Dressing matrix for nursing radioactive skin damage, preparation method and application thereof, and dressing for nursing radioactive skin damage

Abstract

The present invention belongs to the field of biomedicine technology, and is specifically related to a dressing matrix for nursing radioactive skin damage and its preparation method and application, and a dressing for nursing radioactive skin damage. The present invention is based on poloxamer and hyaluronic acid as raw materials. Hyaluronic acid has good biocompatibility and biodegradability, plays an important role in anti-inflammatory, cell proliferation and wound repair, and poloxamer belongs to a thermosensitive nonionic triblock copolymer. Hydrophilic polyoxyethylene segments prevent hyaluronic acid from degrading, and its hydrophobic polyoxypropylene segments can also be used to encapsulate poloxamer and medicinal ingredients. Poloxamer is mixed with hyaluronic acid to have good gel properties, sustained release capacity, and thermosensitive characteristics are applicable to skin wounds. Its water retention and antibacterial properties can also promote wound recovery, improve the effect of medicinal ingredients against radiation skin damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a dressing matrix for nursing radioactive skin damage, a preparation method and application thereof, and a dressing for nursing radioactive skin damage. Background Art

[0002] Radiotherapy is the treatment of malignant tumors and some benign diseases using one or more types of ionizing radiation. The most commonly used direct ionizing particles are electrons, while the most commonly used indirect ionizing particles are photons. The energy range of the X-rays used is 1-25 MV. Currently, radiotherapy technology is becoming increasingly mature and has become an important part of treatment for 40% of patients with curable malignant tumors.

[0003] However, despite breakthroughs in the accuracy and standardization of radiotherapy, complications caused by radiotherapy remain a major challenge in clinical treatment and care. Numerous studies have shown that patients receiving radiotherapy may experience local reactions and systemic symptoms. Of particular note is that nearly 85% to 95% of cancer patients experience varying degrees of radiation-induced skin injury (RSI), which not only increases the risk of infection and seriously affects the quality of life of patients, but also brings tremendous mental stress and financial burden to patients, and even affects treatment and interrupts the progress of radiotherapy.

[0004] In recent years, clinical guidelines have advocated the use of moist healing dressings for the prevention and treatment of radiation-induced skin injuries. However, scientifically validated research evidence for moist dressings specifically targeting this clinical condition is lacking. Selecting appropriate materials, combined with exosomes, for treatment based on the wound characteristics of radiation-induced skin injuries will become an effective treatment and care option in the future. Radiation reduces the expression of various growth factors and their receptors in damaged tissue, leading to prolonged healing of the injured area. Traditional wound care dressings have limited healing potential and can damage new tissue during removal, causing unnecessary bleeding. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies of the existing technology, provide a dressing matrix for the care of radiation skin damage, a preparation method and application thereof, and a dressing for the care of radiation skin damage, so as to reduce the incidence of radiation skin damage, alleviate the pain of patients, improve the quality of care for radiation skin damage, and increase social benefits and economic benefits.

[0006] In order to achieve the above object, the present invention provides a dressing matrix for nursing radiation skin damage, comprising poloxamer and hyaluronic acid;

[0007] The mass ratio of the poloxamer to the hyaluronic acid is 2:8.

[0008] Preferably, the molecular weight of the hyaluronic acid is 1w~150wDa;

[0009] The poloxamer includes poloxamer 407.

[0010] Preferably, the porosity of the dressing matrix is ​​72.3% and the pore size is 50-90 μm.

[0011] The present invention also provides a method for preparing the dressing matrix for treating radiation-induced skin damage described in the above technical solution, comprising the following steps:

[0012] performing a first mixing of poloxamer and water to obtain a first product;

[0013] The first product and hyaluronic acid are mixed for a second time to obtain the dressing matrix for nursing radiation-induced skin damage.

[0014] Preferably, the temperature of the first mixing is 4°C and the time is 12h;

[0015] The mass volume ratio of the poloxamer to water is 1 g:4 mL.

[0016] The present invention also provides the use of the dressing matrix for nursing radiation skin damage described in the above technical solution in the preparation of products for nursing radiation skin damage.

[0017] The present invention also provides a dressing for nursing radiation-induced skin damage, comprising a dressing matrix and an active ingredient;

[0018] The dressing matrix is ​​the dressing matrix for nursing radiation skin damage described in the above technical solution;

[0019] The active ingredients include human umbilical cord mesenchymal stem cell exosomes.

[0020] Preferably, the dressing matrix and active ingredient are packaged independently.

[0021] Preferably, the mass ratio of the poloxamer to the active ingredient is 2×10 6 :1.

[0022] Preferably, the method for preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0023] performing a first centrifugation on the culture medium of human umbilical cord mesenchymal stem cells to obtain a first supernatant;

[0024] performing a second centrifugation on the first supernatant to obtain a second supernatant;

[0025] performing a third centrifugation on the second supernatant to obtain a third supernatant;

[0026] performing a fourth centrifugation on the third supernatant to obtain a precipitate; the precipitate is human umbilical cord mesenchymal stem cell exosomes;

[0027] The first centrifugation was performed at a speed of 500 g and a time of 5 min;

[0028] The second centrifugation was performed at a speed of 2000 g and for 30 min;

[0029] The third centrifugation was performed at a speed of 10,000 g for 60 min.

[0030] The fourth centrifugation was performed at a speed of 120,000 g and for 70 min.

[0031] Beneficial effects:

[0032] The present invention provides a dressing matrix for the care of radiation-induced skin injuries, comprising poloxamer and hyaluronic acid; the mass ratio of the poloxamer to the hyaluronic acid is 2:8. Poloxamer is composed of a hydrophobic polyoxypropylene (PPO) chain with hydrophilic polyoxyethylene (PEO) attached to both ends. It is a thermosensitive nonionic triblock copolymer approved by the U.S. Food and Drug Administration (FDA) as a food additive and pharmaceutical raw material. Hyaluronic acid is a natural macromolecular glycosaminoglycan with excellent biocompatibility and biodegradability, playing an important role in anti-inflammatory, cell proliferation, and wound repair. The present invention uses poloxamer and hyaluronic acid as raw materials. The hydrophilic polyoxyethylene segments of the poloxamer prevent hyaluronic acid degradation, while the hydrophobic polyoxypropylene segments can also be used to entrap the poloxamer and medicinal ingredients. Furthermore, the mixture of poloxamer and hyaluronic acid exhibits excellent gelling properties and sustained release capacity, and its thermosensitive properties are suitable for skin wounds. Its water-retention and antibacterial properties can also promote wound recovery and enhance the effectiveness of medicinal ingredients in combating radiation-induced skin injuries.

[0033] The present invention combines the above-mentioned dressing matrix with human umbilical cord mesenchymal stem cell exosomes to prepare a dressing for the care of radiation skin damage. The human umbilical cord mesenchymal stem cell exosomes can enhance the proliferation and migration of endothelial cells and the formation of angiogenic tubes, reduce scar formation, and are convenient to store. Using human umbilical cord mesenchymal stem cell exosomes as the active ingredient can greatly reduce the risks associated with cell application and effectively resist radiation skin damage, helping to reduce the incidence of radiation skin damage, alleviate patient pain, ensure the integrity of patient treatment, and improve patient survival rate and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0035] Figure 1 Morphological image of human umbilical cord mesenchymal stem cells (100×), scale bar: 50 μm;

[0036] Figure 2 This is the result of flow cytometry immunophenotyping of human umbilical cord mesenchymal stem cells;

[0037] Figure 3 The state of human umbilical cord mesenchymal stem cell exosomes obtained by centrifugation in the tube;

[0038] Figure 4 The morphology of human umbilical cord mesenchymal stem cell exosomes under scanning electron microscopy. The accompanying photos show the electron microscopy results under different fields of view.

[0039] Figure 5-1 and Figure 5-2 The results of the human umbilical cord mesenchymal stem cell exosome concentration and particle size (NTA) test;

[0040] Figure 6 This is the quantitative detection result of BCA protein in human umbilical cord mesenchymal stem cell exosomes;

[0041] Figure 7 Western Blot detection of human umbilical cord mesenchymal stem cell exosome-specific protein expression results;

[0042] Figure 8 is the phase transition temperature of the dressing matrix with different molecular weights;

[0043] Figure 9 is the phase transition time of dressing matrices with different molecular weights; A is a picture of the dressing matrix changing from gel state to liquid state; B is the measured phase transition time;

[0044] Figure 10 The changes of the moisturizing rate of dressing matrices with different molecular weights over time;

[0045] Figure 11 is the swelling rate of dressing matrices with different molecular weights;

[0046] Figure 12 is the degradation rate of dressing matrices with different molecular weights;

[0047] Figure 13 The microstructure of the dressing matrix obtained in Example 2; A is the scanning electron microscopy result, and B is the cryo-electron microscopy result;

[0048] Figures 14-1 to 8 The results of cryo-electron microscopy analysis of points, lines, and surfaces of the dressing matrix obtained in Example 2 are shown.

[0049] Figure 15The cytotoxicity evaluation results of the dressing matrix obtained in Example 2 on HaCaT and HUVEC;

[0050] Figure 16 Effect of the dressing matrix obtained in Example 2 on hemolysis (**: compared with the Triton X 100 group, p < 0.01);

[0051] Figure 17 The effect of the dressing matrix obtained in Example 2 on skin performance;

[0052] Figure 18 The effect of the dressing matrix obtained in Example 2 on blood cell count;

[0053] Figure 19 The effect of the dressing matrix obtained in Example 2 on the histological morphology of organs;

[0054] Figure 20 The effect of the dressing matrix obtained in Example 2 on the skin histological morphology;

[0055] Figure 21 This is the appearance and structure of the nursing dressing obtained in Example 6;

[0056] Figure 22 The body weight changes of mice in each group;

[0057] Figure 23 The wound conditions of mice; A is a photo of wound changes, and B is the proportion of residual wound area;

[0058] Figure 24 is the content of inflammatory factors in mouse serum; A is the content of TNF-α, B is the content of IL-1β, and C is the content of IL-6. *: compared with the control group, p < 0.05; **: compared with the control group, p < 0.01; #: compared with the exosome group, p < 0.05;

[0059] Figure 25 The expression of CD31 in the skin tissue of mice in each group; **: compared with the control group, p < 0.01;

[0060] Figure 26 The expression of IL-1β in the skin tissue of mice in each group; **: compared with the control group, p < 0.01;

[0061] Figure 27 The expression of IL-6 in the skin tissue of mice in each group; **: compared with the control group, p < 0.01;

[0062] Figure 28 The expression of TNF-α in the skin tissue of mice in each group; **: compared with the control group, p < 0.01. DETAILED DESCRIPTION

[0063] The present invention provides a dressing matrix for nursing radiation-induced skin damage, comprising poloxamer and hyaluronic acid, wherein the mass ratio of the poloxamer to the hyaluronic acid is 2:8.

[0064] In the present invention, the porosity of the dressing matrix is ​​preferably 72.3%; the pore size of the dressing matrix is ​​preferably 50-90 μm, more preferably 60-80 μm, more preferably 65-75 μm, and most preferably 70 μm. The pore structure of the dressing matrix network is uniform and dense, with a narrow distribution, which can effectively load the active ingredient.

[0065] In the present invention, the molecular weight of the hyaluronic acid is preferably 1w-150w Da, more preferably 10w-80w Da or 130w-150w Da, more preferably 10w-40w Da, and most preferably 10w-20w Da. Hyaluronic acid is a natural macromolecular glycosaminoglycan with excellent biocompatibility and biodegradability, and plays an important role in anti-inflammation, cell proliferation, and wound repair.

[0066] In the present invention, the poloxamer preferably includes poloxamer 407. Poloxamer 407 is a thermosensitive nonionic triblock copolymer composed of a hydrophobic polyoxypropylene (PPO) chain with hydrophilic polyoxyethylene (PEO) attached to both ends. It has been approved by the U.S. Food and Drug Administration (FDA) as a food additive and pharmaceutical raw material.

[0067] In the present invention, the mass ratio of poloxamer to hyaluronic acid is 2:8. By limiting the mass ratio of poloxamer to hyaluronic acid, the present invention can fully dissolve the two substances and form a stable hydrogel, which is conducive to the subsequent loading of mesenchymal stem cell exosomes.

[0068] The hydrophilic polyoxyethylene segments of Poloxamer 407 described in this invention prevent hyaluronic acid degradation, while the hydrophobic polyoxypropylene segments encapsulate the poloxamer and active ingredients. The dressing matrix, derived from poloxamer and hyaluronic acid, has been tested for biosafety in the laboratory and demonstrated non-cytotoxicity to both HaCaT and HUVEC cells, excellent biocompatibility, and no significant hemolysis, providing a suitable microenvironment for radiation-induced skin damage.

[0069] The present invention also provides a method for preparing the dressing matrix for treating radiation-induced skin damage described in the above technical solution, comprising the following steps:

[0070] performing a first mixing of poloxamer and water to obtain a first product;

[0071] The first product and hyaluronic acid are mixed for a second time to obtain the dressing matrix for nursing radiation-induced skin damage.

[0072] The present invention involves a first mixing of poloxamer and water to obtain a first product. In the present invention, the temperature for the first mixing is preferably 4°C; the time for the first mixing is preferably 12 hours. The mass-to-volume ratio of poloxamer to water is preferably 1 g:4 mL. The first mixing method is preferably stirring. The stirring speed is not strictly required and can be selected at a conventional speed. Preliminary mixing of the poloxamer and water can further maximize the excellent properties of both.

[0073] After obtaining the first product, the present invention performs a second mixing of the first product and hyaluronic acid to obtain the dressing matrix for the care of radiation-induced skin damage. In the present invention, the temperature of the second mixing is preferably 4°C.

[0074] The preparation method of the present invention is simple and time-saving. The obtained dressing matrix can improve the effect of the active ingredients in reducing the incidence of radiation skin damage, relieve patients' pain, ensure the integrity of patients' treatment, and improve patients' survival rate and quality of life; it is beneficial to improving the nursing quality of radiation skin damage, reducing nursing risks and professional pressure; it helps to promote the development of medical materials towards high technology and industrialization, and brings certain social benefits and economic returns.

[0075] The present invention also provides the use of the dressing matrix for nursing radiation skin damage described in the above technical solution in the preparation of products for nursing radiation skin damage.

[0076] The present invention also provides a dressing for nursing radiation-induced skin damage, comprising a dressing matrix and an active ingredient;

[0077] The dressing matrix is ​​the dressing matrix for nursing radiation skin damage described in the above technical solution;

[0078] The active ingredients include human umbilical cord mesenchymal stem cell exosomes.

[0079] In the present invention, the dressing matrix and active ingredient in the radiation-induced skin injury care dressing are preferably packaged separately. Separately packaging the dressing matrix and active ingredient and then mixing them during use can prevent the dressing matrix from affecting the activity and efficacy of the active ingredient, further enhancing the active ingredient's effectiveness against radiation-induced skin injury.

[0080] In the present invention, the mass ratio of the poloxamer to the active ingredient is preferably 2×10 6 : 1. The mass ratio of poloxamer to active ingredient is limited in the present invention so that the two substances can be fully dissolved and form a hydrogel with stable properties, which is conducive to loading mesenchymal stem cell exosomes.

[0081] In the present invention, the active ingredient includes human umbilical cord mesenchymal stem cell exosomes. The method for preparing human umbilical cord mesenchymal stem cell exosomes described herein preferably comprises the following steps: performing a first centrifugation on a culture medium of human umbilical cord mesenchymal stem cells to obtain a first supernatant; performing a second centrifugation on the first supernatant to obtain a second supernatant; performing a third centrifugation on the second supernatant to obtain a third supernatant; and performing a fourth centrifugation on the third supernatant to obtain a precipitate; the precipitate is the human umbilical cord mesenchymal stem cell exosomes. The speed of the first centrifugation is preferably 500g, and the time is preferably 5 minutes. The speed of the second centrifugation is preferably 2000g, and the time is preferably 30 minutes. The speed of the third centrifugation is preferably 10,000g, and the time is preferably 60 minutes. The speed of the fourth centrifugation is preferably 120,000g, and the time is preferably 70 minutes. The human umbilical cord mesenchymal stem cell exosomes of the present invention are convenient to store, can reduce the risks associated with cell application, and can enhance the proliferation and migration of endothelial cells and the formation of angiogenic tubes, reduce scar formation, and have an anti-radiation skin damage effect.

[0082] The present invention mixes human umbilical cord mesenchymal stem cell exosomes with the above-mentioned dressing matrix, which has excellent physical and chemical properties and a good ability to promote skin regeneration. It can provide a suitable microenvironment for radiation-induced skin damage. At the same time, the animal model of radiation-induced skin damage shows that the dressing provided by the present invention has no toxic reaction to mice, can reduce the expression of TNF-α, IL-1β and IL-6 proteins, and alleviate inflammatory reactions. Compared with the use of human umbilical cord mesenchymal stem cell exosomes alone, it has a more obvious anti-radiation skin damage effect.

[0083] The present invention has no strict requirements on the preparation method of the dressing for nursing radiation skin damage, and the dressing matrix and the active ingredient can be mixed.

[0084] To further illustrate the present invention, the following detailed description of the radiation-induced skin injury care dressing matrix, its preparation method and application, and the radiation-induced skin injury care dressing provided by the present invention is provided in conjunction with the accompanying drawings and examples. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0085] Example 1

[0086] Preparation and identification of exosomes from human umbilical cord mesenchymal stem cells

[0087] (1) Isolation and culture of human umbilical cord mesenchymal stem cells

[0088] After obtaining permission from the ethics committee and informed consent from the mother, fresh umbilical cords from normal and healthy fetuses were obtained under sterile conditions. The fresh umbilical cords were placed in a culture dish, and sterile ophthalmic forceps and ophthalmic scissors were used to remove the amniotic membrane and blood vessels from the umbilical cords, and the cords were cut into pieces of approximately 1 mm in size. 3 Place the tissue blocks into a sterile culture dish and allow the tissue blocks to cover 70% of the bottom area. Add serum-free culture medium into the culture dish, shake left and right for 1 minute to evenly disperse the tissue blocks, and place in a 5% CO2 incubator and culture at 37°C. On the 5th day, replace half of the culture medium and continue culturing. On the 15th day, the cells grow to about 50% of the culture dish. Pour out the tissue blocks and collect the cells for subculture (recorded as P1 generation). Thereafter, replace the culture medium every 3 days, and observe the growth status of the stem cells under an inverted microscope. When the cells grow to 80% to 90% of the culture dish by attaching to the wall, add an appropriate amount of PBS buffer to wash twice, digest with protein trypsin for 1 to 2 minutes, and observe the changes in cell morphology under a microscope. When the cells begin to shrink and the cells are no longer connected, stop digestion with serum-containing culture medium, collect the cell suspension, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add human mesenchymal cell serum-free culture medium to the precipitate and resuspend it. Inoculate and culture for 2 to 3 days, and observe its morphology under a microscope. The results are as follows Figure 1 shown.

[0089] according to Figure 1 It can be seen that human umbilical cord mesenchymal stem cells exhibit a typical spiral adherent growth morphology, are slender and spindle-shaped, and are uniform in size and shape.

[0090] (2) Identification of human umbilical cord mesenchymal stem cells

[0091] The second generation (i.e., P2) human umbilical cord mesenchymal stem cells isolated and cultured in step (1) were taken to prepare a cell suspension. After counting the cells, 1×10 5 The cells were divided into test tubes. Antibodies to CD29, CD34, CD44, CD45, CD73, CD90, CD105, and HLA-DR were added and incubated at 2-8°C for 30 minutes. The cells were washed twice with PBS buffer, each time for 5 minutes. The immunophenotype was detected by flow cytometry. The results were as follows: Figure 2 shown.

[0092] according to Figure 2 It can be seen that CD29, CD44, CD73, CD90 and CD105 are highly expressed in isolated and cultured human umbilical cord mesenchymal stem cells, with a positive rate (the main peak is between 10 and 10 4 Meanwhile, CD34, CD45 and HLA-DR were all lowly expressed, with a negative rate of less than 1%.

[0093] (3) Extraction of exosomes from human umbilical cord mesenchymal stem cells

[0094] When the cell density of the third-generation human umbilical cord mesenchymal stem cells in the culture dish reaches 70% to 80%, the cell culture supernatant is removed, centrifuged at 500×g for 5 minutes, the supernatant is collected and transferred to a new 50 mL centrifuge tube, centrifuged at 2000×g for 30 minutes, the supernatant is collected and transferred to a new 50 mL centrifuge tube, centrifuged at 10000×g for 60 minutes, the supernatant is collected, filtered with a 0.22 μm sterile filter, added to an ultra-high-speed centrifuge tube, centrifuged at 120000×g for 70 minutes at 4°C, and the supernatant is carefully removed after centrifugation to obtain human umbilical cord mesenchymal stem cell exosomes, as shown in FIG. Figure 3 The exosomes were resuspended in sterile PBS to obtain a solution containing human umbilical cord mesenchymal stem cell exosomes.

[0095] (4) Transmission electron microscopy observation of exosome morphology

[0096] After repeatedly and gently blowing the solution containing human umbilical cord mesenchymal stem cell exosomes prepared in step (3), 20 μL was added dropwise to the copper mesh for natural adsorption for 5 to 10 minutes. Excess droplets were removed with a filter paper strip, and after drying slightly, 20 μL of 2% phosphotungstic acid solution was added dropwise to the copper mesh. The solution was allowed to stand for 3 to 5 minutes, and excess droplets were removed with a filter paper strip. The solution was dried under an incandescent lamp and observed and photographed under a transmission electron microscope. The results are as follows: Figure 4 shown.

[0097] according to Figure 4 It can be seen that the obtained exosomes are uniform in size and are complete membrane vesicles with round or oval shapes.

[0098] (5) Exosome concentration and particle size (NAT) test

[0099] Use a particle size detector to measure the particle size of human umbilical cord mesenchymal stem cell exosomes in the solution containing human umbilical cord mesenchymal stem cell exosomes prepared in step (3). Specifically: dilute the exosomes to be tested with sterile PBS, slowly inject 1 mL of the sample to be tested into the sample pool, and put the laser module back into the base. At the same time, put the thermometer probe into the copper hole of the laser module panel. After adjusting the focal length, select the appropriate Screen Gain and Camera Level. Select the required measurement method in the SOP, adjust the various measurement parameters, and complete the test according to the on-screen prompts. The results are as follows: Figure 5-1 and Figure 5-2 shown.

[0100] according to Figure 4 It can be seen that the main peak of the particle size of human umbilical cord mesenchymal stem cell exosomes is 131.9nm ( Figure 5-1 ), ranging from 55 to 159 nm ( Figure 5-2 In B), the original concentration was 8.34×10 8particles / mL (number of particles per milliliter). All particles are in irregular Brownian motion ( Figure 5-2 Middle C). The results showed that the extracted exosomes were within the relevant range.

[0101] (6) Quantitative determination of exosomes

[0102] Dissolve 25 mg of BSA in 1 mL of protein standard solution to obtain a 25 mg / mL protein standard solution, which is further diluted to 0.5 mg / mL to obtain the standard. Add the standard to a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 μL per well, respectively. Add protein standard solution to each well to make up to 20 μL. Dilute the exosome protein sample to be tested within the standard curve range according to the kit instructions and add 20 μL per well to a 96-well plate. Set up three replicates for each sample.

[0103] Prepare BCA reagent and copper sulfate solution at a volume ratio of 50:1. After thorough mixing, add 200 μL to each well and incubate at 37°C for 30 minutes. Measure the absorbance of each well at a wavelength of 562 nm. Draw a standard curve based on the measured values ​​and calculate the protein concentration. The results are shown in Table 1 and Figure 6 shown.

[0104] Table 1 Absorbance measurement results and standard curves of samples in each treatment group

[0105]

[0106]

[0107] According to Table 1 and Figure 6 The standard curve can be used to calculate the protein concentration in the test sample (i.e., exosomes) to be 2.38 μg / μL, indicating that the exosomes can meet subsequent needs.

[0108] (7) Western Blot verification of exosomes

[0109] The solution containing human umbilical cord mesenchymal stem cell exosomes prepared in step (3) was placed in protein lysis buffer, allowed to stand for 20 minutes, and centrifuged at 12000 rpm for 30 minutes. The protein marker and the prepared sample to be tested (i.e., the supernatant after centrifugation) were added to the grooves of the precast gel respectively, and the electrophoresis instrument was set to 80V for 30 minutes and 120V for 40 minutes to 60 minutes. The electrophoresis was terminated when the bromophenol blue dropped to the bottom edge of the glass plate. The gel was carefully removed and the protein marker was used as the basis for cutting the gel according to the protein molecular weight of the sample measured in step (6). The cut nitrocellulose membrane was soaked in methanol for 30 seconds to 60 seconds, and then the sponge, filter paper, gel, and nitrocellulose membrane were clamped and placed in the electrophoresis tank. The constant current was set to 260 to 300 mA and the membrane was transferred for 1 to 1.5 hours. During the transfer process, an ice bag was placed to cool down and keep the gel moist. After the transfer was completed, the nitrocellulose membrane was washed in TBST for 2 minutes, transferred to the blocking solution and blocked on a shaker for 2 hours. Wash the nitrocellulose membrane 3 times with TBST, 5 minutes each time, add the prepared primary antibody solution, including CD9 (1:1000), CD63 (1:1000), CD81 (1:1000), and incubate overnight at 4°C on a shaker. The next day, wash the nitrocellulose membrane 3 times and place it in the secondary antibody solution (penicillin-streptomycin mixture), incubate at room temperature for 60 minutes, and wash the membrane 3 times with TBST, 5 minutes each time. Place the washed nitrocellulose membrane in the darkroom of the developer, add the developer, and collect the exposure picture. The results are as follows: Figure 7 shown.

[0110] according to Figure 7 It can be seen that CD9, CD63 and CD81 are all positive in the exosomes of human umbilical cord mesenchymal stem cells.

[0111] Example 2

[0112] Preparation of dressing matrix

[0113] (1) Stir 2 g of Poloxamer 407 with 8 mL of deionized water at 4 °C for 12 h to obtain a colorless, transparent liquid.

[0114] (2) using 10w-20w Da of hyaluronic acid and mixing it with the colorless transparent liquid in step (1) to obtain a dressing matrix with good gel properties;

[0115] Example 3

[0116] Same as Example 2, the only difference is that 10w-20w Da hyaluronic acid is replaced by 1w Da hyaluronic acid.

[0117] Example 4

[0118] Same as Example 2, the only difference is that 10w-20w Da hyaluronic acid is replaced with 20w-40w Da hyaluronic acid.

[0119] Example 5

[0120] Same as Example 2, the only difference is that 10w-20w Da hyaluronic acid is replaced with 40w-80w Da hyaluronic acid.

[0121] Example 6

[0122] Same as Example 2, the only difference is that 10w-20w Da hyaluronic acid is replaced with 130w-150w Da hyaluronic acid.

[0123] Test Example 1

[0124] (1) Detection of phase transition temperature and phase transition time of dressing matrices with different molecular weights

[0125] The dressing matrices obtained in Examples 2 to 6 were used as samples, and the inversion method was used to detect the phase change temperature and phase change time of different dressing matrices. Specifically: the dressing matrices obtained in Examples 2 to 6 were placed at 4°C overnight to allow the poloxamer 407 and sodium hyaluronate in the dressing matrices to be further fully dissolved and mixed. The next day, each sample was placed in an EP tube, and the constant temperature water bath was gradually heated from 10°C, and the water temperature was monitored in real time using a thermometer. Every time the temperature in the water bath increased by 0.5°C, the state of the hydrogel dressing matrix was observed. When the solution did not flow after the EP tube was inverted for 30 seconds, it could be considered that it had completely become a gel, and the temperature at this moment was recorded as the phase change temperature. The experiment was repeated 3 times under the same conditions and the average value was taken. In order to better simulate the phase change of the dressing matrix in the human body, the time it takes for the hydrogel to change from a liquid state to a gel state at a temperature of 37°C was also measured. The hydrogel morphology was observed in real time during the measurement process, and the time required when the solution was completely gelled was recorded, which was the phase change time. The experiment was repeated 3 times under the same conditions and the average value was taken. The results are shown in Tables 2 and 3. Figures 8-9 shown.

[0126] Table 2 Phase transition temperature and phase transition time of different dressing matrices

[0127]

[0128] According to Table 2 and Figures 8-9As can be seen, the phase transition temperature of the dressing matrix ranges from 23.5°C to 25.3°C, decreasing with increasing hyaluronic acid molecular weight. At 37°C, the dressing matrix transitions from a gel to a liquid state, with a measured phase transition time ranging from 57.75s to 63.75s. This phase transition time decreases with increasing hyaluronic acid molecular weight. Considering the physicochemical properties, wound characteristics, and the anti-inflammatory properties of hyaluronic acid, the dressing matrix obtained in Example 2 is more likely to remain on the skin surface.

[0129] (2) Water retention performance test of dressing matrices with different molecular weights

[0130] Good moisturizing properties can maintain a moist microenvironment at the wound site for a longer period of time, and contribute to the formation of wound epithelialization. In order to clarify the water retention performance of the dressing matrix at 37°C, the dressing matrix obtained in Examples 2 to 6 was used as a sample, and a columnar hydrogel sample with a diameter of 12 mm and a thickness of 8 mm was prepared according to the mold, and placed in a 24-well plate and shaken in a constant temperature incubator at 37°C. The weight of the hydrogel was weighed at 0, 2, 4, 6, 8, 10, 12, 24, and 48 h, and the original mass of the dressing matrix was recorded as M0 (g), and the mass at different times was recorded as Mt (g). At the same time, 3 repetitions were set up, 4 parallel experiments were carried out, and the average value was taken. The water retention performance (Waterholding ratio) of the dressing matrix was calculated according to the following formula, and the results are shown in Table 4 and Figure 10 shown.

[0131]

[0132] Table 4 Moisturizing rate of different dressing matrices (%)

[0133] time A1 (Example 3) A2 (Example 2) A3 (Example 4) A4 (Example 5) A5 (Example 6) 0 100 100 100 100 100 2 65 70 75 76.5 78 4 45 49 54 57 60 6 30 38 42 47 51 8 28 32 38 42 46 10 27 30 32 36 40 12 25 27 29 32 36 24 23 25 27 28 30 48 20 21 23 24 23

[0134] According to Table 4 and Figure 10 As can be seen, the moisture retention rate of each group gradually decreased over time, and the lower the molecular weight of the hyaluronic acid, the lower the water retention rate of the dressing matrix formed. At 6 hours, the water retention rate of the dressing matrix formed by hyaluronic acid with a molecular weight of 1w Da was 30%. Within 4 hours, the water retention rate of the nursing dressing matrix of all molecular weights was 50% or above. Therefore, the water retention performance of the dressing matrix of each molecular weight is good and meets the dressing application standards.

[0135] (3) Swelling rate test of dressing matrices with different molecular weights

[0136] The good swelling property of the hydrogel is conducive to absorbing the exudate produced in the wound area and avoiding the growth of bacteria on the surface. The dressing matrix obtained in Examples 2 to 6 is used as a sample. A cylindrical hydrogel sample with a diameter of 12 mm and a thickness of 8 mm is prepared according to the mold. It is soaked in 37°C water and allowed to stand for 24 hours until the hydrogel reaches a swelling equilibrium state. The hydrogel is taken out from the solution, the surface moisture is gently wiped off with filter paper, and the weight of the hydrogel at this moment is measured with an electronic balance. The mass Ws (g) of the swollen hydrogel is measured after freeze-drying. Three repetitions are set up and the average value is taken. The swelling ratio (Swelling ratio) is calculated using the following formula, and the results are shown in Tables 5 and Figure 11 shown.

[0137]

[0138] Table 5 Swelling rate of different dressing matrices (%)

[0139]

[0140]

[0141] According to Table 5 and Figure 11 It can be seen that the dressing matrix with a hyaluronic acid molecular weight of 1w Da exhibited the highest swelling rate, approximately 650%, while the swelling rates of the other dressing matrices decreased significantly with increasing hyaluronic acid molecular weight. When the hyaluronic acid molecular weight was between 130w and 150w Da, the swelling rate of the dressing matrix reached 120%, and the swelling rates of the dressing matrices of various molecular weights were good.

[0142] (4) Degradation rate detection of dressing matrices with different molecular weights

[0143] The dressing matrix obtained in Examples 2 to 6 was used as a sample, and a cylindrical hydrogel sample with a diameter of 12 mm and a thickness of 8 mm was prepared according to the mold, and the weight at this time was recorded as W0. The prepared samples were placed in PBS and PBS containing 100 U / mL hyaluronidase, respectively, and incubated in a 37°C incubator. PBS and degradation solution were replaced regularly during the observation period. The weight of the dressing matrix was measured every day, and the initial sample weight was calculated as W0, and the weight after degradation was calculated as Wd. The degradation rate (Degradation) was calculated according to the following formula, and the results are shown in Tables 6 and Figure 12 shown.

[0144]

[0145] Table 6 Degradation rate of different dressing matrices (%)

[0146]

[0147]

[0148] According to Table 6 and Figure 12 It can be seen that when incubated at 37°C, the degradation rate of the dressing matrix is ​​inversely correlated with the molecular weight of the hyaluronic acid. Specifically, the higher the molecular weight of the hyaluronic acid, the slower the degradation of the dressing matrix. For a hyaluronic acid molecular weight of 1w Da, 100% degradation was achieved by day 8. A dressing matrix made with hyaluronic acid of 130w-150w Da did not fully degrade until day 12.

[0149] Test Example 2

[0150] (1) Scanning electron microscopy and cryo-electron microscopy were used to characterize the microscopic morphology of the dressing matrix. The results are as follows: Figure 13-14-1 ~14-8.

[0151] according to Figure 13 as well as Figures 14-1 to 14-8 It can be seen that the pore structure on the dressing matrix network is relatively uniform and dense, with a porosity of 72.3% and a pore size of 50-90 μm, which is narrowly distributed and can provide a good scaffold for loading related proteins or drugs; the main components of the dressing matrix are C and O elements ( Figure 14-1 ), with an average content of 66.70% and 33.13%, and also contains a small amount of Na and K elements ( Figure 14-2 ~5), cryo-electron microscopy line ( Figure 14-6 and Figure 14-7 ) and noodles ( Figure 14-8 The analysis in Figures E and F) also confirmed the above results, indicating that the dressing matrix obtained by the present invention has a stable structure and safe ingredients.

[0152] (2) Effect of dressing matrix on cell viability

[0153] The biocompatibility of the dressing matrix was tested using the CCK-8 assay. Specifically, cells were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum and 1% double-streptomycin (penicillin-streptomycin mixture) in a 5% CO2, 37°C incubator. Prior to the experiment, the dressing matrix obtained in Example 2 was purified by alternately soaking in PBS and 75% ethanol multiple times. The purified dressing matrix was then sterilized by immersing it in 75% ethanol for 12 hours to obtain a dressing matrix extract, which was then placed in sterile PBS for later use.

[0154] HUVEC cells and HaCaT cells were inoculated into 96-well plates, placed in a cell culture incubator and cultured for 24 hours, and a blank control group without cells was set up. After the cells adhered to the wall, the culture medium was discarded, and after washing twice with PBS, the cells were divided into a nursing dressing matrix (GEL) group and a control (Ctr) group. 200 μL of dressing matrix extract was added to the nursing dressing matrix group, and 200 μL of 2% fetal bovine serum high-glucose DMEM was added to the control group. Four replicates were set up for each treatment. The 96-well plate was placed in a cell culture incubator and incubated for 48 hours. 20 μL of CCK-8 solution was added to each well, and the cells were incubated in the cell culture incubator for another 1 hour. The absorbance (optical density, OD) value at 450 nm was detected with an enzyme reader, and the cell survival rate was calculated according to the following formula. The results are shown in Figure 2. Figure 15 shown.

[0155] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%, where

[0156] As: absorbance of the nursing dressing matrix (GEL) group;

[0157] Ac: absorbance of control (Ctr) group;

[0158] Ab: absorbance of blank control group.

[0159] according to Figure 15 It can be seen that compared with the control group, the cell survival rates of the nursing dressing matrix groups corresponding to the two cells were both 100%, indicating that the dressing matrix obtained by the present invention has no cytotoxicity to HaCaT and HUVEC cells and has good biocompatibility.

[0160] (3) Effect of dressing matrix on hemolysis

[0161] Experimental (GEL) group, positive control (TritonX-100) group, and negative control (Ctr) group were set up. Whole blood was collected from healthy 6-week-old BALB / c mice, centrifuged at 1000 rpm for 10 min, and then washed three times with PBS to obtain red blood cells, which were then diluted and suspended in PBS.

[0162] In the experimental group, 2 mL of the diluted red blood cell suspension and 0.5 mL of the dressing matrix extract obtained in step (2) were added to a 24-well cell culture plate; in the positive control group, 2 mL of the diluted red blood cell suspension and 1% Triton X-100 were added to a 24-well cell culture plate; in the negative control group, 2 mL of the diluted red blood cell suspension was added to a 24-well cell culture plate;

[0163] The experimental group, positive control group and negative control group were all incubated in a shaker at 37°C for 1 hour, centrifuged at 1000 rpm for 10 minutes, and the supernatant was added to a 96-well cell culture plate. Five parallel groups were set up and the absorbance at 540 nm was measured. The absorbance value of the experimental group was ODt, the absorbance value of the positive control group was ODp, and the absorbance value of the negative control group was ODn. The hemolysis ratio was calculated using the following formula. The results are shown in the figure. Figure 16 shown.

[0164]

[0165] according to Figure 16 It can be seen that the supernatant of the negative control group and the experimental group is clear and light yellow, while the supernatant of the positive control group is red ( Figure 16 Figure (A) shows that neither the negative control group nor the experimental group experienced significant hemolysis. The hemolysis rate in the negative control group was 0, the hemolysis rate in the experimental group was 0.82%, and the hemolysis rate in the positive control group was 100%, indicating a significant difference between the experimental and positive control groups (p < 0.01).

[0166] (4) Effects of dressing matrix on the skin and organ tissues of hemolytic mice

[0167] Twenty healthy 6-week-old BALB / c mice were randomly divided into experimental (GEL) group and blank control (Ctr) group, with half male and half female in each group. Before the skin toxicity test, the mice were depilated with a small animal shaver in the range of 1.5×1.5 cm. 2 . After 24 hours of hair removal, the dressing matrix obtained in Example 2 was evenly applied to the hair removal area on the back of the mice in the experimental group and covered with gauze or plastic wrap. The control group was smeared with PBS. After 24 hours, the residue on the back of the mice was gently wiped off. The changes in the skin, hair and limb activities of the mice were observed every day for 7 consecutive days. During the experiment, the mice had normal activities, no abnormal reactions in appetite and defecation, no obvious changes in skin and hair, and no deaths occurred. The skin of the mice in the experimental group did not show redness, swelling or ulceration at 0 hours, 48 ​​hours, 72 hours and 7 days after the dressing matrix was applied ( Figure 17 ).

[0168] After 7 days of observation, blood was collected from the mice for blood cell count; heart, liver, spleen, lung, kidney and skin tissues were collected and fixed with 10% formaldehyde for H&E staining. Figures 18-20 shown.

[0169] according to Figure 18 It can be seen that the number of white blood cells (WBC) in the blood of mice in the experimental group was 5.87±0.86×10 9 / L, and the control group was 5.4±0.72×10 9The number of red blood cells (RBC) in the blood of mice in the experimental group was 9.63±0.76×10 12 / L, and the control group was 9.68±1.36×10 12 / L; the amount of hemoglobin (HGB) in the blood of the experimental group mice was 153±12.49g / L, and that in the control group was 152.67±9.07g / L; the amount of platelets (PTL) in the blood of the experimental group mice was 8440±65.02×10 9 / L, and the control group was 827.33±109.21×10 9 There was no statistical difference in the counts of white blood cells (WBC), red blood cells (RBC), and

[0170] according to Figure 19 It can be seen that there was no significant difference in the histological morphology of the heart, liver, spleen, lung, and kidney of the mice in the control group and the experimental group. The dressing matrix provided by the present invention did not produce toxic reactions in the mice.

[0171] according to Figure 20 It can be seen that no abnormal changes occurred in the skin tissues of the mice in the control group and the experimental group, and the dressing matrix provided by the present invention did not cause skin toxicity to the mice.

[0172] Example 6

[0173] Preparation of nursing dressings

[0174] The human umbilical cord mesenchymal stem cell exosomes obtained in Example 1 were dissolved in PBS to obtain a 100 μg / mL exosome solution; 10 μL of the exosome solution was added to the dressing matrix obtained in Example 2 to obtain a nursing dressing. During the experiment, the dressing was prepared and used immediately. The appearance and structure of the obtained nursing dressing are as shown below. Figure 21 As shown. Figure 21 It can be seen that the nursing dressing exhibits a transparent texture.

[0175] Test Example 2

[0176] Experimental study on the effectiveness of radiation-induced skin damage in mice

[0177] 24 mice were selected and randomly divided into a control group (Ctr), a nursing dressing matrix group (GEL), an exosome group (EXO), and a nursing dressing group (GEL@EXO), with 6 mice in each group. One day before the experiment, the mice in each treatment group were weighed, anesthetized with an intraperitoneal injection of 1% sodium pentobarbital, and the hair on the back of the mice was shaved, and care was taken to protect their skin. On the day of the experiment, the same anesthesia method was used, and the mice were placed on the workbench of the linear accelerator, and the non-irradiated parts were shielded with lead plates. The 6MeV electron beam of the electron linear accelerator was used to irradiate the depilated area of ​​the mice, and the irradiation range was 1.5×1.5cm 2 The dose rate was 100 cGy / min, and the irradiation dose was 4 Gy. After irradiation, a full-thickness skin loss wound was created on the buttocks of the mice to establish a mouse radiation-complex skin injury model.

[0178] The control group was given 1 mL of PBS, the nursing dressing matrix group was given 1 mL of the dressing matrix obtained in Example 2, the exosome group was given 1 mL of human umbilical cord mesenchymal stem cell exosomes obtained in Example 1, and the nursing dressing group was given the nursing dressing obtained in Example 3. The intervention was continued for 3 days.

[0179] (1) The wound diameter, skin, hair, and limb activity were observed 0 to 14 days after the last intervention in each treatment group, the wound score was evaluated, and the mice were weighed. The results are shown in Tables 7 and 8. Figures 22-23 shown.

[0180] Table 7 Body weight changes of mice in each group

[0181] Time(day) Ctr GEL Exo GEL@Exo 0 21.33 21.23 21.3 21.3 3 19.37 19.77 20.5 20.13 5 18.13 18.8 19.2 19.27 7 17.63 18.33 19.67 19.9 14 17.93 18.73 19.93 21.03

[0182] Note: The data in the table are average values.

[0183] Table 8 Ratio of residual wound area in each group of mice

[0184] Time(day) Ctr GEL Exo GEL@Exo 0 100 100 100 100 3 85 70 65 50 7 60 50 45 25 14 45 39 30 15

[0185] According to Tables 7-8 and Figures 22-23 As can be seen, the weight of mice in the nursing dressing matrix group was slightly higher than that in the control group, and the change trend was essentially the same as that in the control group. The lowest weight in both the exosome and nursing dressing groups was on day 5, followed by a gradual increase. The weight of mice in the nursing dressing group was the highest among all groups at all time periods. All mice in this experiment survived the radiation skin injury model, but compared with normal mice, they showed lethargy, decreased appetite, and decreased activity. The majority of mice showed no significant changes in the skin on their buttocks and backs on the day of modeling. On day 3 after treatment, the irradiated skin of mice in all groups showed significant erythema and dryness, even scabs. Among the four groups of mice, the skin of mice in the nursing dressing group healed the fastest, followed by the exosome group. The wound healing rate of mice in the control group was the slowest.

[0186] (2) 14 days after the last intervention in each treatment group, the changes of inflammatory factors such as TNF-α, IL-1β, and IL-6 in the serum of mice were detected by enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 9 and Figure 24 shown.

[0187] Table 9 Contents of inflammatory factors in mouse serum

[0188] Time(day) Ctr GEL Exo GEL@Exo TNF-α 9.80 8.64 7.10 5.23 IL-1β 8.39 7.04 6.27 5.29 IL-6 8.19 7.06 5.93 4.99

[0189] According to Table 9 and Figure 24 As can be seen, compared with the control group, the expression levels of TNF-α in both the nursing dressing matrix group and the exosome group were significantly decreased. Compared with the exosome group, the expression level of TNF-α in the nursing dressing group was further reduced. The changing trends of IL-1β and IL-6 in the serum of mice in each group were similar to those of TNF-α. Thus, the nursing dressing provided by the present invention can effectively reduce the levels of TNF-α, IL-1β, and IL-6 in mice with radiation damage.

[0190] (3) 14 days after the last intervention in each treatment group, the expression of CD31, TNF-α, IL-1β, and IL-6 in the full-thickness skin tissue of mice was observed by immunohistochemical staining to further explore the mechanism by which the nursing dressing exerts a protective effect on radiation-induced skin damage. The results are shown in Tables 10 and Figures 25-28 shown.

[0191] Table 10 Contents of related factors in full-thickness skin tissue of mice

[0192] Time(day) Ctr GEL Exo GEL@Exo CD31 1 1.26 3.8 6.12 TNF-α 1 0.71 0.31 0.18 IL-1β 1 0.93 0.45 0.26 IL-6 1 0.89 0.31 0.21

[0193] According to Table 10 and Figures 25-28 It can be seen that compared with the control group, the angiogenesis marker protein CD31 ( Figure 25 ) were up-regulated, and the difference was statistically significant. Compared with the control group, TNF-α ( Figure 26 ), IL-1β( Figure 27 ) and IL-6 ( Figure 28 ) expression levels were significantly decreased, with statistically significant differences. In the recovery of radiation-induced skin damage in mice, both exosomes and the nursing dressing significantly upregulated CD31 protein expression, promoting angiogenesis, and reduced inflammatory responses by decreasing TNF-α, IL-1β, and IL-6 protein expression. The nursing dressing had a more pronounced anti-damage effect.

[0194] Based on the above content, it can be seen that the dressing matrix provided by the present invention can improve the effect of the active ingredients, effectively reduce the content of TNF-α, IL-1β and IL-6 caused by radiation damage, increase the expression of protein CD31, reduce the incidence of radiation skin damage, and improve the healing speed of radiation skin damage wounds.

[0195] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a dressing matrix for nursing radiation skin damage, characterized in that: The steps include: performing a first mixing of poloxamer and water to obtain a first product; performing a second mixing of the first product and hyaluronic acid to obtain the dressing matrix for nursing radiation-induced skin damage; The mass volume ratio of the poloxamer to water is 1 g:4 mL; The mass ratio of poloxamer to hyaluronic acid is 2:8; The molecular weight of the hyaluronic acid is 1w-150wDa.

2. The method for preparing a dressing matrix for nursing radiation skin damage according to claim 1, wherein: The poloxamer includes poloxamer 407.

3. The preparation method according to claim 1, characterized in that The porosity of the dressing matrix is ​​72.3%, and the pore diameter is 50-90 μm.

4. The preparation method according to claim 1, characterized in that The temperature of the first mixing is 4°C and the time is 12h; The mass volume ratio of the poloxamer to water is 1 g:4 mL.

5. Use of the dressing matrix for the care of radiation-induced skin damage obtained by the preparation method according to any one of claims 1 to 4 in the preparation of products for the care of radiation-induced skin damage.

6. A dressing for nursing radiation-induced skin damage, characterized in that: including a dressing base and active ingredients; The dressing matrix is ​​a dressing matrix for nursing radiation skin damage obtained by the preparation method according to any one of claims 1 to 4; The active ingredients include human umbilical cord mesenchymal stem cell exosomes.

7. The radiation-induced skin injury nursing dressing according to claim 6, characterized in that: The dressing matrix and active ingredients are packaged independently.

8. The radiation-induced skin injury nursing dressing according to claim 6, characterized in that: The mass ratio of the poloxamer to the active ingredient is 2×10 6 :

1.

9. The radiation-induced skin injury nursing dressing according to claim 6, characterized in that: The method for preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps: performing a first centrifugation on the culture medium of human umbilical cord mesenchymal stem cells to obtain a first supernatant; performing a second centrifugation on the first supernatant to obtain a second supernatant; performing a third centrifugation on the second supernatant to obtain a third supernatant; performing a fourth centrifugation on the third supernatant to obtain a precipitate; the precipitate is human umbilical cord mesenchymal stem cell exosomes; The first centrifugation was performed at a speed of 500 g and a time of 5 min; The second centrifugation was performed at a speed of 2000 g and for 30 min; The third centrifugation was performed at a speed of 10,000 g for 60 min. The fourth centrifugation was performed at a speed of 120,000 g and for 70 min.

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