Preparation method and application of mesenchymal stem cell exosome rhinitis spray

Abstract

The invention discloses a preparation method and application of a mesenchymal stem cell exosome rhinitis spray, a membrane lipid vesicle exosome with the diameter of 70-170nm is adopted, the vesicle contains multiple RNA (Ribonucleic Acid), miRNA (Micro Ribonucleic Acid), related effect protein and the like with biological activity, the occurrence of inflammation can be effectively inhibited, and damage repair can be promoted. The selected exosome is small in size, capable of loading signal substances such as lipid, protein and nucleic acid, convenient to absorb and release and free of immunogenicity, and the technology that the exosome is wrapped with temperature-sensitive gel is adopted, so that it is guaranteed that the exosome has activity in vitro for a long time. According to the product, poloxamer 407 / poloxamer 188 / glycerol temperature-sensitive gel is used as a carrier, so that the exosome can be uniformly sprayed on an affected part, and the exosome is adsorbed to the nasal cavity by utilizing the gel forming characteristic at 34 + / -1 DEG C and acts, so that the exosome resides and is continuously and slowly released, and the problem that the medicine is difficult to apply to the nasal cavity and resides in the nasal cavity is solved by adopting the scheme.

Description

Technical Field

[0001] The invention belongs to the technical field of drugs for treating allergic rhinitis, and specifically relates to a preparation method and application of a mesenchymal stem cell exosome rhinitis spray. Background Art

[0002] Allergic rhinitis (AR) is a non-infectious inflammatory disease that occurs in the nasal mucosa. It refers to the symptoms of hypersecretion, swelling of the nasal mucosa, paroxysmal sneezing and nasal itching that occur in atopic individuals after contact with allergens. Some patients will have related symptoms such as reversible hyposmia and allergic conjunctivitis. A European study showed that allergic rhinitis often begins to develop in childhood or adolescence, with a prevalence of more than 5% in children under 3 years old, about 9% in children aged 6-7 years old, and even higher in children aged 13-14 years old, up to 15%; the prevalence of allergic rhinitis in adults varies from region to region, and the prevalence of allergic rhinitis between regions is about 46%, which has become one of the most common chronic health problems in the world. It has a certain impact on patients' sleep, learning efficiency, work productivity and social life, and is also one of the important causes of psychological diseases such as anxiety and depression in patients, while adding to the economic burden on the country and individuals.

[0003] At present, the main methods for treating allergic rhinitis include drug therapy, physical therapy, surgical treatment and lifestyle adjustment. In terms of drug therapy, nasal spray hormones, oral Chinese patent medicines, mucus promoters and antibiotics are often used to reduce inflammation, promote secretion discharge and relieve symptoms. Physical therapy includes massage, hot compress, acupuncture, saline nasal washing and ultrashort wave therapy, etc., which aim to improve nasal ventilation function, promote local blood circulation and accelerate the absorption of inflammation. For patients with severe conditions or ineffective drug treatment, surgical treatment such as endoscopic nasal surgery and partial turbinate resection are effective options that can eliminate lesions and restore nasal physiological functions. However, these methods also have certain limitations. Although drug therapy can relieve symptoms, it is often difficult to cure chronic rhinitis and sinusitis, and long-term use may produce drug resistance, and even more severe symptoms may appear after stopping the drug. Although physical therapy is non-invasive and can improve local symptoms, the effect is relatively slow, and the effect may not be obvious for some patients. Although surgical treatment can cure the disease, the operation itself has certain risks and the cost is relatively high. Some patients may not be able to accept it due to physical conditions or economic reasons.

[0004] Mesenchymal stem cell-based therapy is an emerging treatment method in recent years. Mesenchymal stem cells (MSC) have the potential for self-renewal and multidirectional differentiation, and can repair damaged areas through immune regulation and paracrine effects, creating a good environment for tissue regeneration. Studies have shown that mesenchymal stem cells and related exosome products show good therapeutic potential for allergic rhinitis. Summary of the invention

[0005] In order to solve the deficiencies of drug therapy, physical therapy, surgical treatment and lifestyle conditioning in the prior art, the present invention aims to provide a method for preparing a mesenchymal stem cell exosome nasal spray and its application. The applicant has found that using poloxamer 407 (P407) and poloxamer 188 (P188) as exosome loading materials to obtain a temperature-sensitive exosome hydrogel enhances the retention of mesenchymal stem cell exosomes in the nasal cavity and sustained sustained release. This solution solves the problem of difficulty in applying drugs to the nasal cavity and difficulty in retention. The mesenchymal stem cell exosome nasal spray prepared by the present invention has the functions of immunomodulation and anti-allergy, can repair the nasal microenvironment well, improve the symptoms of allergic rhinitis, and overcome the side effects and poor therapeutic effects of existing allergic rhinitis treatment drugs.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for preparing a mesenchymal stem cell exosome nasal spray comprises the following steps: 1) Inoculation: sterilize the bioreactor by high pressure, dry it, add sterile culture medium (serum-free culture medium), add microslides at a rate of 20 mL / slide, and mix well; After resuscitation, the concentration of mesenchymal stem cells was 1×10 6 / mL cell suspension, the ratio of cells to microslide during inoculation is 500,000 / slide, the inoculation density is 10 mL / slide, and the culture medium (serum-free culture medium) is supplemented to the required volume; The day of inoculation was designated as D0, and the bioreactor parameters were set to 35 rpm for 5 min; 0 rpm for 2 h of intermittent stirring culture, and the glucose concentration in the culture medium was monitored using a blood glucose meter to determine the volume of the replacement solution; 24 hours after inoculation, D1 was used to measure the glucose content of the culture medium. The volume of the replacement solution was determined according to the glucose concentration of the culture medium. The bioreactor parameters were set to 50 rpm for constant stirring culture. 48 hours after inoculation is D2, and samples are taken for fluorescent staining to observe cell growth; the glucose content of the culture medium is measured, and the volume of the replacement solution is determined according to the glucose concentration of the culture medium. The bioreactor parameters are set to 50rpm uniform stirring culture; D3 is 72 hours after inoculation. Samples are taken for fluorescent staining to observe cell growth. If the microcarriers are full of cells, the microcarriers are replenished with 20 mL of supplementary culture medium per microcarrier. D4 is 96 hours after inoculation. Samples are taken for fluorescent staining to observe cell growth. If the microcarriers are full of cells, the microcarriers are replenished with 40 mL of supplementary culture medium per piece. The bioreactor parameters are set to 50 rpm for uniform stirring culture. D4 is 120 hours after inoculation. Samples are taken for fluorescence staining observation (cell fluorescence staining observation operation procedure) to observe cell growth, and the supernatant is collected to end the culture. 2) Extraction of exosomes: The supernatant collected in the previous step was treated with a tangential flow ultrafiltration system, using a 100 kD ultrafiltration membrane package. The ultrafiltration equipment settings were: temperature 4°C, operating pressure 2 Bar, flow rate control 200 mL / min, and the supernatant was concentrated at a ratio of 100:1 to obtain a concentrate; Use a low-temperature ultracentrifuge and set the temperature at 4°C. Centrifuge the above concentrate at 300×g for 10 min, 2000×g for 10 min, 10000×g for 30 min, and 100000×g for 30 min. g Gradient centrifugation was performed by centrifugation for 70 min × 2 times. The pale yellow precipitate attached to the bottom wall of the centrifuge tube was the exosome. Sterile PBS was used to gently pipette and mix until the exosomes reached 5 mL, so that the exosomes were fully resuspended and stored in a -80 ℃ refrigerator. 3) Identification of exosomes: The morphology of exosomes was observed using a transmission electron microscope, which showed a completely round or quasi-round structure. The size of the exosomes was analyzed using a nanoparticle tracking analyzer (Nanosight), which showed that the particle size was 70-170 nm and the particle concentration was 5.0×10 6 Particles / mL (diluted 1000 times); Western blotting was used to detect the expression of specific membrane proteins TSG101 and CD63 in exosomes, but no negative marker Calnexin protein was expressed; 4) Concentrated exosomes: The concentration of the exosome extract after identification in the previous step was tested according to the requirements of the BCA (Bicinchonininc Acid) protein quantification kit, and the exosome concentration was concentrated to 500 mg / mL to obtain an exosome concentrate; 5) Prepare poloxamer thermosensitive gel: according to the proportion, mix poloxamer 407 20g / L, poloxamer 188 5g / L, and glycerol 10g / L, stir at 4°C with the stirrer set at 500 rpm for 30 min, and then place at 33±1°C for 5 min to form gel, with the pH value being 6.00-6.30, to obtain poloxamer thermosensitive gel; 6) Preparing exosome spray: adding the exosome concentrate, β-glucan and gelatin obtained in step 4) to the poloxamer thermosensitive gel obtained in step 5) to make the exosome concentrate content in the poloxamer thermosensitive gel be 500 mg / L, β-glucan 6 g / L, and gelatin 0.1 g / L, and then using a pH adjuster to adjust the pH to 6-6.3 to prepare the exosome rhinitis thermosensitive gel, and dispense it into 5 ml spray bottles to obtain the mesenchymal stem cell exosome rhinitis spray.

[0007] In the present invention: In step 1), at D4, if the microcarriers can still carry cells, measure the glucose content of the culture medium and determine the volume of the replacement solution based on the glucose concentration of the culture medium.

[0008] The pH adjuster described in step 5) is selected from phosphate buffer solution; β-glucan as an anti-inflammatory agent; gelatin as a system protectant and exosome protectant.

[0009] The present invention also relates to a mesenchymal stem cell exosome rhinitis spray, which is obtained by using the above-mentioned preparation method of the mesenchymal stem cell exosome rhinitis spray and has the following technical indicators: ① Each 1000 ml of the mesenchymal stem cell exosome nasal spray contains: Mesenchymal stem cell exosomes 500mg, Poloxamer 407 20g, Poloxamer 188 5g, Glycerin 10g, β-Glucan 6g, Gelatin 0.1g, The balance is deionized water; ② The exosomes in the mesenchymal stem cell exosome nasal spray have good dispersibility. Laser confocal microscopy showed that the exosomes were bright and dispersed, without aggregation, which is conducive to the safety of drug administration; ③ The mesenchymal stem cell exosome rhinitis spray is temperature-sensitive. It is in a flowing liquid state at 4°C and in a gel state at 33°C±1°C, which is conducive to the retention of drugs in the nasal cavity when the spray is used; ④ The mesenchymal stem cell exosome nasal spray has a good drug sustained-release effect. The cumulative release rate of exosomes within 2 hours was 20.9%, the cumulative release rate within 8 hours was 54.5%, and the cumulative release rate within 24 hours was 76.9%, indicating that the nasal spray can have a good sustained-release effect after forming a gel during nasal administration, avoiding nasal irritation caused by repeated administration.

[0010] The present invention also relates to an application of a mesenchymal stem cell exosome rhinitis spray for preparing a spray product for treating rhinitis.

[0011] Compared with the prior art, the present invention has the following beneficial effects: 1. The preparation method of the mesenchymal stem cell exosome nasal spray described in the present invention uses membrane lipid vesicle exosomes with a diameter of 70-170nm. The vesicles contain a variety of biologically active RNAs, miRNAs and related effector proteins, which can effectively inhibit the occurrence of inflammation and promote damage repair.

[0012] 2. The mesenchymal stem cell exosome rhinitis spray described in the present invention is a spray for treating rhinitis symptoms by utilizing the characteristics of mesenchymal stem cell exosomes to inhibit inflammatory pathways; poloxamer 407, poloxamer 188, and glycerol form a thermosensitive gel, which has the advantages of easy preparation, low irritation, and good biocompatibility; water-soluble thermosensitive gel can also improve the stability of exosomes, storage time, and duration of relief; thermosensitive gel preparations can make drugs adhere to the surface of nasal mucosa, prolong the residence time of drugs, and achieve the purpose of controlled drug release, with the dual advantages of thermosensitive gel and nasal administration. The drug is administered in the form of an aqueous gel spray, which prolongs the residence of the drug and the release rate of exosomes, reduces frequent irritation in the nasal cavity, and is convenient and hygienic without foreign body sensation.

[0013] 3. The mesenchymal stem cell exosome nasal spray described in the present invention comprises the following steps: a large number of mesenchymal stem cells are cultured and expanded in a bioreactor, the supernatant is collected, and the stem cell exosomes are collected by a tangential flow ultrafiltration system; a poloxamer 407 / poloxamer 188 / glycerol thermosensitive gel carrier is prepared, and the thermosensitive gel and the mesenchymal stem cell exosomes are mixed and packaged according to the formula ratio to prepare the nasal spray, which is simple to operate and has safe product ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a transmission electron microscopy image of exosomes derived from mesenchymal stem cells described in an embodiment of the present invention; Figure 2 This is a diameter analysis diagram of exosome nanoparticles derived from mesenchymal stem cells according to an embodiment of the present invention; Figure 3 This is a Western blotting analysis of the surface proteins (TSG101, CD63) of exosomes derived from mesenchymal stem cells and the negative marker Calnexin described in the embodiments of the present invention; Figure 4 This is a diagram of exosome distribution analysis of the water solvent of the mesenchymal stem cell exosome nasal spray described in an embodiment of the present invention under an inverted laser confocal microscope; Figure 5 This is a flow analysis diagram of the water solvent of the mesenchymal stem cell exosome nasal spray described in the embodiment of the present invention at different temperatures (4°C, 34°C); Figure 6 This is a graph showing the release analysis of exosomes in a PBS buffer solution when the mesenchymal stem cell exosome nasal spray is in gel form according to an embodiment of the present invention; Figure 7 This is a graph showing the rhinitis symptom score analysis after 7 days of nasal administration of the mesenchymal stem cell exosome nasal spray described in an embodiment of the present invention to mice; Figure 8This is a graph analyzing the expression levels of inflammatory factors in the serum of mice after 7 days of nasal administration of the mesenchymal stem cell exosome nasal spray described in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] The present invention will be more fully understood through the following detailed description, which should be read in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are only exemplary of the present invention, which can be embodied in various forms. Therefore, the specific functional details disclosed herein should not be interpreted as limiting, but only as the basis of the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in different ways in virtually any appropriate detailed embodiment.

[0016] The present invention is further described in detail below by way of examples, but these examples should not be considered as limiting the present invention.

[0017] Example: A method for preparing a mesenchymal stem cell exosome nasal spray comprises the following steps: 1) Inoculation: sterilize the bioreactor by high pressure, dry it, add sterile culture medium (serum-free culture medium), add microslides at a rate of 20 mL / slide, and mix well; After resuscitation, the concentration of mesenchymal stem cells was 1×10 6 / mL cell suspension, the ratio of cells to microslide during inoculation is 500,000 / slide, the inoculation density is 10mL / slide, and the culture medium (serum-free culture medium) is supplemented to the required volume; The day of inoculation was designated as D0, and the bioreactor parameters were set to 35 rpm for 5 min; 0 rpm for 2 h of intermittent stirring culture, and the glucose concentration in the culture medium was monitored using a blood glucose meter to determine the volume of the replacement solution; 24 hours after inoculation, D1 was used to measure the glucose content of the culture medium. The volume of the replacement solution was determined according to the glucose concentration of the culture medium. The bioreactor parameters were set to 50 rpm for constant stirring culture. 48 hours after inoculation is D2, and samples are taken for fluorescent staining to observe cell growth; the glucose content of the culture medium is measured, and the volume of the replacement solution is determined according to the glucose concentration of the culture medium. The bioreactor parameters are set to 50rpm uniform stirring culture; D3 is 72 hours after inoculation. Samples are taken for fluorescent staining to observe cell growth. If the microcarriers are full of cells, the microcarriers are replenished with 20 mL of supplementary culture medium per microcarrier. 96 hours after inoculation is D4, and samples are taken for fluorescent staining to observe cell growth. If the microcarriers are full of cells, the microslides are replenished with 40 mL / piece of supplementary culture medium. If the microcarriers can still carry cells, the glucose content of the culture medium is measured, and the volume of the supplementary solution is determined according to the glucose concentration of the culture medium. The bioreactor parameters are set to 50 rpm uniform stirring culture; D4 is 120 hours after inoculation. Samples are taken for fluorescence staining observation (cell fluorescence staining observation operation procedure) to observe cell growth, and the supernatant is collected to end the culture. 2) Extraction of exosomes: The supernatant collected in the previous step was treated with a tangential flow ultrafiltration system, using a 100 kD ultrafiltration membrane package, and the ultrafiltration equipment settings were: temperature 4 °C, operating pressure 2 Bar, flow rate control 200 mL / min, and the supernatant was concentrated at a ratio of 100:1 to obtain a concentrated solution; a low-temperature ultracentrifuge was used, the temperature was set to 4 °C, and the above concentrated solution was centrifuged at 300 × g for 10 min, 2 000 × g for 10 min, 10 000 × g for 30 min, and 100 000 × g for 10 min. g Gradient centrifugation was performed by centrifugation for 70 min × 2 times. The pale yellow precipitate attached to the bottom wall of the centrifuge tube was the exosomes. Sterile PBS was used to gently pipette and mix until the exosomes reached 5 mL, so that the exosomes were fully resuspended and stored in a -80 ℃ refrigerator. 3) Identification of exosomes: The morphology of exosomes was observed using a transmission electron microscope, which showed a complete circular or quasi-circular structure. The size of the exosomes was analyzed using a nanoparticle tracking analyzer (Nanosight), which showed that the particle size was 70-170 nm and the particle concentration was 5.0×10 6 Particles / mL (diluted 1000 times); Western blotting was used to detect the expression of specific membrane proteins TSG101 and CD63 in exosomes, but no negative marker Calnexin protein was expressed; 4) Concentrate exosomes: Detect the protein concentration of the exosome extract after identification in step 3) according to the requirements of the BCA protein quantification kit, and concentrate the exosome protein concentration to 500 mg / mL to obtain an exosome concentrate; 5) Prepare poloxamer thermosensitive gel: mix poloxamer 407 20 g / L, poloxamer 188 5 g / L, and glycerol 10 g / L, stir at 4°C, set the stirrer to 500 rpm for 30 min, and obtain poloxamer thermosensitive gel, which is then stored at 4°C. 6) Preparing exosome spray: adding the exosome concentrate obtained in step 4), β-glucan and gelatin to the poloxamer thermosensitive gel obtained in step 5), so that the exosome concentrate protein content in the poloxamer thermosensitive gel is 500 mg / L, β-glucan is 6 g / L, and gelatin is 0.1 g / L, and then using a pH adjuster phosphate buffer solution to adjust the pH to 6-6.3 to prepare an exosome rhinitis thermosensitive gel spray, which is dispensed into 5 mL spray bottles to obtain a mesenchymal stem cell exosome rhinitis spray, and stored at 4°C; 7) Gelation temperature of exosome spray: Place 1 mL of the mesenchymal stem cell exosome nasal spray obtained in step 5) in a 2 mL sample bottle, then place the sample bottle in a heated constant temperature metal bath, raise the temperature at 0.5°C / min, take out the sample bottle every 1 minute and quickly tilt it 45°. It is observed that the temperature at which the exosome thermosensitive gel solution sample cannot flow is 34±1°C; 8) Exosome release of nasal spray: The protein concentration of the mesenchymal stem cell exosome nasal spray obtained in step 2) was determined using a BCA protein quantification kit, and the exosome nasal spray solution was prepared in groups to adjust its concentration to 125ug / mL. After mixing, it was injected into a 24-well plate, 200µL per well, a total of 3 wells, and placed in a 37°C incubator; after gelation, 200µL PBS was added to each well, and the supernatant was taken out at 0, 4, 12, 24, 36, 48, 60, 72, and 96h, respectively, and the protein concentration of the supernatant was determined by the BCA method to obtain the exosome release curve of the mesenchymal stem cell exosome nasal spray in the gel state; the results showed that the cumulative release rate of exosomes within 2 h was 20.9%, the cumulative release rate within 8 h was 54.5%, and the cumulative release rate within 24 h was 76.9%, indicating that the spray has a good sustained release effect after the formation of gel after administration, and can avoid nasal irritation caused by repeated administration.

[0018] Experimental example: Study on the therapeutic effect of exosome nasal spray on mice with allergic rhinitis 1) Establishment of mouse allergic rhinitis model Female 6-8 week old Balb / c mice were housed in an SPF animal room. On Day 0, Day 7 and Day 14, mice were sensitized by intraperitoneal injection of 25 µg of lactalbumin (OVA) and 2 mg of aluminum hydroxide (Al(OH)3) to promote primary sensitization. The control group was intraperitoneally injected with an equal amount of saline. Starting from Day 21, 100 µL of 3% lactalbumin solution was dripped into both sides of the nasal cavity of the mice every day for one week for secondary immunization. The control group was nasally dripped with saline. The scratching and sneezing behaviors of the mice were observed and counted within 10 minutes, and scored according to the behavioral scoring criteria of the rhinitis model. The scoring criteria are as follows: Scratching the nose 1-2 times, sneezing 1-2 times, and runny nose to the nostrils are each scored 1 point; Scratching the nose many times, sneezing 3-10 times, and runny nose beyond the anterior nares were each scored 2 points; Violent scratching of the nose and face, rubbing everywhere, sneezing more than 10 times, and runny nose are each worth 3 points; The scores of each symptom were added together, and a score of 5 or more indicated that the model was successfully established; 2) Therapeutic effect of exosome nasal spray on OVA-induced allergic rhinitis in mice After successful modeling, AR model mice were divided into model blank group (Model), no exosome spray group (N-Exos), positive control group (P-Drug, mometasone furoate nasal spray), low-dose exosome spray group (L-Exos), and high-dose exosome group (H-Exos), with 4 mice in each group; a normal control group (Control) was also set up without any treatment. The blank group was given 20µL of normal saline per nostril, the positive control group was given 20µL of mometasone furoate nasal spray per nostril, and the exosome spray group was given 20µL of exosome spray, among which the exosome concentration in the low-dose group was 1 / 2 of that in the high-dose group; AR mice in each group were given continuous medication for 7 days, and 3% OVA solution was given nasal drops every day to maintain sensitization, and the mice were observed and counted for 10 minutes of behavior on the 8th to 10th day, and the levels of inflammatory factors in the serum of mice in each group were detected; 3) Therapeutic results of exosome nasal spray on OVA-induced allergic rhinitis in mice Female Balb / c mice were sensitized by intraperitoneal injection of OVA for three consecutive weeks. In the fourth week, 3% OVA solution was used for nasal challenge. The behavioral observation and statistical scoring of mice within ten minutes after the last three OVA challenges showed that the number of nose scratching, sneezing and runny nose in the model group mice increased significantly, and the AR score was significantly higher than that in the control group ( p <0.0001, Figure 7 Control vs. Model), and the scoring results showed that the AR model mice were successfully established; After one week of 3% OVA stimulation, the AR model mice were successfully modeled. The nasal cavity of each group of mice was intervened by exosome-free spray, mometasone furoate nasal spray, low-dose exosome spray, and high-dose exosome spray. The behavioral observation statistics on the last two days showed that the low-dose exosome spray, high-dose exosome spray, and mometasone furoate nasal spray groups were able to improve the number of mice scratching their noses and sneezing; compared with mometasone furoate nasal spray, high-dose exosome spray had a better improvement on the symptoms of mice scratching their noses and sneezing ( Figure 7 ); At the same time, the expression levels of allergic inflammatory cytokines in the serum of each group of mice were detected. The results showed that compared with the model group and the group without exosome spray, the IL-2 level in each treatment group was significantly increased ( Figure 8A), IL-4 ( Figure 8 B) IL-17 Figure 8 C) TNF-α ( Figure 8 D) The level in mouse serum was significantly reduced, indicating that mesenchymal stem cell exosomes can inhibit the expression of inflammation in rhinitis mice through immunomodulatory effects, and the expression level of inflammatory factors in the high-dose exosome group is closer to that in the normal group of mice.

[0019] Summary and discussion: Technical advantages of this application: 1. Using bioreactor and tangential flow ultrafiltration system, stem cell exosomes are enriched in large quantities with high purity and controllable concentration, which is convenient for mass production and quality control.

[0020] 2. Use membrane lipid vesicle exosomes with a diameter of 70-170nm (see Figure 2 The exosomes of mesenchymal stem cells are described in the particle size analysis chart and the identification section (Figure 2). They contain a variety of biologically active RNA, miRNA, DNA and proteins, and have similar properties to mesenchymal stem cells. In addition, the exosomes of mesenchymal stem cells are tiny in size, can be loaded with lipids, proteins, nucleic acids and other signal substances, are easy to absorb and release, and have no immunogenicity.

[0021] 3. A thermosensitive gel carrier is used to combine mesenchymal stem cell exosomes so that the product can be evenly sprayed in the nasal cavity. The temperature of the nasal cavity is used to quickly adsorb the gel in the nasal cavity, allowing the exosomes to reside and continuously release. This solution solves the problem of difficulty in applying the drug to the nasal cavity and difficulty in retaining the drug.

[0022] 4. The technology of encapsulating exosomes with thermosensitive gel is used, and the temperature of the nasal cavity is used to transform the spray from a flowing liquid to a gel, which can make the exosomes more evenly distributed and more active.

[0023] 5. The water-based spray gel has good affinity; the product is mild and non-irritating, and the application of medicine is convenient and hygienic without any foreign body sensation.

[0024] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a mesenchymal stem cell exosome nasal spray, characterized in that: The following steps are involved: 1) Inoculation: sterilize the bioreactor by high pressure, dry it, add serum-free medium, add microslides at a rate of 20 mL / slide, and mix well; After resuscitation, the concentration of mesenchymal stem cells was 1×10 6 / mL cell suspension, the ratio of cells to microslide during inoculation is 500,000 / slide, the inoculation density is 10 mL / slide, and serum-free medium is supplemented to the required volume; The day of inoculation was designated as D0, and the bioreactor parameters were set to 35 rpm for 5 min; 0 rpm for 2 h of intermittent stirring culture, and the glucose concentration in the culture medium was monitored using a blood glucose meter to determine the volume of the replacement solution; 24 hours after inoculation, D1 was used to measure the glucose content of the culture medium. The volume of the replacement solution was determined according to the glucose concentration of the culture medium. The bioreactor parameters were set to 50 rpm for constant stirring culture. 48 hours after inoculation is D2, and samples are taken for fluorescent staining to observe cell growth; the glucose content of the culture medium is measured, and the volume of the replacement solution is determined according to the glucose concentration of the culture medium. The bioreactor parameters are set to 50rpm uniform stirring culture; D3 is 72 hours after inoculation. Samples are taken for fluorescent staining to observe cell growth. If the microcarriers are full of cells, the microcarriers are replenished with 20 mL of supplementary culture medium per microcarrier. D4 is 96 hours after inoculation. Samples are taken for fluorescent staining to observe cell growth. If the microcarriers are full of cells, the microcarriers are replenished with 40 mL of supplementary culture medium per piece. The bioreactor parameters are set to 50 rpm for uniform stirring culture. D4 was 120 hours after inoculation, and samples were taken for fluorescent staining to observe cell growth, and the supernatant was collected to terminate the culture; 2) Extraction of exosomes: The supernatant collected in the previous step was treated with a tangential flow ultrafiltration system, using a 100 kD ultrafiltration membrane package. The ultrafiltration equipment settings were: temperature 4°C, operating pressure 2 Bar, flow rate control 200 mL / min, and the supernatant was concentrated at a ratio of 100:1 to obtain a concentrate; Use a low-temperature ultracentrifuge at 4°C and centrifuge the concentrate at 300 × g for 10 min, 2000 × g for 10 min, 10000 × g for 30 min, and 100000 × g for 30 min. g Gradient centrifugation was performed by centrifugation for 70 min × 2 times. The pale yellow precipitate attached to the bottom wall of the centrifuge tube was the exosomes. Sterile PBS was used to gently pipette and mix until the exosomes reached 5 mL, so that the exosomes were fully resuspended and stored in a -80 ℃ refrigerator. 3) Identification of exosomes: The morphology of exosomes was observed using a transmission electron microscope, which showed a completely round or quasi-round structure. The size of the exosomes was analyzed using a nanoparticle tracking analyzer, which showed that the particle size was 70-170 nm and the particle concentration was 5.0×10 6 Particles / mL; Western blotting was used to detect the expression of specific membrane proteins TSG101 and CD63 in exosomes, but no negative marker Calnexin protein was expressed; 4) Concentrate exosomes: Detect the protein concentration of the exosome extract after identification in step 3) above according to the requirements of the BCA protein quantification kit, and concentrate the exosome protein concentration to 500 mg / mL to obtain an exosome concentrate; 5) Prepare poloxamer thermosensitive gel: according to the proportion, mix poloxamer 407 20g / L, poloxamer 188 5g / L, and glycerol 10g / L, stir at 4°C with the stirrer set at 500 rpm for 30 min, and then place at 33±1°C for 5 min to form gel, with the pH value being 6.00-6.30, to obtain poloxamer thermosensitive gel; 6) Preparing exosome spray: adding the exosome concentrate obtained in step 4), β-glucan and gelatin to the poloxamer thermosensitive gel obtained in step 5), so that the exosome concentrate protein content in the poloxamer thermosensitive gel is 500 mg / L, β-glucan is 6 g / L, and gelatin is 0.1 g / L, and then using a pH adjuster phosphate buffer solution to adjust the pH to 6-6.3 to prepare an exosome rhinitis thermosensitive gel spray, which is dispensed into 5 mL spray bottles to obtain a mesenchymal stem cell exosome rhinitis spray, and stored at 4°C; The mesenchymal stem cell exosome nasal spray has the following technical indicators: ① Each 1000 ml of the exosome nasal spray contains: Mesenchymal stem cell exosomes 500mg, Poloxamer 407 20g, Poloxamer 188 5g, Glycerin 10g, β-Glucan 6g, Gelatin 0.1g, The balance is deionized water; ② The exosomes in the exosome rhinitis spray have good dispersibility. Laser confocal microscopy showed that the exosomes were bright and dispersed, without aggregation; ③ Exosome rhinitis spray is temperature-sensitive, in the form of a flowing liquid at 4°C and in the form of a gel at 33°C±1°C; ④ The exosome nasal spray has a good drug sustained-release effect. The cumulative release rate of exosomes is 20.9% within 2 hours, 54.5% within 8 hours, and 76.9% within 24 hours.

2. The method for preparing a mesenchymal stem cell exosome nasal spray according to claim 1, characterized in that: The pH adjuster described in step 5) is selected from phosphate buffer solution; β-glucan as an anti-inflammatory agent; gelatin as a system protectant and exosome protectant.

3. A mesenchymal stem cell exosome nasal spray, characterized in that: The preparation method of the mesenchymal stem cell exosome nasal spray according to claim 1 or 2 is obtained, and has the following technical indicators: ① Each 1000 ml of the exosome nasal spray contains: Mesenchymal stem cell exosomes 500mg, Poloxamer 407 20g, Poloxamer 188 5g, Glycerin 10g, β-Glucan 6g, Gelatin 0.1g, The balance is deionized water; ② The exosomes in the exosome rhinitis spray have good dispersibility. Laser confocal microscopy showed that the exosomes were bright and dispersed, without aggregation; ③ Exosome rhinitis spray is temperature-sensitive, in the form of a flowing liquid at 4°C and in the form of a gel at 33°C±1°C; ④ The exosome rhinitis spray has a good drug sustained-release effect. The cumulative release rate of exosomes is 20.9% within 2 hours, 54.5% within 8 hours, and 76.9% within 24 hours.

4. The use of a mesenchymal stem cell exosome nasal spray according to claim 3, characterized in that: Used to prepare spray products for treating rhinitis.

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