Chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell derived exosome and preparation method of chitosan-hyaluronic acid hydrogel

Abstract

The invention belongs to the technical field of medicines, and particularly relates to chitosan-hyaluronic acid hydrogel (UCMSCs-exo-coated CS-HAhydrogel) loaded with an umbilical cord mesenchymal stem cell derived exosome and a preparation method of the chitosan-hyaluronic acid hydrogel (UCMSCs-exo-coated CS-HAhydrogel) loaded with the umbilical cord mesenchymal stem cell derived exosome. The UCMSCs-exo-coated CS-HA hydrogel prepared by the preparation method disclosed by the invention can be used for remarkably promoting the repair of a full-thickness skin defect wound surface of a C57BL / 6 mouse. The CS and the HA are effectively combined to construct a CS-HAhydrogel drug delivery system so as to promote the in-vitro utilization rate of the exosome and promote the healing of the wound surface. The CS-HAhydrogel has various biological activities, can be used as a drug carrier for drug delivery and controlled release, improves the curative effect and safety of drugs, and has wide application in the field of biomedicine.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and specifically relates to a chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body, consisting of the epidermis, dermis and subcutaneous tissue, and plays a very important role in resisting the invasion of pathogens. A wound in which the skin tissue is damaged by external factors is called a wound. Difficult wound healing has always been a common problem in the medical system. Its treatment is difficult, long and expensive. It is a disease that seriously endangers the physical and mental health of the people and reduces the quality of life. It also increases the burden on families and society and occupies a large amount of medical resources. Studies have shown that traditional therapies for wound healing include negative pressure wound therapy, gauze dressings, hyperbaric oxygen therapy, etc. However, these treatments can cause damage to wound tissue and limited tissue activity, and cannot meet the needs of patients. Therefore, designing and developing an effective wound healing therapy has become the main research goal.

[0003] In recent years, stem cell exosome therapy has become the mainstream of wound healing research. Exosomes are one of the paracrine components of cells, rich in proteins, lipids, nucleic acids and carbohydrate conjugates. Exosomes can reshape the extracellular matrix and transmit signals and molecules to other cells. At the same time, many shortcomings of stem cells are avoided because exosomes are stable and easy to store. In addition, they are not rejected by the immune system, have a homing effect, and the dosage is easy to control. However, the in vitro inactivation rate of exosomes is high, and they cannot act on the wound for a long time to promote wound healing. Studies have shown that biomaterial scaffolds with good biocompatibility can be used as drug release carriers to improve the utilization rate of drugs in vitro to promote wound healing. According to relevant literature reports, CS is widely present in shellfish and is obtained by deacetylation of extracted chitin. The presence of amino groups in the CS structure can change the entry and exit of substances in the cell membrane and is an excellent biological carrier for drug transport. In addition, CS has multiple biological activities such as antibacterial, promoting wound healing and lowering blood sugar. It also has the function of regulating lipid substances and immune response and is non-toxic. HA is a natural bioactive substance existing in humans and animals, mainly in the intercellular matrix. As the main component of the extracellular matrix, it has high adhesion, can protect cells and tissues, and has functions such as regulating proteins, regulating the permeability of blood vessel walls, and promoting wound healing. Based on this, research and development is conducted on how to improve the in vitro utilization rate of exosomes to accelerate the repair of full-thickness skin defects and promote wound healing. Summary of the invention

[0004] In order to comprehensively solve the above problems, the present invention proposes a chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and a preparation method thereof, which combines UCMSCs-exo, CS and HA for the repair of full-thickness skin defects, which not only effectively promotes the repair of the wound surface but also improves the efficacy and safety of the drug.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and a preparation method thereof, comprising:

[0007] Step 1: Preparation of UCMSCs-exo. Collect the supernatant of UCMSCs cultured for 2 to 8 generations, separate UCMSCs-exo by ultra-high-speed centrifugation, use 2000g centrifugation for 10min to initially remove cells and cell debris; after absorbing the supernatant, use 10000g high-speed centrifugation for 30min to remove organelles, etc., and keep the supernatant; use 100000g high-speed centrifugation for 100min to keep the precipitate; use PBS to wash and resuspend the precipitate and repeat the previous step once. The obtained precipitate is resuspended in 1mL of PBS solution and filtered through a 0.22μm filter. After measuring the concentration, take it out or store it at -80℃.

[0008] Step 2: Identification of UCMSCs-exo. (1) TEM: Before electron microscopy, the exosome samples that have been resuspended and packaged should be taken out in advance and thawed in a 4°C refrigerator. After thawing, an equal volume of 4% paraformaldehyde was added to fix the exosomes, and then electron microscopy was performed according to the following method. Take 10μL of the fixed sample and drop it on the copper grid for transmission electron microscopy. Let it settle for 3 minutes. Use filter paper to absorb the floating liquid from the edge. During this process, pay attention to gentle operation and pay attention to absorbing the floating liquid. Try to operate under sterile conditions to avoid damaging the exosome sample or causing background contamination. Then rinse with PBS three times. Then take 10μL of phosphotungstic acid and drop it on the copper grid. Let it settle for 1 minute. Use filter paper to absorb the floating liquid. This process also needs to be done gently and under sterile conditions as much as possible. Dry at room temperature for 2 minutes, image on the machine, and control the electron microscope operating voltage to 80-120KV. Finally, look for typical exosome structures and take pictures. (2) Western blot: Detection of exosome marker proteins CD9, CD63, and TSG101: Take 50 μL of exosome sample, add 100 μL of lysis buffer and lyse on ice for 1 h. Take an appropriate amount of the mixed solution after lysis, add protein loading buffer, place in a 100°C metal bath for 10 min, cool and store at -20°C as the electrophoresis sample. Prepare protein electrophoresis gel, take 15 μL of exosome electrophoresis sample and add it to the gel lane and add it to the adjacent lane, 5 μL of protein Maker, the strip runs through the compression gel at a voltage of 80 V, and runs through the separation gel at 120 V. Remove the gel in the lane where the sample is located, cut the matching nitrocellulose film, and assemble it in order in the transfer tank. Transfer the membrane at 300 mA according to the molecular weight of the protein. Remove the transferred membrane and put it into the incubation box, and block it with milk blocking solution at room temperature for 1 h. Discard the blocking solution, cut the membrane according to the different molecular weights of CD9, CD63, and TSG101, and incubate with the corresponding antibody diluent at 4°C overnight. Take out the corresponding membrane and put it into the antibody incubation box and wash it with PBST buffer 3 times, 10 minutes each time. According to the source of the antibody, incubate it at room temperature for 1 hour with the corresponding secondary antibody diluent. Then wash it with PBST buffer 3 times, 10 minutes each time. Prepare an appropriate amount of ECL color developer (A solution: B solution = 1:1), soak the membrane in the color developer for several minutes in the dark, observe it in a gel imager and take pictures to record.

[0009] Step 3: Preparation of CS-HA hydrogel. Preparation was performed by physical crosslinking method. 5.85g CS was mixed with 60.75g physiological saline and stirred evenly, then 1.65g acetic acid was added and stirred to dissolve, and 6g HA was added and stirred to dissolve, and stirring was stopped when the hydrogel was in a gel state, and finally CS-HA hydrogel was obtained.

[0010] Step 4: Identification of CS-HAhydrogel. CS-HAhydrogel was identified using a scanning electron microscope. A small piece of freeze-dried gel sample was sprayed with gold to prepare a sample, and then the sample was placed in a SEM and observed and photographed at an accelerating voltage of 10 kV.

[0011] Step 5: Preparation of UCMSCs-exo@CS-HAhydrogel. The UCMSCs-exo extracted by centrifugation was loaded into the prepared CS-HAhydrogel and added to the centrifuge tube and rotated for 3 minutes to ensure that the exosomes in the gel were evenly distributed. 2 , 360-480 nm) for 10 s to obtain UCMSCs-exo@CS-HAhydrogel.

[0012] Furthermore, the UCMSCs-exo in step 1 was extracted at room temperature.

[0013] Furthermore, the exosomes extracted in step 2 need to be identified before subsequent related experiments can be carried out.

[0014] Furthermore, in step 3, CS-HAhydrogel is prepared at room temperature, and then the characterization experiment of step 4 is carried out, and then subsequent related experiments are carried out.

[0015] Furthermore, in step 5, the preparation of UCMSCs-exo@CS-HAhydrogel includes loading the UCMSCs-exo extracted by centrifugation into the prepared CS-HAhydrogel. The UCMSCs-exo@CS-HAhydrogel of the present invention is used to promote the repair of full-thickness skin defects in C57BL / 6 mice.

[0016] Furthermore, the concentration of exosomes was determined by the BCA method, and the concentration was 0.2 mg / mL.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Loading UCMSCs-exo@CS-HAhydrogel can promote the proliferation of L929 fibroblasts, accelerate wound healing, and has good biocompatibility and safety;

[0019] 2. CS-HA hydrogel has a good three-dimensional porous structure and is suitable for delivering UCMSCs-exo for repairing full-thickness skin defects in C57BL / 6 mice;

[0020] 3. Through the cell live-death staining experiment, it was observed that CS-HAhydrogel has good biocompatibility. In addition, CS-HAhydrogel can keep the activity of UCMSCs-exo in vitro and promote wound healing;

[0021] 4.UCMSCs-exo@CS-HAhydrogel has a certain antibacterial effect and can inhibit the proliferation of Escherichia coli and Staphylococcus aureus.

[0022] 5.UCMSCs-exo@CS-HAhydrogel can promote the deposition of collagen fibers, promote the formation of new blood vessels, and accelerate wound healing.

[0023] 6.UCMSCs-exo@CS-HAhydrogel can inhibit the expression of COX2 and INOS pro-inflammatory factors and has a certain anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0025] In the attached picture:

[0026] Figure 1 A is a transmission electron microscopy identification image of exosomes in step 2 of Example,

[0027] Figure 1 B is a Western blot identification image of exosomes in step 2 of Example.

[0028] Figure 2 C is the concentration and diameter identification diagram of UCMSCs-exo analyzed by nanoparticle tracking.

[0029] Figure 3 To evaluate the proliferation-promoting ability and safety of UCMSCs-exo on L929 fibroblasts through cell CCK8 cell proliferation experiment and toxicity experiment. Figure 3 A is the evaluation of the proliferation ability of UCMSCs-exo on L929 fibroblasts at 24 h; Figure 3 B is the evaluation of the proliferation ability of UCMSCs-exo on L929 fibroblasts at 48 h; Figure 3 C is the evaluation of the proliferation ability of UCMSCs-exo on L929 fibroblasts at 72 h; Figure 3 D is the safety assessment of UCMSCs-exo on L929 fibroblasts at different concentrations.

[0030] Figure 4C is the cell biocompatibility evaluation of the UCMSCs-exo@CS-HAhydrogel of the present invention.

[0031] Figure 5 A is the evaluation of the antibacterial ability of UCMSCs-exo@CS-HAhydrogeld of the present invention against Escherichia coli and Staphylococcus aureus; Figure 5 B is the antibacterial ability of UCMSCs-exo@CS-HA hydrogeld of the present invention against Escherichia coli; Figure 5 C is the antibacterial ability of UCMSCs-exo@CS-HA hydrogeld of the present invention against Staphylococcus aureus.

[0032] Figure 6 A is the evaluation of the expression of collagen fibrillin factors and inflammatory factors by Western blot experiment of UCMSCs-exo@CS-HAhydrogeld of the present invention; Figure 6 B is a diagram showing that UCMSCs-exo@CS-HAhydrogel of the present invention promotes the formation of type I collagen; Figure 6 C is a diagram showing that UCMSCs-exo@CS-HAhydrogel of the present invention promotes the formation of type III collagen; Figure 6 D is a graph showing that UCMSCs-exo@CS-HAhydrogel of the present invention reduces the expression of inflammatory factor COX2; Figure 6 E is a graph showing that UCMSCs-exo@CS-HAhydrogel of the present invention reduces the expression of inflammatory factor iNOS;

[0033] Attached Figure 7 This is the SME image of the hydrogel obtained from the sample in Measurement Example 1.

[0034] Attached Figure 8 This is a graph showing the rheological test results of the hydrogel obtained in Measurement Example 2.

[0035] Attached Fig. 9 This is a graph showing the drug sustained-release test results obtained in Determination Example 3.

[0036] Attached Fig.10 A is the wound healing pictures of mice at different treatment times;

[0037] Attached Fig.10 B is the calculation of the healing rate of mouse wounds;

[0038] Attached Fig.10 C is the HE staining observation image of UCMSCs-exo@CS-HAhydrogel.

[0039] Attached Fig.11A is the Masson staining result of UCMSCs-exo@CS-HAhydroged;

[0040] Attached Fig.11 B is the immunohistochemical result of UCMSCs-exo@CS-HAhydroged;

[0041] Attached Fig.11 C is the quantification graph of Masson staining results of UCMSCs-exo@CS-HAhydroged;

[0042] Attached Fig.11 D is the quantitative graph of the positive expression of vascular endothelial cell adhesion factor CD31 in UCMSCs-exo@CS-HAhydroged;

[0043] Attached Fig.11 E is the quantitative graph of the positive expression of vascular endothelial growth factor VEGF in UCMSCs-exo@CS-HA hydroged;

[0044] Attached Fig.11 F is the quantification of the positive expression of hypoxia-inducible factor HIF-1α in UCMSCs-exo@CS-HA hydroged.

[0045] Attached Fig.12 The preparation process and mechanism diagram of UCMSCs-exo@CS-HAhydroged. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0047] Example 1

[0048] A chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and a preparation method thereof, comprising:

[0049] Step 1: Preparation of UCMSCs-exo. Collect the supernatant of UCMSCs cultured for 2 to 8 generations, separate UCMSCs-exo by ultra-high-speed centrifugation, use 2000g centrifugation for 10min to initially remove cells and cell debris; after absorbing the supernatant, use 10000g high-speed centrifugation for 30min to remove organelles, etc., and keep the supernatant; use 100000g high-speed centrifugation for 100min to keep the precipitate; use PBS to wash and resuspend the precipitate and repeat the previous step once. The obtained precipitate is resuspended in 1mL of PBS solution and filtered through a 0.22μm filter. After measuring the concentration, take it out or store it at -80℃.

[0050] Step 2: Identification of UCMSCs-exo. (1) TEM: Before electron microscopy, the exosome samples that have been resuspended and packaged should be taken out in advance and thawed in a 4°C refrigerator. After thawing, an equal volume of 4% paraformaldehyde was added to fix the exosomes, and then electron microscopy was performed according to the following method. Take 10μL of the fixed sample and drop it on the copper grid for transmission electron microscopy. Let it settle for 3 minutes. Use filter paper to absorb the floating liquid from the edge. During this process, pay attention to gentle operation and pay attention to absorbing the floating liquid. Try to operate under sterile conditions to avoid damaging the exosome sample or causing background contamination. Then rinse with PBS three times. Then take 10μL of phosphotungstic acid and drop it on the copper grid. Let it settle for 1 minute. Use filter paper to absorb the floating liquid. This process also needs to be done gently and under sterile conditions as much as possible. Dry at room temperature for 2 minutes, image on the machine, and control the electron microscope operating voltage to 80-120KV. Finally, look for typical exosome structures and take pictures. (2) Western blot: Detection of exosome marker proteins CD9, CD63, and TSG101: Take 50 μL of exosome sample, add 100 μL of lysis buffer and lyse on ice for 1 h. Take an appropriate amount of the mixed solution after lysis, add protein loading buffer, place in a 100°C metal bath for 10 min, cool and store at -20°C as the electrophoresis sample. Prepare protein electrophoresis gel, take 15 μL of exosome electrophoresis sample and add it to the gel lane and add it to the adjacent lane, 5 μL of protein Maker, the strip runs through the compression gel at a voltage of 80 V, and runs through the separation gel at 120 V. Remove the gel in the lane where the sample is located, cut the matching nitrocellulose film, and assemble it in order in the transfer tank. Transfer the membrane at 300 mA according to the molecular weight of the protein. Remove the transferred membrane and put it into the incubation box, and block it with milk blocking solution at room temperature for 1 h. Discard the blocking solution, cut the membrane according to the different molecular weights of CD9, CD63, and TSG101, and incubate them with the corresponding antibody diluent at 4°C overnight. Take out the corresponding membrane and put it into the antibody incubation box and wash it 3 times with PBST buffer, 10 minutes each time. According to the source of the antibody, incubate it at room temperature for 1 hour with the corresponding secondary antibody diluent. Then wash it 3 times with PBST buffer, 10 minutes each time. Prepare an appropriate amount of ECL color developer (A solution: B solution = 1:1), soak the membrane in the dark with the color developer for several minutes, observe it in a gel imager and take pictures and record it. In addition, the concentration of exosomes was determined by the BCA method, and the concentration was 0.2 mg / mL.

[0051] Step 3: Preparation of CS-HA hydrogel. Preparation was performed by physical crosslinking method. 5.85g CS was mixed with 60.75g physiological saline and stirred evenly, then 1.65g acetic acid was added and stirred to dissolve, and 6g HA was added and stirred to dissolve, and stirring was stopped when the hydrogel was in a gel state, and finally CS-HA hydrogel was obtained.

[0052] Step 4: Identification of CS-HAhydrogel. CS-HAhydrogel was identified using a scanning electron microscope. A small piece of freeze-dried gel sample was sprayed with gold to prepare a sample, and then the sample was placed in a SEM and observed and photographed at an accelerating voltage of 10 kV.

[0053] Step 5: Preparation of UCMSCs-exo@CS-HAhydrogel. The UCMSCs-exo extracted by centrifugation was loaded into the prepared CS-HAhydrogel and added to the centrifuge tube and rotated for 3 minutes to ensure that the exosomes in the gel were evenly distributed. 2 , 360-480 nm) for 10 s to obtain UCMSCs-exo@CS-HAhydrogel.

[0054] Measurement Example 1

[0055] Performance evaluation determination: structural characterization and morphology observation of UCMSCs-exo@CS-HAhydrogel.

[0056] Step 1: Cut the CS-HA hydrogel into 0.5 cm (length) × 0.2 cm (width) × 0.1 cm (thickness) size and immerse it in liquid nitrogen solution for supercritical drying. Soak for 3 minutes to remove the water in the gel, and immediately put it into a freeze dryer until it is completely dried and then sprayed with gold for use. The SEM acceleration voltage is 15KV.

[0057] Step 2: Freeze-dry the CS-HA hydroge that has adsorbed heavy metal ions to remove excess water. Take a certain amount of the ground sample and mix it with dry KBr to make a tablet. Then use a Fourier transform infrared spectrometer to obtain the sample's wavelength in the range of 4000-400 cm -1 FT-IR in the range.

[0058] Attached Figure 7 The SME diagram of the hydrogel obtained from the sample of Example 1 was obtained from the attached Figure 7 It can be seen that the hydrogels exhibit a porous structure and a three-dimensional structure with holes connected to each other, which is conducive to the transmission and release of drugs.

[0059] Measurement Example 2

[0060] Among them, the multifunctionality evaluation of UCMSCs-exo@CS-HAhydrogel includes:

[0061] Step 1: monitoring the rheological properties of the hydrogel at 25 °C, rheometer (MCR 302, Anton Parr, Austria);

[0062] Step 2: CS-HA hydrogel was mixed thoroughly and loaded onto a preheated stage. The parallel plate geometry strain rate was set to 0.1%, the frequency was 6.28 rads-1, and the time sweep was performed until the high point of the storage modulus G' was reached.

[0063] Attached Figure 8 The rheological test result of the hydrogel obtained in Example 2 is shown in the figure. Figure 8 It can be seen that the hydrogel has certain elasticity, good strength and flexibility.

[0064] Measurement Example 3

[0065] The drug adsorption and sustained release tests of UCMSCs-exo@CS-HAhydrogel include:

[0066] Step 1: Labeling and purification of exosomes: Take 5 μL of EvlINK505 probe marker and 150 μL of exosomes and incubate them in the dark at room temperature for 30 minutes. Then use a 100KD ultrafiltration tube for ultrafiltration separation and purification. The centrifugation conditions are 4°C, 7000-10000g, 15 minutes, and repeat 4 times.

[0067] Step 2: Load UCMSCs-exof into CS-HAhydrogelz (1:1).

[0068] Step 3: Place the labeled exosomes and hydrogel in the upper transwell chamber of a 24-well plate, and add 1.2 mL of release medium (PBS solution containing 10% fetal bovine serum) to the lower chamber. Then place the chamber in a 37°C incubator and rotate at 60 r / min. Take 400 μL of the lower chamber solution at a predetermined time point (replenish fresh release medium in time), use 488 nm as the excitation wavelength and 525 nm as the emission wavelength, measure the sample fluorescence intensity, and calculate the cumulative release rate.

[0069] Attached Fig. 9 This is a graph showing the drug sustained-release test results obtained in Determination Example 3.

[0070] Animal Testing:

[0071] Forty healthy male C57BL / 6 mice weighing 18-22 g were selected to establish a wound model (n=10). After isoflurane inhalation anesthesia, the mice were placed in a prone position, their limbs were fixed, and they were disinfected with 75% ethanol. A circular full-thickness skin defect with a diameter of 0.8 cm was prepared on the back of the mice. Gauze was used to compress the wound to stop bleeding. The mice were divided into 4 groups using a random number table: control group, UCMSCs-exo group, CS-HAhydrogel group, and UCMSCs-exo@CS-HAhydrogel group. Intervention was performed immediately after injury. After the wound was modeled, all mice were housed in single cages. Drug intervention treatment was given in the first three days. At different time points on days 0, 3, 5, 7, 9, and 11, pictures of the mouse wounds were taken at the same height, and the wound healing rate was calculated. See Table 1. Fig.10 A and Fig.10 B.

[0072] After the 5th and 11th days of treatment, 3 mice were randomly selected from each group. After anesthesia, tissue blocks including the entire wound were cut from 5 mm away from the edge of the wound under sterile conditions, and serum was collected. The tissue sections were prepared after fixation with 40g / L paraformaldehyde and paraffin embedding for later use. HE staining, Masson staining and immunohistochemistry were then performed to observe the effect of UCMSCs-exo@CS-HAhydrogel on wound tissue, and to explore whether it promotes wound tissue regeneration, collagen fiber deposition and new blood vessel regeneration. Fig.10 C.

[0073] Preliminary conclusions were drawn: The wound healing rates from high to low were UCMSCs-exo@CS-HA hydrogel group, CS-HA hydrogel group, UCMSCs-exo group, and control group. UCMSCs-exo@CS-HAhydrogel can promote collagen deposition in wound tissue and accelerate wound healing. The expression levels of collagen fibrillary proteins from high to low were UCMSCs-exo@CS-HAhydrogel group, CS-HA hydrogel group, UCMSCs-exo group, and control group. In addition, UCMSCs-exo@CS-HAhydrogel can promote the regeneration of new capillaries in wound tissue. The expression levels of new blood vessels from high to low were UCMSCs-exo@CS-HA hydrogel group, CS-HAhydrogel group, UCMSCs-exo group, and control group.

[0074] Beneficial Effects

[0075] The present invention effectively combines CS and HA to construct a CS-HAhydrogel drug delivery system to promote the in vitro utilization of exosomes to promote wound healing. CS-HAhydrogel has multiple biological activities and can be used as a drug carrier for drug delivery and controlled release, improving the efficacy and safety of drugs, and has a wide range of applications in the biomedical field. UCMSCs-exo is a subcellular vesicle component with a size of 30-150nm, and is a new type of carrier for intercellular communication that regulates the biological behavior of skin cells. UCMSCs-exo has been shown to promote angiogenesis, and more importantly, it can also affect macrophage polarization, inhibit M1 macrophage polarization and promote M2 macrophage polarization, thereby reducing the release of proinflammatory factors and inflammatory responses. In addition, UCMSCs-exo can also improve tissue repair by inhibiting inflammation at the wound site and reshaping the immune microenvironment.

[0076] Based on this, the present invention physically combines CS-HAhydrogel and UCMSCs-exo to prepare UCMSCs-exo@CS-HAhydroge to improve the in vitro utilization rate of UCMSCs-exo to accelerate the repair of full-thickness skin defects and promote wound healing.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and a preparation method thereof comprising: Step 1: Preparation of umbilical cord mesenchymal stem cell derived exosomes (UCMSCs-exo): Collect the supernatant of umbilical cord mesenchymal stem cells (UCMSCs) cultured for 2 to 8 generations, separate UCMSCs-exo by ultra-high speed centrifugation, and use 2000g centrifugation for 10 min to initially remove cells and cell debris; after absorbing the supernatant, use 10000g high-speed centrifugation for 30 min to remove organelles and keep the supernatant; use 100000g high-speed centrifugation for 100 min to keep the precipitate; Wash and resuspend the precipitate with PBS and repeat the previous step once. Resuspend the precipitate with 1 mL of PBS solution and filter through a 0.22 μm filter. Determine the concentration and use or store at -80°C. Step 2: Identification of UCMSCs-exo: (1) Transmission electron microscopy (TEM): Before TEM detection, the exosome samples that have been resuspended and packaged should be taken out in advance and thawed in a 4°C refrigerator. After thawing, an equal volume of 4% paraformaldehyde is added to fix the exosomes, and then TEM detection is performed according to the following method: 10 μL of the fixed sample is added to the copper grid dedicated to the transmission electron microscope, and the sedimentation is carried out for 3 minutes. The floating liquid is removed from the edge with filter paper, and then rinsed with PBS for 3 times. Then 10 μL of phosphotungstic acid is added to the copper grid, and the sedimentation is carried out for 1 minute. The floating liquid is removed with filter paper, and the grid is dried at room temperature for 2 minutes. The image is taken on the machine, and the operating voltage of the electron microscope is controlled at 80-120 KV. Finally, the typical exosome structure is found and photographed; (2) Western blot: Detection of exosome marker proteins CD9, CD63, and TSG101: 50 μL exosome sample, add 100μL lysis buffer and lyse on ice for 1h, take an appropriate amount of the mixed solution after lysis, add protein loading buffer, 100℃ metal bath for 10min, cool and store at -20℃ as electrophoresis sample; prepare protein electrophoresis gel, take 15μL exosome electrophoresis sample and add it to the gel lane and add it to the adjacent lane, 5μL protein Maker, the strip runs through the compression gel at a voltage of 80V, and runs through the separation gel at 120V, remove the gel in the lane where the sample is located, cut the matching nitrocellulose film, assemble it in the transfer tank in order, transfer the membrane at 300mA according to the molecular weight of the protein, remove the transferred membrane and put it into the incubation box, block it with milk blocking solution at room temperature for 1h, discard the blocking solution, cut the membrane according to the different molecular weights of CD9, CD63, and TSG101, and incubate it overnight at 4℃ with the corresponding antibody diluent. Take out the corresponding membrane and put it into the antibody incubation box, wash it with PBST buffer 3 times, 10 minutes each time; according to the source of the antibody, incubate it with the corresponding secondary antibody diluent at room temperature for 1 hour, and then wash it with PBST buffer 3 times, 10 minutes each time. Prepare an appropriate amount of ECL color developer (A solution: B solution = 1:1), soak the membrane with the color developer for several minutes in the dark, observe it in a gel imager and take pictures; Step 3: Preparation of chitosan-hyaluronic acid hydrogel (CS-HAhydrogel): Prepared by physical crosslinking method, 5.85g chitosan powder (CS) and 60.75g physiological saline were mixed and stirred evenly, then 1.65g acetic acid was added and continued to stir and dissolve, and 6g hyaluronic acid powder (HA) was added and continued to stir and dissolve, and stirring was stopped when it became a gel state, and finally CS-HAhydrogel was obtained; Step 4: Identification of CS-HAhydrogel: CS-HAhydrogel was identified using a scanning electron microscope. A small piece of freeze-dried gel sample was sprayed with gold to prepare a sample. The sample was then placed in a S-4800 scanning electron microscope (SEM) and observed and photographed at an accelerating voltage of 10 kV. Step 5: Preparation of UCMSCs-exo@CS-HAhydrogel: Load the UCMSCs-exo extracted by centrifugation into the prepared CS-HAhydrogel, add it to the centrifuge tube and rotate for 3 minutes to ensure that the exosomes in the gel are evenly distributed. 2 , 360-480 nm) for 10 s to obtain UCMSCs-exo@CS-HAhydrogel.

2. The chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and the preparation method thereof according to claim 2, characterized in that: The preparation process of UCMSCs-exo in step 1 was carried out at room temperature.

3. The chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and the preparation method thereof according to claim 2, characterized in that: The identification of UCMSCs-exo in step 2 requires dual identification by TEM and Western blot to observe the normal size and morphology of exosomes and whether they have been successfully extracted.

4. The chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and the preparation method thereof according to claim 2, characterized in that: In step 3, CS-HA hydrogel is prepared by physical cross-linking at room temperature. The obtained sample needs to be subjected to SEM identification in step 4 to determine the three-dimensional porous structure of CS-HA hydrogel, thereby determining that it can be used as a carrier for drug loading.

5. The chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and the preparation method thereof according to claim 2, characterized in that: In step 5, the preparation of UCMSCs-exo@CS-HAhydrogel is obtained by cross-linking the prepared CS-HAhydrogel and the prepared UCMSCs-exo at room temperature.

6. The chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and the preparation method thereof according to claim 2, characterized in that: The concentration of exosomes was determined by the BCA method and the concentration was 0.2 mg / mL.

7. A chitosan-hyaluronic acid hydrogel loaded with umbilical cord mesenchymal stem cell-derived exosomes and a preparation method thereof, characterized in that: The method is prepared by any one of claims 1 to 6.

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