratch area at 0, 12, 24 and 48 hours to evaluate the migration of HaCaT cells. Figure 12 As shown in the results, GS, MSC-exo and GS / exo all promoted the migration of HaCaT cells, among which the GS group showed a moderate promoting effect, while the GS / exo group showed the most obvious migration enhancement effect.

[0047] (6) Cell migration assay Cell migration was assessed using polycarbonate transwell filters with a pore size of 8 μm (NEST), with a total of 4 × 10 5Individual cells were seeded in the upper chamber of a transwell in serum-free culture medium together with GS, MSC-exo prepared in Example 1, and GS / exo prepared in Example 2, and allowed to migrate to the lower chamber. After incubation for 48 hours, the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, and stained with 0.1% crystal violet for 30 minutes before being counted. Finally, the cells were imaged and analyzed using ImageJ software.

[0048] The present invention further evaluates the migration ability of cells by using the transwell method. After 48 hours, the transwell chamber is stained to evaluate the migration of HaCaT cells. Figure 13 As shown in Figure 3, GS, MSC-exo and GS / exo can all promote cell migration, among which GS has a moderate promoting effect, and the migration enhancement effect of GS / exo group is the most significant; Figure 14 As shown, the number of cells migrating through the upper chamber in the GS / exo group was the largest. The transwell experiment further confirmed the promoting effect of GS / exo on cell migration.

[0049] (7) Determination of cellular reactive oxygen species HaCaT cells were cultured at 5×10 5 Cells were plated at a high density in 6-well plates. When they reached approximately 70% confluency, they were incubated in serum-free medium for 12 hours, followed by induction with a high glucose solution for 24 hours. Each well was then treated with GS, MSC-exo prepared in Example 1, and GS / exo prepared in Example 2 for 24 hours. After treatment, the cells were digested with EDTA-free trypsin and washed twice with pre-chilled PBS. Reactive oxygen species (ROS) levels were measured using a ROS detection kit (BeyotimeS0033S) according to the manufacturer's instructions. The washed cells were resuspended in 1 mL of DCFH-DA (10 μmol / L) and incubated at 37°C for 20 minutes. The cells were then centrifuged at 1000 × g for 5 minutes and washed three times with PBS. Finally, the cells were analyzed using the FITC channel of a flow cytometer (Beckman A00-1-1102).

[0050] In vitro evaluation of the effects of GS / exo on cell survival and inflammation showed that the level of reactive oxygen species (ROS) increased significantly in diabetic wounds, which would lead to delayed wound healing. The ROS level was detected by flow cytometry using DCFH-DA fluorescent probe. Figure 15 As shown, the fluorescence intensity of the model group was significantly higher than that of the control group ( p <0.05), the ROS generation in the drug-treated group was significantly lower than that in the model group ( p<0.05); GS / exo can effectively inhibit the production of ROS in HaCaT cells, thereby alleviating high glucose-induced damage.

[0051] (8) Cell apoptosis experiment HaCaT cells were cultured at 5×10 5 The cells were placed at a density of 100 μg / mL in a 6-well plate. When the cells reached a confluence of approximately 70%, they were treated in serum-free medium for 12 hours and then cultured in a high glucose solution for 24 hours to induce modeling. Each well was treated with GS, MSC-exo prepared in Example 1, and GS / exo prepared in Example 2 for 24 hours. After treatment, the cells were digested with EDTA-free trypsin and washed twice with pre-cooled PBS. Cell apoptosis was quantified using a FITC Annexin V apoptosis detection kit (BD Pharmingen). The washed cells were resuspended in 600 μL of 1× binding buffer and incubated with 5 μL of PI and 5 μL of annexin V-FITC at room temperature for 15 minutes. 400 μL of 1× binding buffer was then added to each tube, and the samples were analyzed using a PE and FITC channel flow cytometer (Beckman A00-1-1102).

[0052] Annexin V / PI double staining was performed by flow cytometry to analyze cell viability in more detail. Figure 16 As shown in Figure 2, the apoptosis rate of rats in the model group was significantly higher than that in the control group ( p <0.05); compared with the model group, the cell apoptosis in the drug-treated group was effectively reduced ( p <0.05); among them, the GS / exo group showed the most significant inhibition of cell apoptosis. Thus, the GS / exo provided by the present invention effectively inhibited high glucose-induced apoptosis in HaCaT cells.

[0053] (9) Mitochondrial membrane potential change experiment HaCaT cells were cultured at 5×10 5The cells were placed at a density of 400 μg / mL in a 6-well plate. When the cells reached a confluence of approximately 70%, they were allowed to stand in serum-free medium for 12 hours, and then a high glucose solution was added for 24 hours to induce modeling. Each well was treated with GS, MSC-exo prepared in Example 1, and GS / exo prepared in Example 2 for 24 hours, respectively. After treatment, the cells were digested with EDTA-free trypsin and washed twice with pre-cooled PBS. According to the manufacturer's instructions, the mitochondrial membrane potential was assessed using a mitochondrial membrane potential assay kit (Beyotime C2006). The washed cells were resuspended in 0.5 mL of JC-1 solution and incubated at 37°C for 20 minutes. The cells were centrifuged at 600 g for 5 minutes and washed with binding buffer. Finally, the cells were analyzed using a FITC channel flow cytometer (Beckman A00-1-1102).

[0054] Cell apoptosis is accompanied by changes in mitochondrial membrane potential. The qualitative evaluation of mitochondrial membrane potential changes is as follows: Figure 17 As shown, compared with the control group, the model group had a higher proportion of cells with mitochondrial membrane depolarization and a lower mitochondrial membrane potential ( p <0.05); on the contrary, in the GS / exo treatment group, the percentage of cells with intact mitochondrial membranes increased, and the red / green relative fluorescence intensity gradually increased. The mitochondrial membrane potential in the GS / exo experimental group recovered most significantly ( p <0.05) (10) Determination of inflammatory factors HaCaT cells were plated at 1 × 10 6 The cells were cultured at a density of 1 cm in a 6 cm culture dish. When the cells reached a confluence of approximately 70%, they were allowed to stand in serum-free medium for 12 hours, and then a high glucose solution was added for 24 hours to induce modeling. Each plate was treated with GS, MSC-exo prepared in Example 1, and GS / exo prepared in Example 2 for 24 hours, respectively. The cells were lysed using RIPA lysis buffer (Beyotime) and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and the levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β were detected according to the kit instructions (Beyotime).

[0055] The results are as follows Figure 18As shown in the results, compared with the control group, high glucose stimulation of HaCaT cells and HUVEC significantly increased the levels of TNF-α, IL-6, and IL-1β, while the levels of TNF-α, IL-6, and IL-1β in the GS / exo group were significantly reduced. Compared with the control group, high glucose stimulation of HaCaT cells and HUVEC significantly decreased the levels of TGFβ and IL-10, while the levels of TGFβ and IL-10 in the GS / exo group were significantly increased. It can be seen that the GS / exo provided by the present invention can reduce the inflammatory response of diabetic skin defects by inhibiting the release of proinflammatory cytokines and promoting cell survival.

[0056] (11) Establishment of a complete skin defect model in diabetic mice Six-week-old ICR mice were acclimated for 7 days and fasted for 12 hours before being intraperitoneally injected with 150 mg / kg of streptozotocin (STZ). Blood glucose levels were randomly measured on the 7th day, and a random blood glucose level of 16.7 mmol / L indicated a successful diabetic mouse model. Blood glucose levels in the different groups of mice were measured using a glucometer, and the results showed that the blood glucose levels in the diabetic mice were significantly higher than those in the control group. Thirty-six successfully induced diabetic mice were randomly divided into the Con group, the GS group, the MSC-exo group, and the GS / exo group. The backs of the mice were shaved, disinfected, and circular skin excision wounds (6 mm in diameter) were created. To minimize the effects of skin contraction on wound healing, an inner diameter rubber ring and skin adhesive were used to secure the wound site.

[0057] To verify the in vivo healing effects of the four groups (Con group, GS group, MSC-exo group, and GS / exo group), the Con group, GS group, MSC-exo group, and GS / exo group were applied to a full-thickness skin defect model. The wound healing process was recorded by photography, and wound images were collected on days 0, 7, and 14 after treatment. ImageJ software was used to quantify and statistically analyze the wound area (mm²) and healing rate. Figure 19 As shown, 14 days after wounding, the wounds in the GS / exo group were almost completely healed, with new skin covering the original open wound, no scar tissue on the surface, and surrounding hair regeneration. This indicates that the GS-loaded mesenchymal stem cell exosomes (GS / exo) provided by the present invention can accelerate skin wound healing, promote the formation of new skin, and promote hair regeneration.

[0058] Wound tissues were collected on the 7th and 14th days after treatment for immunohistochemical analysis, H&E staining, and Masson staining. The specific steps are as follows: H&E staining steps: Skin tissues were collected from mice with skin defect models on the 7th and 14th days after the above treatments, and immediately fixed with 4% paraformaldehyde and dehydrated stepwise with gradient ethanol (75%, 85%, 90%, 95%, 100%) for 5 min. After dehydration, the samples were cleared with xylene and embedded in paraffin. 4 μm thick sections were prepared and flattened in warm water. They were dried in a 60°C oven for 3 h and stored at room temperature for later use. When dewaxing the sections, the samples were first immersed in xylene I and II solutions for 5 min each, and then hydrated with reverse gradient ethanol (100%, 95%, 80%, 70%) for 5 min. The tissue sections were stained with hematoxylin for 5 min, rinsed with distilled water until there was no chromatin residue in the background, differentiated with 1% hydrochloric acid ethanol solution for 3 s, and quickly rinsed with running water to terminate the reaction. The sections were stained with eosin solution for 3 s. min, showing a characteristic pink color; after staining, the sections were dehydrated again with gradient ethanol (70%, 80%, 95%, 100%) for 3 min, and finally transparentized with xylene, mounted with optical resin and covered with a coverslip. After the mounting agent was completely solidified, the samples were prepared for microscopic morphological observation.

[0059] Masson staining steps: Skin tissue was collected from mice with skin defect models on days 7 and 14 after the above treatments. The tissue was immediately fixed with 4% paraformaldehyde and dehydrated in a gradient of ethanol (75%, 85%, 90%, 95%, and 100%) for 5 minutes. After dehydration, the samples were cleared with xylene and embedded in paraffin. 4-μm-thick sections were prepared and flattened in warm water. They were dried in a 60°C oven for 3 hours and stored at room temperature until further use. The sections were immersed in Massona solution for approximately 15 hours and heated at 37°C for 30 minutes. Massona solution (D) and solution (F) were preheated, and equal amounts of Massona solution (B) and solution (C) were mixed and stained for 1 minute. The sections were then washed with running water, treated with differentiation solution, washed with water, immersed in Massona solution for 7 minutes, treated with Massona solution for 1 minute, stained in Massona solution (F) for 10 seconds, treated with differentiation solution, and mounted using a gradient of alcohol.

[0060] The results of H&E staining and Masson staining are as follows Figure 20-21As shown, on the 7th day, all tissues in the Con group, GS group, and MSC-exo group showed varying degrees of inflammatory response, although the inflammatory cell infiltration in the treated group was reduced compared with the control group; on the 14th day, the wounds in the Con group, GS group, and MSC-exo group were not completely healed, and the red exposed wound in the center was still covered by the tumor epithelium; however, using the GS-loaded mesenchymal stem cell exosomes provided by the present invention (GS / exo group), the wound area was significantly reduced, the epithelial tissue covered the tumor skin, there was no obvious scar formation, the new skin covered the original open wound, there was no scar tissue on the surface, the surrounding hair regenerated, and the wound was almost completely healed.

[0061] In summary, the present invention utilizes the natural extracellular vesicles MSC-exo secreted by MSCs and combines it with GS having antibacterial properties, so that they work synergistically with each other to promote the healing of skin tissue defects.

[0062] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for preparing GS-loaded mesenchymal stem cell exosomes, characterized in that: The preparation method comprises the following steps: S1: Mesenchymal stem cells were digested using acoustofluidic nano-directional coupling technology, and the mesenchymal stem cells were transferred to serum-free OPTI-MEM medium for culture. The culture medium was allowed to stand, and the supernatant was centrifuged three times. The supernatant was filtered through a 0.2 μm syringe, concentrated using a 100 kDa MWCO ultrafiltration, and centrifuged at 4000 × g for 30 min to obtain mesenchymal stem cell exosomes. S2: GS was mixed with the mesenchymal stem cell exosomes obtained in S1 through acoustofluidic nano-directional coupling technology to obtain GS-loaded mesenchymal stem cell exosomes.

2. The preparation method according to claim 1, characterized in that The processing steps of the acoustofluidic nano-directional coupling technique described in S1 and S2 are as follows: centrifugation at 1000×g for 10 min, 5 The cells were resuspended in serum-free OPTI-MEM medium at a density of 10 cells / mL and treated with an ultrasonic spray generator.

3. The preparation method according to claim 2, characterized in that The flow rate of the ultrasonic spray generator treatment was 1.4 L / h.

4. The preparation method according to claim 2, characterized in that The frequency of the ultrasonic spray generator is 15 kHz.

5. The preparation method according to claim 1, wherein The standing time described in S1 is 48 h.

6. The preparation method according to claim 1, characterized in that The conditions for the three centrifugal treatments in S1 are: the first centrifugal treatment condition is centrifugation at 500 rpm for 30 min, the second centrifugal treatment condition is centrifugation at 2000 rpm for 30 min, and the third centrifugal treatment condition is centrifugation at 10000 rpm for 60 min.

7. The preparation method according to claim 1, characterized in that The mixing volume ratio of GS and mesenchymal stem cell exosomes described in S2 was 1:

60.

8. A mesenchymal stem cell exosome loaded with GS, characterized in that: The GS-loaded mesenchymal stem cell exosomes are obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the GS-loaded mesenchymal stem cell exosomes according to claim 8 in the preparation of a drug for promoting the healing of skin tissue defects.

10. The use according to claim 9, characterized in that The skin tissue defects are caused by diabetes and its complications.

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