Resveratrol-loaded exosome as well as preparation method and application thereof

Abstract

The invention provides a resveratrol-loaded exosome and a preparation method thereof, and belongs to the technical field of biological medicine manufacturing. The preparation method of the resveratrol-loaded exosome comprises the following steps: performing 3D culture on human umbilical cord or hair follicle-derived mesenchymal stem cells under a low-oxygen condition, collecting supernate, and separating by a differential ultracentrifugation method to obtain the exosome of the mesenchymal stem cells; the method comprises the following steps: mixing a resveratrol solution containing a surfactant with the MSCs-Exos, and carrying out co-incubation and ultrafiltration to obtain the resveratrol loaded MSCs-Exos. According to the invention, the surfactant is added, so that not only are the entrapment rate and the drug loading rate of the MSCs-Exos loaded resveratrol greatly improved, but also the activity of the MSCs-Exos is maintained, the common effect of the MSCs-Exos and the resveratrol is fully exerted, and a synergistic effect is achieved. The resveratrol loaded MSCs-Exos disclosed by the invention has an effect of treating diabetes mellitus.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical manufacturing, and in particular relates to resveratrol-loaded exosomes, a preparation method thereof, and applications thereof. Background Art

[0002] Extracellular vesicles are important mediators of intercellular communication. They contain transmembrane and membrane-anchored proteins, which can promote endocytosis of target cells, thereby facilitating the delivery of their contents and playing an important role in physiological and pathological processes. Extracellular vesicles can be divided into three types: exosomes (Exos), microvesicles, and apoptotic bodies. Among them, the intrinsic biochemical properties of Exos give it great potential as a powerful drug delivery system. Exos drug delivery can reduce the metabolic consumption of drugs before reaching the lesion site, avoid the occurrence of adverse events such as systemic toxicity caused by the low efficiency of drug delivery to the lesion site, and compared with the direct use of traditional drugs, Exos drug delivery has better targeting and efficacy in treating diseases.

[0003] The key to the drug delivery technology of Exos is the selection of drug delivery method. The appropriate drug delivery method can greatly improve the loading rate of Exos for drugs, thereby achieving the effect of reducing toxicity and increasing efficacy. Exos drug delivery methods can be divided into two categories. One is the exogenous drug delivery pathway: first extract and purify Exos, and then load the therapeutic drug into Exos; the other is the endogenous drug delivery pathway: the drug enters the donor cell of Exos through various methods, enters Exos during the formation process of Exos, and then Exos is secreted from the donor cell, and the drug-loaded Exos is obtained by separation and purification. The endogenous drug delivery pathway is complicated to operate and the final drug loading rate is difficult to control, and the drug will affect the secretion of cells and their Exos. For example, chemical transfection method can make the target gene expressed in the cell through transfection, which is suitable for loading gene drugs, but the experiment is difficult, the cycle is long, the transfection efficiency is low, and the gene expression effect is poor. Therefore, in practical applications, the exogenous drug delivery pathway is more widely used. At present, the commonly used exogenous drug delivery methods of Exos include electroporation, ultrasound and co-incubation. The electroporation method is to form micropores on the surface of Exos through short and high electric pulses, so that drugs can migrate to them. Generally, nucleic acids and nanomaterials are loaded. However, the loading efficiency is relatively low, and it is easy to destroy the integrity of the lipid membrane of Exos. Special electroporation equipment and matching electroporation buffer are required, which is costly and complicated to operate. The ultrasonic method is to use an ultrasonic homogenizer to ultrasonicate Exos and drugs. The mechanical shear force of ultrasound opens the membrane of Exos, allowing the drug to enter Exos. The loading efficiency is high, and small chemical molecules, proteins, and nanomaterials can be loaded. However, membrane damage caused by mechanical shear stress can easily cause deformation and destruction of the Exos lipid membrane, leakage of contents, and loss of Exos activity. At the same time, ultrasound needs to optimize specific ultrasonic frequency, time and other parameters for each drug. The operation is complicated, ultrasound equipment is required, and the cost is high. The co-incubation method is to incubate the drug with Exos under suitable conditions so that Exos can be loaded with small molecule drugs and nanomaterials. This method is simple to operate, does not require special equipment, is low in cost, and is easy to scale up and apply. More importantly, the co-incubation method does not destroy the integrity of the Exos membrane and can well maintain the content and activity of the original contents of Exos. However, the efficiency of loading drugs on Exos using the co-incubation method is currently low, with a loading rate of about 10%.

[0004] Drug-loaded Exos have attracted much attention. On the one hand, Exos are natural nanoscale drug carriers with great development potential in drug delivery systems. On the other hand, some types of cell-derived Exos are also drugs themselves, which can play an active role in the treatment of many diseases. This is mainly because Exos contain a large amount of proteins, nucleic acids and lipids of the mother cells, which participate in the regulation of the biological functions of the target cells. The activity of Exos, that is, the content and quality stability of the contents, is crucial for regulating target cell communication, immune response, cell regeneration and differentiation. Mesenchymal stem cells (MSCs) are a type of adult stem cells from multiple tissues with self-renewal and multidirectional differentiation potential. After transplantation, MSCs can migrate and home to damaged tissues, and by secreting immunomodulatory factors, growth factors, extracellular vesicles and other bioactive substances, they play anti-inflammatory, anti-viral, anti-apoptotic, anti-fibrotic, angiogenesis and immune regulation effects, and show good efficacy in the treatment of autoimmune diseases and tissue and organ repair. More and more studies have shown that mesenchymal stem cells exert their effects mainly through their paracrine active substances. As the main component of MSCs paracrine, Exos not only has the same therapeutic effect as MSCs, but also has the advantages of small size, strong tissue permeability, can pass through multiple biological barriers, long circulation half-life, low immunogenicity, and high stability. Resveratrol (RSV) is a polyphenolic compound with a wide range of biological activities, mainly derived from the traditional Chinese medicine Polygonum cuspidatum. As a natural antioxidant, RSV can chelate free radicals, eliminate or reduce the production of free radicals, reduce lipid peroxidation, regulate the activity of antioxidant-related enzymes, and reduce ROS damage to mitochondria. A large number of experimental studies have shown that RSV improves the body's anti-fatigue and anti-aging abilities through various mechanisms, and has achieved significant effects in neuroprotection, anti-inflammatory, antiviral, anti-diabetic, and cardiovascular protection. However, RSV is generally administered orally, rapidly metabolized in the body, with a half-life of 8 to 14 minutes, generating glucuronic acid and sulfate conjugates, and a bioavailability of only 2%. RSV has poor water solubility and inconvenient administration methods, which affect its pharmacological effects and severely limit its clinical use. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a resveratrol-loaded exosome and a preparation method thereof, which can greatly improve the encapsulation rate and drug loading rate, maintain the activity of mesenchymal stem cell exosomes (MSCs-Exos), increase the administration mode of resveratrol, and allow intravenous administration to improve its bioavailability. The resveratrol-loaded MSCs-Exos of the present invention can reduce blood sugar in diabetic mice and improve diabetes-related symptoms.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing resveratrol-loaded exosomes, comprising: 3D culturing mesenchymal stem cells derived from human umbilical cord or hair follicles under the condition of an oxygen concentration of 2% to 10%, collecting the supernatant, and separating to obtain MSCs-Exos by a differential ultracentrifugation method; mixing a resveratrol solution containing a surfactant with the MSCs-Exos, and then incubating and ultrafiltrating to obtain the resveratrol-loaded MSCs-Exos; the surfactant comprises saponin, Triton X-100 or Tween.

[0008] Preferably, the rotation frequency of the 3D culture is 5 to 10 min / h, the rotation speed is 30 to 50 rpm, the oxygen concentration is 2% to 10%, the temperature is 36 to 38° C., and the CO2 concentration is 4% to 6%.

[0009] Preferably, the centrifugal force and time of the differential centrifugation are 300g, 10 min, 2000g, 30 min, 12000g, 30 min, 120000g, 4 h, respectively.

[0010] Preferably, the mass volume concentration of the surfactant is 0.5%, the concentration of the resveratrol solution is 5 mg / mL, and the volume ratio of the surfactant to the resveratrol solution is 20-40:1.

[0011] Preferably, the concentration of the MSCs-Exos is 450 μg / mL.

[0012] Preferably, the volume ratio of the resveratrol solution containing a surfactant to MSCs-Exos is 1 to 3:1.

[0013] Preferably, the incubation temperature is 36-38° C., the rotation speed is 70-90 rpm, and the time is 8-16 hours.

[0014] Preferably, the ultrafiltration comprises: transferring the incubated mixed solution to a 100 kDa ultrafiltration tube, centrifuging at 4°C and 3000 g for 15 min, adding physiological saline, and centrifuging at 4°C and 3000 g for 15 min.

[0015] The present invention also provides MSCs-Exos loaded with resveratrol obtained by the above method.

[0016] The present invention also provides the use of the MSCs-Exos in preparing a drug for treating diabetes.

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

[0018] The present invention 3D cultured human umbilical cord or hair follicle derived MSCs under hypoxic conditions, separated the MSCs-Exos in the culture supernatant by differential ultracentrifugation, and adopted a surfactant synergistic co-incubation method to load MSCs-Exos with resveratrol to obtain MSCs-Exos loaded with resveratrol. The addition of a surfactant during co-incubation significantly improved the encapsulation rate and drug loading rate of MSCs-Exos loaded with resveratrol, reduced the use of MSCs-Exos, reduced costs, and also increased the efficiency of encapsulation. More importantly, the method of surfactant synergistic co-incubation can effectively prevent the leakage of MSCs-Exos contents, maintain the activity of MSCs-Exos, give full play to the common effect of MSCs-Exos and resveratrol, and play a synergistic role in the treatment of diseases. The MSCs-Exos loaded with resveratrol prepared by the present invention can be intravenously administered to improve its bioavailability, expand the clinical application of resveratrol, significantly reduce blood sugar in diabetic model mice, improve diabetes-related symptoms, and the effect is comparable to commercially available semaglutide (Semaglutide). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A flow chart of preparing resveratrol-loaded exosomes MSCs-Exos & RSV by the surfactant-co-incubation method of the present invention;

[0020] Figure 2 The characteristics of MSCs-Exos and MSCs-Exos&RSV; A is the transmission electron microscopy image of MSCs-Exos and MSCs-Exos&RSV (scale, 100 nm), B is the particle size distribution diagram of MSCs-Exos and MSCs-Exos&RSV, and C is the immunoblot of the marker proteins of MSCs-Exos and MSCs-Exos&RSV;

[0021] Figure 3 The relative expression comparison results of highly abundant miR-21-5p, miR-122-5p and miR-146a-5p in MSCs-Exos and MSCs-Exos&RSV;

[0022] Figure 4 Comparison of encapsulation efficiency and drug loading rate of different methods; A is the comparison of encapsulation efficiency of different drug loading methods, and B is the comparison of drug loading rate of different encapsulation methods; the results are expressed as Mean±SD (n=3), *p<0.05, **p<0.01, ***p<0.001;

[0023] Figure 5The effect of MSCs-Exos&RSV in treating type 2 diabetes; A is food intake, B is food utilization, C is fasting body weight, D is fasting blood glucose, E is fasting insulin, F is insulin resistance index, G is the number of islets, H is the islet area ratio, and I is a representative image of HE staining of the pancreas of type 2 diabetic mice; data are expressed as mean ± SEM, *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0024] The present invention provides a resveratrol-loaded exosome and a preparation method thereof, comprising: 3D culturing mesenchymal stem cells derived from human umbilical cord or hair follicles under the condition of an oxygen concentration of 2% to 10%, collecting the supernatant, and separating to obtain MSCs-Exos by a differential ultracentrifugation method; mixing a resveratrol solution containing a surfactant with the MSCs-Exos, and then incubating and ultrafiltrating to obtain the resveratrol-loaded MSCs-Exos; the surfactant comprises saponin, Triton X-100 or Tween.

[0025] The surfactant of the present invention can increase the permeability of the Exos membrane, thereby improving the efficiency of the drug entering the Exos when co-incubated with the Exos, thereby improving the encapsulation rate and the drug loading rate.

[0026] The present invention adds a surfactant during incubation, which not only greatly improves the encapsulation rate and drug loading rate of MSCs-Exos loaded with resveratrol, but also maintains the activity of MSCs-Exos, fully exerts the common effect of MSCs-Exos and resveratrol, and plays a synergistic role.

[0027] The oxygen concentration of the hypoxia described in the present invention is preferably 2% to 10%, more preferably 5%.

[0028] The rotation frequency of the 3D culture of the present invention is preferably 5-10 min / h, more preferably 5 min / h, the rotation speed is preferably 30-50 min / h, more preferably 30 rpm, the temperature is preferably 36-38°C, more preferably 37°C, and the CO2 concentration is preferably 4%-6%, more preferably 5%.

[0029] During the culture process, when the cells enter the logarithmic growth phase, it is preferred to gently rinse once with PBS, replace the Exos-free culture medium, and culture for 72 hours, and then collect the culture supernatant.

[0030] The differential centrifugation of the present invention preferably includes: centrifuging the supernatant at 300g and 4°C for 10 minutes to remove dead cells; centrifuging at 2000g and 4°C for 30 minutes to remove cell debris; centrifuging at 12000g and 4°C for 30 minutes to remove apoptotic bodies; filtering the culture medium supernatant with a 0.22μm filter; centrifuging at 120000g and 4°C for 4 hours, discarding the supernatant, and resuspending the precipitate with physiological saline to obtain the MSCs-Exos.

[0031] The mass volume concentration of the surfactant of the present invention is preferably 0.5%, the preferred concentration of the resveratrol solution is 5 mg / mL, and the volume ratio of the surfactant to the resveratrol solution is preferably 20-40:1, more preferably 32.3:1.

[0032] The concentration of the MSCs-Exos of the present invention is preferably 450 μg / mL, and the volume ratio of the resveratrol solution containing a surfactant to the MSCs-Exos is preferably 1 to 3:1, more preferably 1:1.

[0033] The incubation temperature of the present invention is preferably 36-38°C, more preferably 37°C, the rotation speed is preferably 70-90 rpm, more preferably 70 rpm, and the time is preferably 8-16, more preferably 12 hours.

[0034] The ultrafiltration of the present invention preferably comprises: transferring the incubated mixed solution to a 100 kDa ultrafiltration tube, centrifuging at 4°C and 3000 g for 15 min, adding physiological saline, and centrifuging at 4°C and 3000 g for 15 min.

[0035] The present invention also provides resveratrol-loaded exosomes obtained by the above method.

[0036] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1

[0040] Preparation of MSCs-Exos&RSV by surfactant-assisted co-incubation

[0041] 1. Primary culture of human umbilical cord MSCs

[0042] (1) Place an umbilical cord of about 10 cm in length in a culture dish and repeatedly rinse the periphery of the umbilical cord and the umbilical cord blood vessels with PBS (with 5% penicillin-streptomycin) for 3 to 5 times to remove residual cord blood. During the cleaning process, make sure that the umbilical cord is always immersed in PBS.

[0043] (2) Three blood vessels can be clearly seen at the end of the umbilical cord. One relatively thick one is the umbilical vein, and two relatively thin ones are umbilical arteries, which are distributed on both sides of the umbilical vein. Use one forceps to clamp one end of the umbilical cord and another forceps to clamp one end of one of the arteries. Gently peel off the artery along the spiral of the umbilical cord. Use the same method to peel off the other artery and vein. The remaining gelatinous tissue is the Wharton's jelly tissue of the umbilical cord. Rinse the Wharton's jelly tissue block repeatedly with PBS (5% penicillin-streptomycin) 3 to 5 times.

[0044] (3) Use a sterile surgical blade to cut the Wharton's jelly tissue into small pieces of 1 to 3 mm square, place them evenly on a culture dish, add a small amount of MSCs culture medium, and place them in a CO2 incubator for culture.

[0045] (4) The above tissue blocks were replaced with MSCs culture medium every 3 days. After culturing for about 7 to 10 days, long spindle-like cells grew around the tissue blocks. As the culture time prolonged, the number of cells gradually increased and fused. The degree of fusion was about 80%. The cells were rinsed with PBS once and digested with an appropriate amount of trypsin (0.25%)-EDTA for 3 to 5 minutes. Most of the cells became round and floated under the microscope. An appropriate amount of culture medium was added to terminate the digestion. The cell suspension was collected in a centrifuge tube, the culture dish was rinsed with PBS once, and the cells were transferred to a centrifuge tube together. The cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. The cells were resuspended in culture medium, counted with a hemocytometer, and inoculated into a new culture dish at 8,000 to 10,000 cells per square centimeter. An appropriate amount of culture medium was added and the dish was placed in a CO2 incubator for 37°C and 5% CO2 culture. This was the first generation of cells (P1). According to the above steps, human umbilical cord MSCs were expanded.

[0046] 2. 3D hypoxia culture of MSCs

[0047] (1) Weigh 1 g of microcarriers into a 100 mL spinner flask and add 50 mL of MSCs culture medium;

[0048] (2) Digest the third generation MSCs cultured on the flat surface, count and centrifuge them, and take 5×10 6 Cells were counted at 500 / cm 2 The density was mixed with the carrier and suspended in 50 mL of MSCs culture medium;

[0049] (3) Mix the microcarriers and cell suspension in a rotating bottle;

[0050] (4) The rotating flask was placed in a cell three-gas incubator for culture. The bioreactor mode was set to: rotation 5 min / h, speed 30 rpm, and the incubator was set to: 37°C, 5% CO2, 5% O2;

[0051] (5) When the cells attached to the microcarriers enter the logarithmic growth phase, they are gently rinsed once with PBS and replaced with Exos-free culture medium. After 96 h of culture, the culture medium supernatant is collected.

[0052] 3. Isolation of MSCs-Exos

[0053] (1) The culture supernatant was centrifuged at 300g and 4°C for 10 min to remove dead cells.

[0054] (2) Centrifuge at 2000 g and 4 °C for 30 min to remove cell debris;

[0055] (3) Centrifuge at 12000g, 4°C for 30 min to remove apoptotic bodies;

[0056] (4) Filter the culture supernatant with a 0.22 μm filter to remove extracellular vesicles larger than 200 nm;

[0057] (5) Centrifuge at 120,000 g, 4 °C for 4 h and discard the supernatant;

[0058] (6) After resuspending the precipitate in each ultracentrifuge tube with 200 μL of physiological saline, MSCs-Exos were obtained. 5 μL was taken and quantified using a BSA kit. The remaining MSCs-Exos were stored in a -80°C refrigerator.

[0059] 4. MSCs-Exos loaded with resveratrol:

[0060] (1) The above MSCs-Exos were diluted to 450 μg / mL with normal saline;

[0061] (2) Prepare 0.5% (W / V) Tween solution in saline: weigh 0.05 g of Tween, add 10 mL of saline, mix well, and fully dissolve;

[0062] (3) Prepare 5 mg / mL resveratrol stock solution: weigh 2.5 mg of resveratrol and add 0.5 mL of anhydrous ethanol, mix well until the solution becomes colorless and transparent;

[0063] (4) preparing a mixed solution of resveratrol and Tween: taking 0.1 mL of 5 mg / mL resveratrol solution and adding it into 3.23 mL of 0.5% (W / V) Tween saline solution, and mixing well;

[0064] (5) 450 μg / mL MSCs-Exos and the mixed solution of resveratrol and Tween were mixed in equal volumes;

[0065] (6) Place the mixed centrifuge tube in a constant temperature shaker at 37°C and 70 rpm for 12 h;

[0066] (7) Remove unloaded free resveratrol and Tween by ultrafiltration: Transfer the mixed solution after incubation of the above MSCs-Exos and resveratrol to a 100 kDa ultrafiltration tube, centrifuge at 4°C, 3000 g for 15 min, discard the outer layer of liquid, add 15 mL of normal saline to the inner tube, centrifuge at 4°C, 3000 g for 15 min, add 100 μL of normal saline to the inner tube to dissolve the MSCs-Exos, and obtain MSCs-Exos & RSV.

[0067] Example 2

[0068] The specific implementation is the same as that of Example 1, except that step 1 is:

[0069] Primary culture of human hair follicle MSCs

[0070] (1) Under sterile conditions, about 20 single hair follicles were obtained from the back of the human head and the hair follicles were rinsed three times with PBS containing 5x double antibody;

[0071] (2) Use sterile ophthalmic scissors to cut the root tissue of the hair follicle, clamp it into the well plate with sterile ophthalmic forceps, add MSCs culture medium, and place it in a CO2 incubator at 37°C and 5% CO2;

[0072] (3) Observe under the microscope every day. When the cell confluence is about 80%, rinse once with PBS, add an appropriate amount of trypsin (0.25%)-EDTA to digest for 3-5 minutes. Under the microscope, most of the cells become round and float. Add an appropriate amount of culture medium to stop digestion. Collect the cell suspension into a centrifuge tube, rinse the culture dish once with PBS, transfer it to a centrifuge tube, centrifuge at 300g for 5 minutes, and discard the supernatant. Resuspend the cells in the culture medium, count them with a hemocytometer, and inoculate them into a new culture dish at 8000-10000 cells per square centimeter. Add an appropriate amount of culture medium, place in a CO2 incubator, and culture at 37°C and 5% CO2. This is the first generation of cells (P1). According to the above steps, expand human hair follicle MSCs.

[0073] Example 3

[0074] The specific implementation method is the same as that of Example 1, except that step 4 (2) is as follows: prepare a 0.5% (W / V) saponin solution in physiological saline: weigh 0.05 g of saponin, add 10 mL of physiological saline, mix well, and fully dissolve.

[0075] Example 4

[0076] The specific implementation is the same as that of Example 1, except that step 4 (2) is as follows: prepare a 0.5% (V / V) solution of Triton X-100 in physiological saline: take 0.05 mL of Triton X-100, add 10 mL of physiological saline, and mix thoroughly.

[0077] Example 5

[0078] The specific implementation is the same as Example 1, except that step 2 (4) is: the rotating bottle is placed in a cell three-gas incubator for culture, the bioreaction mode is set to: rotation 5min / h, speed 30rpm, and the incubator is set to: 37°C, 5% CO2, 2% O2.

[0079] Example 6

[0080] The specific implementation is the same as that of Example 1, except that step 2 (4) is: the rotating bottle is placed in a cell three-gas incubator for culture, the bioreaction mode is set to: rotation 5min / h, speed 30rpm, and the incubator is set to: 37°C, 5% CO2, 10% O2.

[0081] Example 7

[0082] The specific implementation is the same as Example 1, except that step 2 (4) is: the rotating bottle is placed in a cell three-gas incubator for culture, the bioreaction mode is set to: rotation 5min / h, speed 20rpm, and the incubator is set to: 37°C, 5% CO2, 5% O2.

[0083] Example 8

[0084] The specific implementation is the same as that of Example 1, except that step 2 (4) is: the rotating bottle is placed in a cell three-gas incubator for culture, the bioreaction mode is set to: rotation 5min / h, speed 40rpm, and the incubator is set to: 37°C, 5% CO2, 2% O2.

[0085] Example 9

[0086] The specific implementation is the same as that of Example 1, except that step 2 (4) is: the rotating bottle is placed in a cell three-gas incubator for culture, the bioreaction mode is set to: rotation 10min / h, speed 30rpm, and the incubator is set to: 37°C, 5% CO2, 2% O2.

[0087] Example 10

[0088] The specific implementation is the same as that of Example 1, except that step 2 (4) is as follows: prepare a solution of resveratrol and a surfactant: add 0.1 mL of a 5 mg / mL resveratrol solution into 1.9 mL of a 0.5% (W / V) surfactant saline solution and mix well.

[0089] Embodiment 11

[0090] The specific implementation is the same as that of Example 1, except that step 2 (4) is as follows: prepare a solution of resveratrol and a surfactant: add 0.1 mL of a 5 mg / mL resveratrol solution into 3.9 mL of a 0.5% (W / V) surfactant saline solution and mix well.

[0091] Example 12

[0092] The specific implementation is the same as that of Example 1, except that step 2 (5) is as follows: 450 μg / mL MSCs-Exos is mixed with the mixed solution of the above-mentioned resveratrol and surfactant at a volume ratio of 2:1.

[0093] Embodiment 13

[0094] The specific implementation is the same as that of Example 1, except that step 2 (5) is as follows: 450 μg / mL MSCs-Exos is mixed with the mixed solution of the above-mentioned resveratrol and surfactant at a volume ratio of 3:1.

[0095] Embodiment 14

[0096] The specific implementation is the same as that of Example 1, except that step 2 (6) is as follows: placing the mixed centrifuge tube into a constant temperature shaker at 37° C. and 80 rpm for 12 hours.

[0097] Embodiment 15

[0098] The specific implementation is the same as that of Example 1, except that step 2 (6) is as follows: placing the mixed centrifuge tube into a constant temperature shaker at 37° C. and 90 rpm for 12 hours.

[0099] Example 16

[0100] The specific implementation is the same as that of Example 1, except that step 2 (6) is as follows: placing the mixed centrifuge tube into a constant temperature shaker at 37° C. and 70 rpm for 8 hours.

[0101] Embodiment 17

[0102] The specific implementation is the same as that of Example 1, except that step 2 (6) is as follows: placing the mixed centrifuge tube into a constant temperature shaker at 37° C. and 70 rpm for 16 hours.

[0103] Comparative Example 1

[0104] The specific implementation method of the ultrasonic method is the same as that of Example 1, except that step 4 is:

[0105] (1) The above MSCs-Exos were diluted to 450 μg / mL with normal saline;

[0106] (2) Prepare 5 mg / mL resveratrol stock solution: weigh 2.5 mg of resveratrol and add 0.5 mL of anhydrous ethanol, and mix until the solution becomes colorless and transparent;

[0107] (3) Prepare 50 μg / mL resveratrol solution: take 0.1 mL of 5 mg / mL resveratrol stock solution, add it into 10 mL of normal saline solution, and mix well;

[0108] (4) 150 μg / mL MSCs-Exos and 50 μg / mL resveratrol solution were mixed in equal volumes using an ultrasonic homogenizer with a 0.25-inch tip under the following conditions: 20% amplitude, 6 cycles, 2 min between each cycle, on / off 30 s, 3 min duration. After ultrasonic treatment, the mixture was incubated at 37°C for 1 h to allow the outer membrane to recover.

[0109] (5) Ultrafiltration method to remove unloaded free resveratrol: The mixed solution after incubation of the above MSCs-Exos and resveratrol was transferred to a 100 kDa ultrafiltration tube. After centrifugation at 4°C and 3000 g for 15 min, the outer layer of liquid was discarded, 15 mL of normal saline was added to the inner tube, and the mixture was centrifuged at 4°C and 3000 g for 15 min. 100 μL of normal saline was added to the inner tube to dissolve the MSCs-Exos to obtain MSCs-Exos & RSV.

[0110] Comparative Example 2

[0111] Co-incubation method

[0112] The specific implementation is the same as that of Example 1, except that no surfactant is added.

[0113] Test Example 1

[0114] The morphological characteristics of MSCs-Exos and MSCs-Exos & RSV in Example 1 were observed using transmission electron microscopy. Figure 2 As shown in A.

[0115] The particle size distribution of MSCs-Exos and MSCs-Exos&RSV was detected by NanoFCM nanoflow cytometry. The results are as follows Figure 2 As shown in B.

[0116] Immunoblotting was used to analyze the expression of CD63, CD81 and TSG101 in MSCs-Exos and MSCs-Exos&RSV. Figure 2 As shown in C.

[0117] Depend on Figure 2 It can be seen that the MSCs-Exos loaded with resveratrol (MSCs-Exos & RSV) has the same morphology as MSCs-Exos, both are spherical and have a typical double-layer membrane structure. Under the microscope, MSCs-Exos & RSV are slightly larger than MSCs-Exos, but the diameter is about 30-150nm. The results of nanoflow cytometry also show that the particle size distribution curves of MSCs-Exos and MSCs-Exos & RSV are between 30 and 150nm, which are consistent with the particle size distribution of exosomes. The average particle size of MSCs-Exos is 70.9nm, and the average particle size of MSCs-Exos & RSV is 80nm. The increase in the average particle size of MSCs-Exos & RSV proves that MSCs-Exos is successfully loaded with resveratrol to a certain extent. The results of immunoblotting show that MSCs-Exos & RSV still express exosome biomarkers, transmembrane protein markers CD63 and CD81, and cytoplasmic protein marker TSG101.

[0118] Test Example 2

[0119] The total RNA of MSCs-Exos and MSCs-Exos & RSV prepared in Example 1 was extracted using Trizol, and qRT-PCR was performed using Bulge-LoopTM miRNAqRT-PCR StarterKit and primers of miR-21-5p, miR-122-5p and miR-146a-5p to detect the expression of representative inclusions miR-21-5p, miR-122-5p and miR-146a-5p in MSCs-Exos and MSCs-Exos & RSV. The results are shown in FIG. Figure 3 shown.

[0120] Depend on Figure 3 It can be seen that compared with MSCs-Exos, the expression abundance of key inclusions miR-21-5p, miR-122-5p and miR-146a-5p of MSCs-Exos&RSV is basically the same. Using the surfactant co-incubation method, MSCs-Exos&RSV can still maintain the activity of exosomes and play a role.

[0121] Test Example 3

[0122] The encapsulation rate and drug loading rate of the exosomes loaded with resveratrol in Example 1 (surfactant synergistic co-incubation method), Comparative Example 1 (ultrasound method), and Comparative Example 2 (co-incubation method) were respectively detected. The specific results are as follows Figure 4 shown.

[0123] The method for detecting the loading rate and the drug loading rate is as follows: establishing a standard curve, diluting a 5 mg / mL resveratrol stock solution with a 0.5% (W / V) surfactant saline solution to 300 μg / mL, 150 μg / mL, 75 μg / mL, 37.5 μg / mL, 18.75 μg / mL, 9.375 μg / mL, and 4.6875 μg / mL, respectively, measuring the OD value at 304 nm, establishing a standard curve, and using an ultrasonic cell lyser at an ultrasonic power of 20%, an ultrasonic power of 10%, and a standard curve. Under the condition of 20s sonication time, the reaction was repeated for 3 times to lyse Exos. After lysis, 200μL of the solution was added to a 96-well plate and the OD value at 304nm was measured. The obtained OD value was substituted into the standard curve to calculate the concentration of resveratrol after MSCs-Exos encapsulated resveratrol. The mass of resveratrol was calculated based on the actual volume obtained after encapsulation. The encapsulation rate was calculated as follows: (mass of resveratrol after encapsulation / mass of resveratrol before encapsulation)×100%, and the drug loading rate was calculated as follows: (mass of resveratrol after encapsulation / mass of Exos)×100%.

[0124] Depend on Figure 4 It can be seen that the surfactant-co-incubation method has a loading rate of about 3 times higher than the co-incubation method, which is 1.5 times that of the ultrasound method, reaching 27%-32%. In terms of drug loading rate, the surfactant-co-incubation method can reach 18%, while the ultrasound method and the co-incubation method are less than 2%. The surfactant-co-incubation method can significantly improve the efficiency of MSCs-Exos loading resveratrol and greatly reduce the amount of MSCs-Exos used.

[0125] Test Example 4

[0126] MSCs-Exos&RSV treatment of diabetic mice

[0127] (1) Establishment of type 2 diabetes mouse model and drug administration

[0128] The type 2 diabetes mouse model was established by inducing a high-fat diet combined with a low dose of streptozotocin (STZ). C57BL / 6J mice were fed a 60% high-fat diet (energy of 5240 kcal / kg, 60% kcal from fat, 20% kcal from protein, and 20% kcal from carbohydrates) for 4 weeks before modeling. The mice were fasted but not water-deprived, and STZ (85 mg / kg, dissolved in 0.1 M citric acid-sodium citrate buffer, pH 4.4) was injected into the left lower abdominal cavity 12 hours later.

[0129] The normal control group was intraperitoneally injected with citric acid-sodium citrate buffer at a dose of 10mL / kg body weight. After the injection, the mice were fasted but not watered for 2 hours before resuming their diet. After 72 hours of the first injection, the second injection was performed according to the above operation. 72 hours after the second injection of STZ, fasting blood glucose was measured, and those with fasting blood glucose greater than 11.1mM were included in the type 2 diabetic mouse model. After the type 2 diabetic mouse model was successfully established, it was fed with 60% high-fat feed for 4 weeks, and then given MSCs-Exos&RSV for 4 weeks.

[0130] After the type 2 diabetes mouse model was successfully established, the mice were randomly divided into 7 groups according to blood sugar and body weight, with 9 mice in each group. The corresponding reagents were injected into the tail vein of each group of mice once every 3 days. Animal groups: NC (normal saline, normal mice, normal control group), MC (normal saline, type 2 diabetes model mice, negative control group), Semaglutide (32.5μg / kg Semaglutide, type 2 diabetes model mice, positive control group), MSCs-Exos-L (13mg / kg MSCs-Exos, type 2 diabetes model mice, low-dose exosome control group), MSCs-Exos-H (26mg / kg MSCs-Exos, type 2 diabetes model mice, high-dose exosome control group), MSCs-Exos&RSV-L (containing 13mg / kg MSCs-Exos and 4mg / kg RSV, type 2 diabetes model mice, low-dose experimental group), MSCs-Exos&RSV-H (containing 26mg / kg MSCs-Exos and 8mg / kg RSV, type 2 diabetes model mice, high-dose experimental group).

[0131] MSCs-Exos&RSV are prepared according to Example 1 of the present invention.

[0132] (2) Detection

[0133] During the drug intervention period, the food consumption of mice in each cage was measured for 3 days, and the food consumption of each cage every 3 days was measured by weighing the remaining food. The weight gain or loss within three days was recorded and measured continuously. After four weeks of drug intervention, the food intake (g / mouse / week) and food utilization rate (%) were calculated respectively. Food utilization rate (%) = total weight gain or loss (g) / total food intake × 100%. The fasting body weight (FBW) and fasting blood glucose concentration (FBG) of mice in each group were measured once every 2 weeks. Before the measurement, mice in each group were fasted for 12 hours, during which they were free to drink water. Blood was collected from the tail tip of mice to measure FBG. After 4 weeks of drug injection, mice in each group were fasted for 12 hours, during which they were free to drink water. Mice were anesthetized with isoflurane, 1 ml of blood was collected, and the blood was left to stand for 2 hours. The supernatant was serum, which was packaged and stored at -80°C until analysis.

[0134] The serum pancreatic islet level (INS) of each group of mice was measured by mouse insulin ELISA kit (H203-1-2, Nanjing Jiancheng, China). The insulin resistance index (HOMA-IR) was calculated, HOMA-IR = INS (mLU / L) × FBG (mmol / L) / 22.5.

[0135] The mouse pancreatic islet tissue was fixed with formalin, sliced, dewaxed, dehydrated, stained with hematoxylin, rinsed and acidified, stained with eosin, routinely dehydrated, transparentized, and sealed. According to the HE staining results of the pancreas, the number of pancreatic islets and the percentage of pancreatic islets in the total pancreatic area were counted.

[0136] Test results such as Figure 5 shown.

[0137] Depend on Figure 5 It can be seen that compared with the NC group, the food intake of the MC group increased significantly, but the food utilization rate and fasting weight decreased significantly, showing the typical "three more and one less" diabetes symptoms. Combined with blood sugar indicators, the fasting blood sugar level of the MC group was significantly higher than that of the NC group, indicating that the type 2 diabetes model was successfully replicated.

[0138] Compared with the MC group, the Semaglutide group significantly improved the food intake, food utilization, fasting blood glucose (9.0±1.95), and fasting body weight (25.8±2.22) of diabetic mice. The MSCs-Exos&RSV-L group and the MSCs-Exos&RSV-H group significantly improved the food intake and fasting blood glucose levels of diabetic mice. The results showed that MSCs-Exos&RSV not only improves the general symptoms of type 2 diabetic mice, but also has the function of lowering blood sugar.

[0139] Measuring the fasting insulin levels of mice revealed that the fasting insulin levels of mice in the MC group increased significantly compared with the NC group. Compared with the MC group, the fasting insulin levels of mice in all drug intervention groups were significantly reduced. Among them, the fasting insulin levels of mice in the MSCs-Exos&RSV-H group and the Semaglutide group were comparable. The results showed that MSCs-Exos&RSV can reduce the insulin levels of mice with type 2 diabetes.

[0140] The change of insulin resistance index (HOMA-IR) is positively correlated with the level of insulin resistance and can be used to evaluate the level of individual insulin resistance. Compared with the NC group, the HOMA-IR of the MC group increased significantly, indicating that the mice in the MC group developed insulin resistance. Compared with the MC group, the HOMA-IR of all drug intervention groups decreased significantly. The results showed that MSCs-Exos & RSV reduced the insulin resistance level of type 2 diabetic mice.

[0141] The HE staining results of the mouse pancreas showed that compared with the NC group, the MC group showed shrinkage and necrosis of pancreatic islet cells, blurred boundaries, reduced number of pancreatic islet cells, and significantly reduced area percentage. Compared with the MC group, the drug intervention group showed clearer boundaries of pancreatic islet cells to varying degrees, less shrinkage of pancreatic islet cells, more complete cells, and reduced degree of degeneration and necrosis of pancreatic islet cells. Among them, after intervention with the positive control drug Semaglutide and MSCs-Exos&RSV-L, the area percentage of pancreatic islet cells increased significantly. After intervention with MSCs-Exos&RSV-H, the area percentage and number of pancreatic islet cells increased significantly. The results show that MSCs-Exos&RSV has a certain repair function on the pancreatic islets of type 2 diabetic mice.

[0142] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing exosomes loaded with resveratrol, characterized in that: include: 3D culture of mesenchymal stem cells derived from human umbilical cord or hair follicles under an oxygen concentration of 2% to 10%, collecting the supernatant, and separating MSCs-Exos by differential ultracentrifugation; mixing a resveratrol solution containing a surfactant with MSCs-Exos, incubating and ultrafiltration, and obtaining resveratrol-loaded MSCs-Exos; The surfactant includes saponin, Triton X-100 or Tween.

2. The preparation method according to claim 1, characterized in that: The rotation frequency of the 3D culture is 5 to 10 min / h, the rotation speed is 30 to 50 rpm, the oxygen concentration is 2% to 10%, the temperature is 36 to 38° C., and the CO2 concentration is 4% to 6%.

3. The preparation method according to claim 1, characterized in that: The centrifugal force and time of the differential centrifugation are 300g, 10 min, 2000g, 30 min, 12000g, 30 min, 120000g, 4 h, respectively.

4. The preparation method according to claim 1, characterized in that: The mass volume concentration of the surfactant is 0.5%, the concentration of the resveratrol solution is 5 mg / mL, and the volume ratio of the surfactant to the resveratrol solution is 20-40:

1.

5. The preparation method according to claim 1, characterized in that: The concentration of the MSCs-Exos is 450 μg / mL.

6. The preparation method according to claim 1, characterized in that: The volume ratio of the resveratrol solution containing a surfactant to MSCs-Exos is 1 to 3:

1.

7. The preparation method according to claim 1, characterized in that: The incubation temperature is 36-38° C., the rotation speed is 70-90 rpm, and the time is 8-16 hours.

8. The preparation method according to claim 1, characterized in that: The ultrafiltration comprises: transferring the incubated mixed solution to a 100 kDa ultrafiltration tube, centrifuging at 4° C. and 3000 g for 15 min, adding physiological saline, and centrifuging at 4° C. and 3000 g for 15 min.

9. Resveratrol-loaded MSCs-Exos obtained by the method according to any one of claims 1 to 8.

10. Use of the resveratrol-loaded MSCs-Exos obtained by the method of any one of claims 1 to 8 or the MSCs-Exos according to claim 9 in the preparation of a drug for treating diabetes.

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