A method for purifying mesenchymal stem cell exosomes, and mesenchymal stem cell exosome particles prepared therefrom and uses thereof

Abstract

The application provides a mesenchymal stem cell exosome purification method, a prepared mesenchymal stem cell exosome particle and an application thereof, and belongs to the technical field of exosomes. Fat mesenchymal stem cells are subjected to expansion culture to stimulate exosome secretion, then gradient centrifugation is performed, supernatant is collected, a protective agent is added, stirring and mixing are uniformly performed, a polymer is added, stirring and incubation are performed, centrifugation is performed, exosome particles are collected, washing is performed, freeze-drying is performed, and the mesenchymal stem cell exosome particles are prepared. The prepared mesenchymal stem cell exosome particles have the characteristics of simple preparation method, low cost, easy industrial application, high yield, good protection effect on exosomes, prolonged preservation period, and slow and controlled release of exosomes, and have wide application in the preparation of anti-inflammatory, antioxidant and skin repair promoting drugs.

Description

Technical Field

[0001] This invention relates to the field of exosome technology, specifically to a method for purifying mesenchymal stem cell exosomes, the resulting mesenchymal stem cell exosome particles, and their applications. Background Technology

[0002] Mesenchymal stem cells (MSCs) have shown great potential in the treatment of various diseases due to their immunomodulatory and anti-inflammatory properties. In recent years, studies have found that exosomes released by MSCs also possess anti-inflammatory effects and, due to their smaller size and lower immunogenicity, are more suitable as therapeutic carriers. However, the efficient isolation and purification of exosomes remains a key challenge for their clinical application.

[0003] Currently, there are various methods for preparing exosomes, including ultrafiltration, reagent-based precipitation, sucrose gradient centrifugation, and ultracentrifugation. Each method has its advantages and disadvantages. For example, ultracentrifugation removes impurities through multiple differential centrifugation steps to obtain high-purity exosomes; although this method yields the highest purity exosomes, making it suitable for high-quality research and clinical applications, its equipment is expensive, the operation is complex, and it is time-consuming. Density gradient centrifugation uses a gradient constructed with isodense substances like sucrose, and centrifugation causes exosomes to settle to a specific layer, thus separating the exosomes; although this method can effectively remove impurities and obtain high-purity exosomes, its operation is relatively cumbersome and requires precise control of the density gradient and centrifugation conditions.

[0004] Therefore, it is necessary to develop a method with better overall performance based on existing methods for the purification of mesenchymal stem cell exosomes. Summary of the Invention

[0005] The purpose of this invention is to provide a purification method for mesenchymal stem cell exosomes, the resulting mesenchymal stem cell exosome particles, and their applications. The preparation method is simple, low-cost, easy to implement for industrial application, and has a high yield. At the same time, it has a good protective effect on exosomes, extending their preservation period. The obtained mesenchymal stem cell exosome particles have the characteristics of sustained-release and controlled-release of exosomes, and have wide applications in the preparation of anti-inflammatory, antioxidant, and skin repair-promoting drugs.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for purifying mesenchymal stem cell exosomes. Adipose-derived mesenchymal stem cells are expanded and cultured to stimulate exosome secretion, followed by gradient centrifugation. The supernatant is collected, a protective agent is added, and the mixture is stirred and mixed evenly. A polymer is added, and the mixture is stirred and incubated. The mixture is then centrifuged again, and the exosome particles are collected, washed, and freeze-dried to obtain mesenchymal stem cell exosome particles.

[0008] As a further improvement to the present invention, the following steps are included:

[0009] S1. Add adipose-derived mesenchymal stem cells to the expansion medium, adjust the cell concentration, culture, centrifuge, and separate the cells and the culture medium;

[0010] S2. Perform gradient centrifugation on the culture medium separated in step S1 and collect the supernatant;

[0011] S3. Add a protective agent to the supernatant obtained in step S2, stir and mix evenly, add a polymer, stir and incubate, centrifuge, collect exosome particles, wash, freeze dry, and obtain mesenchymal stem cell exosome particles.

[0012] As a further improvement of the present invention, the step S1 of adjusting the cell concentration to 10 2 -10 3 The culture conditions are 36-38℃, 4-6 v / v% CO2, culture for 36-48 h, and centrifugation conditions are 250-350g centrifugation for 10-20 min.

[0013] As a further improvement of the present invention, the amplification medium in step S1 is a complete mTeSRTM1 stem cell culture medium with an oxygen content of 0.5-1 v / v%, a pH of 6.6-6.8, and containing 2-4 mmol / L CaCl2 and 10-15 ng / mL IL-1β.

[0014] As a further improvement of the present invention, the gradient centrifugation method in step S2 is to centrifuge at 250-350g for 10-20 min at 3-5℃, collect the supernatant, and then centrifuge at 1850-2150g for 15-25 min.

[0015] As a further improvement of the present invention, in step S3, the mass ratio of the supernatant, protective agent, and polymer obtained in step S2 is 100:0.1-0.2:3-5, the incubation conditions are 3-5℃ for 25-35 min, the centrifugation conditions are 2800-3200g for 10-20 min, and the protective agent is trehalose and sucrose in a mass ratio of 2-3:1-2.

[0016] As a further improvement of the present invention, the preparation method of the polymer in step S3 is as follows:

[0017] T1. Preparation of protamine-RGD peptide conjugate: Protamine was added to water, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, stirred and activated, RGD peptide was added, stirred and reacted, dialyzed, and freeze-dried to obtain protamine-RGD peptide conjugate.

[0018] Preparation of T2.PEG-COOH: Polyethylene glycol was dissolved in dimethyl sulfoxide, and alkaline solution and chloroacetic acid were added. The mixture was stirred and reacted. The solvent was removed under reduced pressure. The product was precipitated in cold diethyl ether, filtered, washed, and dried to obtain PEG-COOH.

[0019] T3. Preparation of the polymer: PEG-COOH was added to water, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the mixture was stirred and activated, protamine-RGD peptide conjugate was added, the mixture was stirred and reacted, dialyzed, and freeze-dried to obtain the polymer.

[0020] As a further improvement of the present invention, in step T1, the mass ratio of protamine, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and RGD peptide is 5-7:2-3:2-3:1-2, and the stirring reaction time is 5-7 hours; in step T2, the polyethylene glycol is polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 1000, and the mass ratio of polyethylene glycol to chloroacetic acid is 0.2-1:1.8- 2. The alkaline solution is a 30-50 wt% NaOH or KOH solution, the stirring reaction time is 8-12 h, and the temperature of the cold ether is 3-5 °C; in step T3, the mass ratio of PEG-COOH, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and protamine-RGD peptide conjugate is 1-1.5:0.1-0.15:0.12-0.17:2-3, and the stirring reaction time is 7-10 h.

[0021] The present invention further protects a mesenchymal stem cell exosome particle obtained by the above-described purification method.

[0022] This invention further protects the use of the above-mentioned mesenchymal stem cell exosome particles in the preparation of drugs with anti-inflammatory, antioxidant and skin repair-promoting properties.

[0023] This invention offers the following beneficial effects: By adding IL-1β to the complete culture medium of mTeSRTM1 stem cells, the secretion of exosomes significantly increases upon stimulation by the inflammatory factor IL-1β. This secretion-promoting effect may be achieved by activating inflammatory signaling pathways such as NF-κB. Furthermore, the hypoxic and low-pH culture environment acts as a synergistic stimulant, activating acid sphingomyelinase and promoting ceramide production in the lipid raft region of the cell membrane, thereby further enhancing the generation and release of exosomes at the biosynthetic level, and thus greatly increasing exosome yield.

[0024] This invention employs gradient centrifugation to first remove other macromolecular debris from the culture medium, retaining exosomes in the supernatant. Then, a polymer-induced precipitation method is used, where a highly hydrophilic polymer interacts with the water molecules surrounding the exosomes, creating a hydrophobic microenvironment that leads to exosome precipitation. Polyethylene glycol (PEG), a commonly used, is a good, non-toxic polymer with the ability to reshape the water solubility of surrounding materials. The process is simple, low-cost, and suitable for processing large-volume samples while improving yield. However, directly using PEG precipitation enriches not only exosomes but also other high-molecular impurities, lacking sample selectivity and resulting in low purity exosomes.

[0025] This invention prepares a polymer by coupling protamine with an RGD peptide, thereby obtaining a protein peptide structure capable of specifically recognizing exosomes. The RGD peptide (Arg-Gly-Asp) can target and bind to integrins (such as αvβ3 and α5β1) on the surface of exosomes. Integrins are common membrane proteins of mesenchymal stem cell exosomes and tumor exosomes. Therefore, the polymer with the RGD peptide can be used for the targeted capture of such exosomes. At the same time, protamine, a positively charged small molecule protein, can bind to the negatively charged surface of exosomes, improving the recovery rate and reducing lipoprotein contamination. The polymer obtained by further coupling with polyethylene glycol not only specifically recognizes exosomes but also reshapes the water solubility of surrounding materials, forming a hydrophobic microenvironment that leads to the targeted precipitation of exosomes.

[0026] Adding a protective agent before exosome precipitation can form a high-viscosity glassy state, inhibiting ice crystal growth, preventing membrane rupture, protecting exosomes from ice crystal damage, and improving antioxidant capacity. At the same time, the addition of sucrose can also reduce costs.

[0027] The mesenchymal stem cell exosome particles prepared by this invention are simple to prepare, low in cost, easy to implement in industrial applications, and have high yield. At the same time, they have a good protective effect on exosomes, extending their preservation period. The prepared mesenchymal stem cell exosome particles have the characteristics of sustained-release and controlled-release of exosomes, and have wide applications in the preparation of anti-inflammatory, antioxidant and skin repair-promoting drugs. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a TEM image of the mesenchymal stem cell exosome particles obtained in Example 1 of the present invention. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] RGD peptide, arginylglycyl aspartate peptide, Arg-Gly-Asp; PEG, polyethylene glycol; adipose-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, Wuhan Pronosei Life.

[0032] Preparation of Polymer in Example 1

[0033] The preparation method is as follows:

[0034] T1. Preparation of protamine-RGD peptide conjugate: 5g of protamine was added to 100mL of water, 2g of N-hydroxysuccinimide and 2g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the mixture was stirred and activated at 0℃ for 30min. 1g of RGD peptide was added, and the mixture was stirred and reacted for 5h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 24h and then freeze-dried to obtain the protamine-RGD peptide conjugate.

[0035] Preparation of T2.PEG-COOH: 0.2g of polyethylene glycol 200 was dissolved in 25mL of dimethyl sulfoxide, 10mL of 30wt% NaOH solution and 1.8g of chloroacetic acid were added, the mixture was stirred for 8h, the solvent was removed under reduced pressure, the product was precipitated in diethyl ether at 3℃ for 1h, filtered, washed and dried to obtain PEG-COOH;

[0036] T3. Preparation of polymer: 1g PEG-COOH was added to 50mL of water, along with 0.1g N-hydroxysuccinimide and 0.12g 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated at 0℃ for 20min. 2g protamine-RGD peptide conjugate was added, and the mixture was stirred and reacted for 7h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 12h and then freeze-dried to obtain the polymer.

[0037] Preparation Example 2 Polymer

[0038] The preparation method is as follows:

[0039] T1. Preparation of protamine-RGD peptide conjugate: 7g of protamine was added to 100mL of water, 3g of N-hydroxysuccinimide and 3g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the mixture was stirred and activated at 0℃ for 30min, 2g of RGD peptide was added, the mixture was stirred and reacted for 7h, dialyzed through a dialysis bag with a pore size of 3000Da for 24h, and then freeze-dried to obtain protamine-RGD peptide conjugate;

[0040] Preparation of T2.PEG-COOH: 1g of polyethylene glycol 1000 was dissolved in 25mL of dimethyl sulfoxide, 10mL of 50wt% KOH solution and 2g of chloroacetic acid were added, the mixture was stirred and reacted for 12h, the solvent was removed under reduced pressure, the product was precipitated in diethyl ether at 5℃ for 1h, filtered, washed and dried to obtain PEG-COOH;

[0041] T3. Preparation of polymer: 1.5g PEG-COOH was added to 50mL of water, followed by 0.15g N-hydroxysuccinimide and 0.17g 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated at 0℃ for 20min. Then, 3g protamine-RGD peptide conjugate was added and the mixture was stirred and reacted for 10h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 12h and then freeze-dried to obtain the polymer.

[0042] Preparation of polymer in Example 3

[0043] The preparation method is as follows:

[0044] T1. Preparation of protamine-RGD peptide conjugate: 6g of protamine was added to 100mL of water, along with 2.5g of N-hydroxysuccinimide and 2.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated at 0℃ for 30min. Then, 1.5g of RGD peptide was added and the mixture was stirred and reacted for 6h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 24h and then freeze-dried to obtain the protamine-RGD peptide conjugate.

[0045] Preparation of T2.PEG-COOH: 0.4g of polyethylene glycol 400 was dissolved in 25mL of dimethyl sulfoxide, 10mL of 40wt% NaOH solution and 1.9g of chloroacetic acid were added, the mixture was stirred and reacted for 10h, the solvent was removed under reduced pressure, the product was precipitated in diethyl ether at 4℃ for 1h, filtered, washed and dried to obtain PEG-COOH;

[0046] T3. Preparation of polymer: 1.3g PEG-COOH was added to 50mL of water, followed by 0.12g N-hydroxysuccinimide and 0.15g 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated at 0℃ for 20min. Then, 2.5g protamine-RGD peptide conjugate was added, and the mixture was stirred and reacted for 8.5h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 12h and then freeze-dried to obtain the polymer.

[0047] Comparative Preparation Example 1

[0048] The difference from Preparation Example 3 is that in step T3, the protamine-RGD peptide conjugate was replaced by an equal mass of protamine.

[0049] The preparation method is as follows:

[0050] Preparation of T1.PEG-COOH: 0.4g of polyethylene glycol 400 was dissolved in 25mL of dimethyl sulfoxide, 10mL of 40wt% NaOH solution and 1.9g of chloroacetic acid were added, the mixture was stirred and reacted for 10h, the solvent was removed under reduced pressure, the product was precipitated in diethyl ether at 4℃ for 1h, filtered, washed and dried to obtain PEG-COOH;

[0051] T2. Preparation of polymer: 1.3g PEG-COOH was added to 50mL of water, followed by 0.12g N-hydroxysuccinimide and 0.15g 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated at 0℃ for 20min. Then, 2.5g protamine was added and the mixture was stirred and reacted for 8.5h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 12h and then freeze-dried to obtain the polymer.

[0052] Comparative Preparation Example 2

[0053] The difference compared to Preparation Example 3 is that steps T2 and T3 were not performed.

[0054] The preparation method is as follows:

[0055] Preparation of protamine-RGD peptide conjugate: 6g of protamine was added to 100mL of water, along with 2.5g of N-hydroxysuccinimide and 2.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The mixture was stirred and activated at 0℃ for 30min. Then, 1.5g of RGD peptide was added, and the mixture was stirred and reacted for 6h. The mixture was dialyzed through a dialysis bag with a pore size of 3000Da for 24h and freeze-dried to obtain the protamine-RGD peptide conjugate, which is the polymer.

[0056] Example 1

[0057] This embodiment provides a method for purifying mesenchymal stem cell exosomes, including the following steps:

[0058] S1. Add adipose-derived mesenchymal stem cells to the expansion culture medium and adjust the cell concentration to 10. 2 -10 3 Cells / mL, 36℃, 4v / v%CO2, cultured for 36h, centrifuged at 250g for 10min to separate cells and culture medium;

[0059] The amplification medium was a complete mTeSRTM1 stem cell culture medium with an oxygen content of 0.5 v / v%, a pH of 6.6, and containing 2 mmol / L CaCl2 and 10 ng / mL IL-1β.

[0060] S2. Centrifuge the culture medium separated in step S1 at 3°C ​​and 250g for 10 min, collect the supernatant, and then centrifuge at 1850g for 15 min to collect the supernatant.

[0061] S3. Add 0.1g of protective agent to 100g of the supernatant obtained in step S2, stir and mix for 10min, add 3g of the polymer prepared in Preparation Example 1, incubate at 3°C ​​with stirring for 25min, centrifuge at 2800g for 10min, collect the exosome particles, wash with deionized water, freeze-dry, and obtain purified mesenchymal stem cell exosome particles. Figure 1 The image shows a TEM image of the obtained mesenchymal stem cell exosome particles. As can be seen from the image, the particle size is between 50-200 nm. The protective agent is trehalose and sucrose in a mass ratio of 2:1.

[0062] Example 2

[0063] This embodiment provides a method for purifying mesenchymal stem cell exosomes, including the following steps:

[0064] S1. Add adipose-derived mesenchymal stem cells to the expansion culture medium and adjust the cell concentration to 10. 2 -10 3 Cells / mL, 38℃, 6v / v%CO2, cultured for 48h, centrifuged at 350g for 20min to separate cells and culture medium;

[0065] The amplification medium was a complete mTeSRTM1 stem cell culture medium with an oxygen content of 1 v / v%, a pH of 6.8, and containing 4 mmol / L CaCl2 and 15 ng / mL IL-1β.

[0066] S2. Centrifuge the culture medium separated in step S1 at 350g for 20 min at 5℃, collect the supernatant, and then centrifuge at 2150g for 25 min to collect the supernatant.

[0067] S3. Add 0.2g of protective agent to 100g of the supernatant obtained in step S2, stir and mix for 10min, add 5g of the polymer prepared in Preparation Example 2, incubate at 5°C with stirring for 35min, centrifuge at 3200g for 20min, collect the exosome particles, wash with deionized water, freeze dry, and obtain purified mesenchymal stem cell exosome particles; the protective agent is trehalose and sucrose in a mass ratio of 3:2.

[0068] Example 3

[0069] This embodiment provides a method for purifying mesenchymal stem cell exosomes, including the following steps:

[0070] S1. Add adipose-derived mesenchymal stem cells to the expansion culture medium and adjust the cell concentration to 10. 2 -10 3 Cells / mL, 37℃, 5v / v%CO2, cultured for 42h, centrifuged at 300g for 15min to separate cells and culture medium;

[0071] The amplification medium was a complete mTeSRTM1 stem cell culture medium with an oxygen content of 0.7 v / v%, a pH of 6.7, and containing 3 mmol / L CaCl2 and 12 ng / mL IL-1β.

[0072] S2. Centrifuge the culture medium separated in step S1 at 300g for 15 min at 4℃, collect the supernatant, and then centrifuge at 2000g for 20 min to collect the supernatant.

[0073] S3. Add 0.15g of protective agent to 100g of the supernatant obtained in step S2, stir and mix for 10min, add 4g of the polymer prepared in Preparation Example 3, incubate at 4℃ with stirring for 30min, centrifuge at 3000g for 15min, collect the exosome particles, wash with deionized water, freeze dry, and obtain purified mesenchymal stem cell exosome particles; the protective agent is trehalose and sucrose in a mass ratio of 2.5:1.5.

[0074] Comparative Example 1

[0075] The difference from Example 3 is that the polymer was prepared by Comparative Preparation Example 1.

[0076] Comparative Example 2

[0077] The difference from Example 3 is that the polymer was prepared by Comparative Preparation Example 2.

[0078] Comparative Example 3

[0079] The difference from Example 3 is that the amplification medium was replaced with an equal mass of mTeSRTM1 stem cell complete culture medium.

[0080] Comparative Example 4

[0081] The difference from Example 3 is that the polymer was replaced with an equal mass of polyethylene glycol 400.

[0082] Comparative Example 5

[0083] The difference from Example 3 is that the polymer was replaced with an equal mass of protamine sulfate.

[0084] Test Example 1

[0085] SD rats were randomly divided into a control group, a model group, and groups 1-3 and 1-5 of the examples, with 10 rats in each group.

[0086] Control group rats were fed a normal diet for 28 days, then fasted for 12 hours with unlimited water, followed by an intraperitoneal injection of physiological saline. Seven days post-injection, the skin on the posterior sides of both paws of the rats was prepared, disinfected, and a circular full-thickness skin defect of 0.5 cm in diameter was created using a magnet. SD rats in other groups (excluding the control group) were fed a high-sugar, high-fat diet for 28 days, then fasted for 12 hours with unlimited water, followed by an intraperitoneal injection of 65 mg / kg streptozotocin solution. Seven days post-injection, the skin on the posterior sides of both paws of the rats was prepared, disinfected, and a circular full-thickness skin defect of 0.5 cm in diameter was created using a magnet. Deionized water (1 mL / rat) was applied daily to the wounds of control and model group rats. In Examples 1-3 and Comparative Examples 1-5, the corresponding prepared mesenchymal stem cell exosome granule solution (10 mg / mL) was applied daily to the wounds (1 mL / rat). Administration was once daily for 7 consecutive days.

[0087] Wound healing rate testing:

[0088] Twenty-four hours after the last administration, photos were taken and the wound area was calculated using ImageJ.

[0089] Wound healing rate = [1 - (wound area 24 hours after the last administration / wound area after modeling)] × 100%.

[0090] Detection of inflammatory factor concentrations:

[0091] The collected wound tissue was added to PBS solution at pH 7.4 to prepare tissue homogenates. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the wound tissue homogenates were detected using a kit.

[0092] The results are shown in Table 1.

[0093] Table 1

[0094]

[0095] As shown in the table above, the mesenchymal stem cell exosome particles prepared in Examples 1-3 of this invention have good effects in promoting wound healing and anti-inflammation.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for purifying mesenchymal stem cell exosomes, characterized in that, Includes the following steps: S1. Add adipose-derived mesenchymal stem cells to the expansion medium, adjust the cell concentration, culture, centrifuge, and separate the cells and the medium; the expansion medium is a complete mTeSRTM1 stem cell medium with an oxygen content of 0.5-1 v / v%, a pH of 6.6-6.8, and containing 2-4 mmol / L CaCl2 and 10-15 ng / mL IL-1β. S2. Perform gradient centrifugation on the culture medium separated in step S1 and collect the supernatant; S3. Add a protective agent to the supernatant obtained in step S2, stir and mix evenly, add a polymer, stir and incubate, centrifuge, collect exosome particles, wash, freeze dry, and obtain mesenchymal stem cell exosome particles. The protective agent is trehalose and sucrose in a mass ratio of 2-3:1-2. The polymer is prepared as follows: T1. Preparation of protamine-RGD peptide conjugate: Protamine was added to water, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the mixture was stirred and activated, RGD peptide was added, the mixture was stirred and reacted, dialyzed, and freeze-dried to obtain protamine-RGD peptide conjugate. T2. Preparation of PEG-COOH: Polyethylene glycol was dissolved in dimethyl sulfoxide, and alkaline solution and chloroacetic acid were added. The mixture was stirred and reacted. The solvent was removed under reduced pressure. The product was precipitated in cold diethyl ether, filtered, washed, and dried to obtain PEG-COOH. T3. Preparation of the polymer: PEG-COOH was added to water, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the mixture was stirred and activated, protamine-RGD peptide conjugate was added, the mixture was stirred and reacted, dialyzed, and freeze-dried to obtain the polymer.

2. The purification method according to claim 1, characterized in that, The step S1 involves adjusting the cell concentration to 10. 2 -10 3 The culture conditions are 36-38℃, 4-6 v / v% CO2, culture for 36-48 h, and centrifugation conditions are 250-350g centrifugation for 10-20 min.

3. The purification method according to claim 1, characterized in that, The gradient centrifugation method described in step S2 is to centrifuge at 250-350g for 10-20 minutes at 3-5℃, collect the supernatant, and then centrifuge at 1850-2150g for 15-25 minutes.

4. The purification method according to claim 1, characterized in that, In step S3, the mass ratio of the supernatant, protective agent, and polymer obtained in step S2 is 100:0.1-0.2:3-5. The incubation conditions are 3-5℃ for 25-35 min, and the centrifugation conditions are 2800-3200g for 10-20 min.

5. The purification method according to claim 1, characterized in that, In step T1, the mass ratio of protamine, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and RGD peptide is 5-7:2-3:2-3:1-2, and the stirring reaction time is 5-7 hours. In step T2, the polyethylene glycol is polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, or polyethylene glycol 1000, and the mass ratio of polyethylene glycol to chloroacetic acid is 0.2-1:1.8-2. The alkaline solution... The solution is a 30-50 wt% NaOH or KOH solution. The stirring reaction time is 8-12 h, and the temperature of the cold ether is 3-5 °C. In step T3, the mass ratio of PEG-COOH, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and protamine-RGD peptide conjugate is 1-1.5:0.1-0.15:0.12-0.17:2-3, and the stirring reaction time is 7-10 h.

6. A mesenchymal stem cell exosome particle prepared by the purification method according to any one of claims 1-5.

7. The use of the mesenchymal stem cell exosome particles as described in claim 6 in the preparation of anti-inflammatory or wound-healing drugs.

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