A preparation method of exosomes encapsulating microRNA and its products and applications

Abstract

The present invention provides a method for preparing exosomes encapsulating microRNA, and its products and applications. The exosomes encapsulating microRNA are directly secreted by mesenchymal stem cells. The exosomes obtained from each 15 mL of adipose tissue are calculated by protein content. The total protein of the exosomes encapsulated by microRNA can reach 95.13 μg (SD=8.43 μg), and the total RNA content can reach 201.25 μg (SD=31.00 μg). The total protein content of the exosomes not encapsulated by microRNA is only 74.83 μg (SD=6.31 μg), and the total RNA content is only 51.67 μg (SD=10.67 μg). The exosomes prepared by this method have more content and a higher total RNA content. The preparation method provided by the present invention can reduce steps, increase yield and reduce costs in drug production.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for preparing exosomes encapsulating microRNA, and a product and application thereof. Background Art

[0002] COPD is a type of chronic bronchitis and / or emphysema characterized by airflow obstruction, which is associated with abnormal inflammatory responses to harmful gases and particles. Clinical symptoms include chronic cough, sputum, shortness of breath or dyspnea. Therapeutic drugs for COPD mainly include glucocorticoids, immunosuppressants, bronchodilators and targeted drugs.

[0003] Patent CN103830299A discloses a pharmaceutical composition for treating chronic obstructive pulmonary disease, which contains total flavonoids and total saponins. The active ingredients are extracted from the Chinese medicines Aster and Coltsfoot to prepare a composition containing total saponins represented by Aster saponin E and total flavonoids represented by rutin. The raw materials of the pharmaceutical composition are easily available, low in cost and easy to industrialize. The pharmaceutical composition can be made into corresponding dosage forms according to needs, providing safe, effective and quality-controlled modern Chinese medicine for clinical treatment.

[0004] Patent CN109498625A discloses a pharmaceutical composition for treating chronic obstructive pulmonary disease, which comprises two active ingredients, namely, thiazine dione from Xanthium sibiricum and glucocorticoid. Both thiazine dione from Xanthium sibiricum and the combination of thiazine dione from Xanthium sibiricum and glucocorticoid can effectively improve the lung function of patients with chronic obstructive pulmonary disease and alleviate related clinical symptoms, wherein the combined use of thiazine dione from Xanthium sibiricum and glucocorticoid can significantly reduce the effective dose of glucocorticoid while maintaining similar therapeutic effects, thereby reducing the use of glucocorticoid and thus reducing the side effects of glucocorticoid.

[0005] Since COPD is a chronic disease of aging and irreversibility, current drugs cannot reverse existing lesions, and the long-term use of glucocorticoids and immunosuppressants can lead to multiple complications and endanger health. COPD patients are in urgent need of therapeutic drugs that can reverse lesions and have high safety. Therefore, it is important to provide a safe and effective drug for the treatment of COPD. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing exosomes encapsulating microRNA, and its products and applications. The exosomes encapsulating microRNA are obtained by direct secretion of mesenchymal stem cells. The yield of exosomes obtained by direct secretion of mesenchymal stem cells is higher. The yield of exosomes obtained from 15 mL of adipose tissue is calculated by protein content. The total protein of exosomes encapsulated by microRNA can reach 95.13 μg (SD=8.43 μg), while the total protein content of exosomes not encapsulated by the microRNA is only 74.83 μg (SD=6.31 μg); the total RNA content of exosomes encapsulated by microRNA can reach 201.25 μg (SD=31.00 μg), while the total RNA content of exosomes not encapsulated by the microRNA is only 51.67 μg (SD=10.67 μg). The exosomes prepared by this method have more content (in terms of protein content) and a higher total RNA content.

[0007] The preparation method provided by the present invention can reduce the steps, increase the yield and reduce the cost in drug production. At the same time, the exosomes encapsulating microRNA have a low risk of tumor formation during treatment because they do not have a complete cell structure, and the treatment is safer; they can improve the aging phenotype of chronic obstructive pulmonary disease, reduce inflammatory cells in lung tissue (such as neutrophils, CD8 + T cells, etc.), reduce the levels of inflammatory factors (such as tumor necrosis factor α, IL-8, etc.) and prevent the process of pulmonary fibrosis.

[0008] The SD rat COPD model was used to evaluate the effect of exosomes containing or not containing microRNA on reducing the level of serum inflammatory factors. Twenty 8-week-old SD rats were selected and the COPD model of rats was established by lipopolysaccharide and smoke induction in the respiratory tract. Peripheral blood was collected before and 1 week after induction to detect the levels of inflammatory factors TNFα, IL-6 and IL-1β in serum. Before induction, the average levels of serum TNFα, IL-6 and IL-1β in 20 SD rats were 45.8p·m·s and 1p·m·s, respectively. g / mL (SD = 9.1pg / mL), 19.9ng / mL (SD = 2.3ng / mL), 27.9pg / mL (SD = 4.1pg / mL); 1 week after induction, the average levels of serum TNFα, IL-6 and IL-1β in 20 SD rats were 284.9pg / mL (SD = 34.9pg / mL), 61.8ng / mL (SD = 6.4ng / mL), 163.8pg / mL (SD = 7.0pg / mL). The mice were randomly divided into two groups, receiving exosome preparations without microRNA encapsulation (cohort 1) and after microRNA encapsulation (cohort 2) respectively. The treatment method was to drip 20μL of the preparation into the respiratory tract twice a day for 10 days. After treatment, serum TNFα, IL-6 and IL-1β levels were measured. The average levels of TNFα, IL-6 and IL-1β in cohort 1 were 204.3pg / mL (SD = 25.4pg / mL), 53.3ng / mL (SD = 4.7ng / mL), and 117.6pg / mL (SD = 14.4pg / mL), respectively; the average levels of TNFα, IL-6 and IL-1β in cohort 2 were 149pg / mL (SD = 13.1pg / mL), 32.5ng / mL (SD = 3.4ng / mL), and 94.6pg / mL (SD = 8.4pg / mL), respectively. Both types of exosome preparations can significantly reduce the level of serum inflammatory factors, but the exosome preparations encapsulating microRNA are more effective, especially in reducing IL-6. In the present invention, on the one hand, exosomes are good drug delivery carriers that carry target microRNAs that regulate gene expression. On the other hand, exosomes themselves also contain macromolecular substances such as proteins, lipids and nucleic acids that can play a corresponding therapeutic role. The two work together to achieve the technical effect of targeted regulation of gene expression and protein synthesis in tissue cells.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for preparing exosomes encapsulating microRNA, the method for preparing exosomes encapsulating microRNA comprising:

[0011] (1) isolating and culturing mesenchymal stem cells, mixing the mesenchymal stem cells with an electroporation working solution to obtain a mixed solution, and mixing a microRNA mother solution with the mixed solution to obtain a pre-transfection solution;

[0012] (2) electroporating the pre-transfection solution in step (1);

[0013] (3) culturing the electroporated mesenchymal stem cells to obtain a culture medium;

[0014] (4) collecting the exosomes encapsulating the microRNA from the culture medium.

[0015] Mesenchymal stem cells are a type of living cells with the ability to self-renew and differentiate, with strong tissue repair ability and anti-inflammatory properties. However, mesenchymal stem cells have the risk of tumorigenesis in clinical applications, which limits their application.

[0016] Preferably, step (1) specifically includes:

[0017] The tissue is digested and cultured with a digestion solution to obtain a mesenchymal stem cell suspension, and the mesenchymal stem cells are obtained by centrifugation. The mesenchymal stem cells are mixed with an electroporation working solution to obtain a mixed solution, and the microRNA mother solution is mixed with the mixed solution to obtain a pre-transfection solution.

[0018] Preferably, the digestion solution is 0.05-0.2% trypsin digestion solution, for example, it can be 0.05%, 0.1%, 0.15% or 0.2%.

[0019] Preferably, the culture medium used in the culture is a serum-free culture medium, the culture time is 30 to 36 hours (for example, 30 hours, 32 hours, 34 hours or 36 hours, etc.), the culture temperature is 37° C., and the carbon dioxide concentration in the culture is 5%.

[0020] Preferably, the temperature of the centrifugal treatment is 0-8°C (for example, it can be 0°C, 4°C, 6°C or 8°C, etc.), the rotation speed of the centrifugal treatment is 1100-1200rpm (for example, it can be 1100rpm, 1150rpm or 1200rpm, etc.), and the time of the centrifugal treatment is 5-8min (for example, it can be 5min, 6min, 7min or 8min, etc.).

[0021] Preferably, the concentration of total RNA added to the mixed solution in step (1) is 0.192-2.88 μg / μL, for example, 0.192 μg / μL, 0.216 μg / μL, 0.264 μg / μL or 0.2888 μg / μL.

[0022] The total amount of microRNA added to the mixed solution is 960-1440 μg (the concentration of microRNA added to the mixed solution is 0.192-0.288 μg / μL), for example, the total amount of microRNA added can be 960 μg, 1080 μg, 1200 μg, 1320 μg or 1440 μg, etc. Preferably, the total microRNA concentration of the microRNA mother solution is 3.2-4.8 μg / μL, for example, it can be 3.2 μg / μL, 3.6 μg / μL, 4.0 μg / μL, 4.4 μg / μL or 4.8 μg / μL, etc., and the OD value of the absorbance at 260 nm wavelength is 0.8-1.2.

[0023] Preferably, the microRNA mother solution includes any one of hsa-mir-218-1, hsa-mir-365a, hsa-mir-29a or hsa-mir-324-5p, or a combination of at least two of them.

[0024] MicroRNA is a type of non-coding single-stranded RNA molecule encoded by endogenous genes, with a length of about 22 nucleotides. MicroRNA participates in the regulation of post-transcriptional gene expression. MicroRNA and other nucleic acid drugs have low cellular uptake efficiency during use, which seriously limits their application in treatment. The present invention transfers the target microRNA into mesenchymal stem cells through electroporation, and after culturing, exosomes encapsulating the target microRNA are isolated, and the exosomes encapsulating the target microRNA can play a role in regulating cellular gene expression.

[0025] Preferably, the ratio of hsa-mir-218-1, hsa-mir-365a, hsa-mir-29a and hsa-mir-324-5p in the microRNA mother solution includes (3-4):(1-2):(1-2):1, for example, it can be 3:1:1:1, 4.5:1.5:1.5:1 or 4:2:2:1, etc.

[0026] Preferably, the volume ratio of the microRNA mother solution to the mixed solution is 3:(400-600), for example, it can be 3:400, 3:500 or 3:450.

[0027] In the present invention, by adjusting the OD value of the mixed solution, the OD value of the microRNA mother solution, the ratio of each component in the microRNA mother solution, and the volume ratio of the microRNA mother solution and the mixed solution, a higher electroporation efficiency can be achieved.

[0028] Preferably, the mesenchymal stem cells include adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells.

[0029] Preferably, the voltage of the electroporation in step (2) is 200-220 V (for example, 200 V, 210 V or 220 V, etc.), the shock duration of the electroporation is 4-6 s (for example, 4 s, 5 s or 6 s, etc.), and the pulse length of the electroporation is 20-25 ms (for example, 20 ms, 21 ms, 22 ms, 23 ms, 24 ms or 25 ms, etc.).

[0030] Preferably, the culture medium used in the culture in step (3) is a serum-free culture medium, the culture time is 20 to 24 hours (for example, it can be 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, etc.), the culture temperature is 37°C, and the carbon dioxide concentration of the culture is 5%.

[0031] Preferably, step (4) specifically includes:

[0032] The culture solution is centrifuged once, twice and three times in sequence to obtain the exosomes encapsulating the microRNA.

[0033] Preferably, the temperature of the centrifugation is 0-8°C (for example, 0°C, 4°C, 6°C or 8°C, etc.), the rotation speed of the centrifugation is 1800-2000rpm (for example, 1800rpm, 1900rpm or 2000rpm, etc.), and the time of the centrifugation is 15-30min (for example, 15min, 20min, 25min or 30min, etc.).

[0034] Preferably, the temperature of the secondary centrifugation is 0-8°C (for example, it can be 0°C, 4°C, 6°C or 8°C, etc.), the speed of the secondary centrifugation is 2500-3000rpm (for example, it can be 2500rpm, 2700rpm or 3000rpm, etc.), and the time of the secondary centrifugation is 30-40min (for example, it can be 30min, 32min, 35min, 37min or 40min, etc.).

[0035] Preferably, the temperature of the three centrifugations is 0-8°C (for example, it can be 0°C, 4°C, 6°C or 8°C, etc.), the rotation speed of the three centrifugations is 11000-12000rpm (for example, it can be 11000rpm, 11500rpm or 12000rpm, etc.), and the time of the three centrifugations is 50-60min (for example, it can be 50min, 52min, 55min, 57min or 60min, etc.).

[0036] The present invention separates exosomes from cell culture fluid by differential centrifugation, first uses a low speed to remove cells and apoptotic debris, increases the speed to remove large vesicles, and finally uses high-speed centrifugation to precipitate exosomes.

[0037] In a second aspect, the present invention provides exosomes encapsulating microRNA, prepared by the method for preparing exosomes encapsulating microRNA according to the first aspect.

[0038] Exosomes derived from mesenchymal stem cells do not have a complete cell structure, so they have higher safety. At the same time, they can improve the aging phenotype of chronic obstructive pulmonary disease, reduce inflammatory cells in lung tissue, reduce the level of inflammatory factors and prevent the process of pulmonary fibrosis. In the present invention, exosomes are good drug delivery carriers on the one hand, loaded with microRNA that has the function of regulating gene expression, and on the other hand, exosomes themselves also have macromolecular substances such as proteins, lipids and nucleic acids, which can play a corresponding therapeutic role. The two work together to play a technical effect of targeted regulation of gene expression and protein synthesis in tissue cells.

[0039] In a third aspect, the present invention provides an exosome composition, comprising the microRNA-encapsulated exosomes and mesenchymal stem cell exosomes described in the second aspect.

[0040] The exosome composition described in the present invention is exosomes secreted by co-culturing mesenchymal stem cells that have been electroporated with microRNA and mesenchymal stem cells that have not been electroporated with microRNA.

[0041] The exosome composition described in the present invention includes exosomes containing target microRNA and exosomes without target microRNA. Exosomes containing target microRNA can play a role in regulating related metabolism and anti-inflammatory repair. Mesenchymal stem cells into which target microRNA is electrotransferred are co-cultured with untreated mesenchymal stem cells, which can also promote the secretion of exosomes of the latter. Exosomes without target microRNA also have macromolecular substances such as proteins, lipids and nucleic acids, which can play the role of exosome anti-inflammatory repair. The two can achieve better therapeutic effects by matching each other. The exosome preparation method increases the exosome yield calculated by protein content by 27.13%, and the exosomes produced by 15mL of mesenchymal stem cells contain 95.13μg (SD=8.43μg) of protein. Compared with directly introducing microRNA into exosomes, the yield of the exosome composition provided by the present invention is increased by 289.47%, and the total RNA content of exosomes produced by 15mL of mesenchymal stem cells reaches 201.25μg (SD=31.00μg).

[0042] In a fourth aspect, the present invention provides a method for preparing the exosome composition according to the third aspect, the method for preparing the exosome composition comprising:

[0043] Electroporated mesenchymal stem cells are prepared according to steps (1) and (2) of the method for preparing exosomes encapsulating microRNA described in the first aspect, and the electroporated mesenchymal stem cells are added to a cell suspension of mesenchymal stem cells that have not been electroporated for mixed culture to obtain a culture fluid, and the exosome composition is collected.

[0044] Preferably, the mixed culture time is 20 to 24 hours (for example, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, etc.), the mixed culture temperature is 37° C., and the carbon dioxide concentration of the mixed culture is 5%.

[0045] In the present invention, the activity of mesenchymal stem cells after electroporation is reduced and the yield of exosomes is limited. Therefore, adding electroporated mesenchymal stem cells to a cell suspension of mesenchymal stem cells that has not been electroporated for mixed culture can increase the yield of exosomes and help reduce costs.

[0046] In a fifth aspect, the present invention provides a pharmaceutical composition, comprising the microRNA-encapsulated exosomes described in the second aspect and / or the exosome composition described in the third aspect.

[0047] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0048] In a sixth aspect, the present invention provides a use of any one or a combination of at least two of the exosomes encapsulating microRNA described in the second aspect, the exosome composition described in the third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug for treating lung diseases.

[0049] Preferably, the pulmonary disease comprises chronic obstructive pulmonary disease.

[0050] Preferably, the dosage form of the drug is an aerosol dosage form.

[0051] The pulmonary inhalation administration method for chronic obstructive pulmonary disease is a local medication. Theoretically, the dosage is small and it acts directly on the target organs. The therapeutic effect of aerosol formulations is expected to be better than systemic oral administration.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The exosomes encapsulated with microRNA in the present invention are directly secreted by mesenchymal stem cells. Compared with the exosomes directly secreted by mesenchymal stem cells after microRNA is electroporated to mesenchymal stem cells, the yield of exosomes obtained from 15 mL of adipose tissue is higher. The total protein of exosomes encapsulated with microRNA can reach 95.13 μg (SD=8.43 μg), while the total protein content of exosomes not encapsulated with the microRNA is only 74.83 μg (SD=6.31 μg); the total RNA content of exosomes encapsulated with microRNA can reach 201.25 μg (SD=31.00 μg), while the total RNA content of exosomes not encapsulated with the microRNA is only 51.67 μg (SD=10.67 μg). The exosomes prepared by this method have a higher content (in terms of protein content) and a higher total RNA content. The preparation method of the present invention can reduce steps and reduce costs in drug production.

[0054] (2) The exosomes derived from mesenchymal stem cells in the present invention have a low risk of tumor formation during treatment because they do not have a complete cell structure, and the treatment is safer; they can improve the aging phenotype of chronic obstructive pulmonary disease, reduce the number of inflammatory cells in lung tissue (such as neutrophils, CD8 + T cells, etc.) and reduce the level of inflammatory factors (such as tumor necrosis factor α, IL-8, etc.) and prevent the process of pulmonary fibrosis. The SD rat model experiment of chronic obstructive pulmonary disease showed that the exosomes derived from mesenchymal stem cells can significantly reduce the level of inflammatory factors in serum, and the exosomes encapsulated by microRNA have better effects (such as Figure 2 ). In the present invention, exosomes are good drug delivery carriers on the one hand, loaded with microRNA that regulates gene expression, and on the other hand, exosomes themselves also contain macromolecular substances such as proteins, lipids and nucleic acids, which can play a corresponding therapeutic role. The two work together to achieve the technical effect of targeted regulation of gene expression and protein synthesis in tissue cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a comparison chart of the total protein and total RNA contents of exosomes encapsulated by microRNA and exosomes not encapsulated by microRNA in Test Example 2.

[0056] Figure 2 This is a statistical chart of the treatment results of SD rats with chronic obstructive pulmonary disease using exosome preparations without microRNA encapsulation and with microRNA encapsulation in Test Example 3. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0059] Source of experimental materials and models of experimental instruments:

[0060] EDTA solution: ThermoFisher EDTA 0.1 mM, pH 8.0, RNase-free, Cat. No. AM9912.

[0061] Trypsin: ThermoFisher GIBCO Trypsin, catalog number R001100.

[0062] PBS solution: Invitrogen PBS-phosphate buffered saline (10×), pH 7.4, RNase-free, catalog number AM9625, diluted to 1× with double distilled water before use.

[0063] Serum-free medium: EX-CELL EBx GRO-I serum-free medium, catalog number 24530C.

[0064] TE buffer: Invitrogen, catalog number: AM9849.

[0065] Transfection kit: Amaxa Lonza Cell Line Nucleofector Kit V, Amaxa Lonza, model: VCA-1003.

[0066] RNA extraction kit: Invitrogen PureLink RNA micro extraction kit, catalog number 12183016.

[0067] Lonza cell nuclear transfection instrument: 4D-NucleofectorTM LV.

[0068] BCA protein concentration assay kit (enhanced): Biyuntian, product number P0010S.

[0069] RIPA lysis buffer: Bio-Tech, catalog number P0013B.

[0070] The preparation steps of adipose-derived mesenchymal stem cells are as follows:

[0071] A digestion solution containing 0.2% trypsin was prepared by diluting trypsin with 0.4% EDTA solution.

[0072] Digest 10-20 g of adipose tissue with 30 mL of trypsin digestion solution at 37°C for 10 min, gently shake the digested cell suspension, and transfer it to a centrifuge tube. Digest the remaining tissue again with 20 mL of trypsin digestion solution at 37°C for 5 min, gently shake the digested suspension, and transfer it to a centrifuge tube.

[0073] The collected digested cell suspension was centrifuged at 1000 rpm for 3 min, the supernatant was discarded, 10 mL of PBS solution was added for shaking and washing, and then centrifuged at 1000 rpm for 3 min, and the supernatant was discarded; the washing step was repeated once to obtain adipose-derived mesenchymal stem cells after digestion and separation.

[0074] Example 1

[0075] This embodiment provides a method for preparing exosomes encapsulating microRNA, and the method for preparing exosomes encapsulating microRNA is as follows:

[0076] The digested and separated adipose-derived mesenchymal stem cells were resuspended in 15 mL of PBS solution, and 10 mL of the solution was cultured in 60 mL of serum-free medium at 37° C. and 5% carbon dioxide concentration for 36 h to obtain a cell suspension.

[0077] Prepare microRNA master solution: dissolve the synthesized microRNA in TE buffer according to the ratio in Table 1 and prepare 2 mL microRNA master solution, wherein the total RNA concentration of the microRNA master solution is 4.0 μg / μL (calculated as 1 according to the absorbance value OD at a wavelength of 260 nm).

[0078] The nucleotide sequence of hsa-mir-218-1 is shown in SEQ ID NO.1, the nucleotide sequence of hsa-mir-365a is shown in SEQ ID NO.2, the nucleotide sequence of hsa-mir-29a is shown in SEQ ID NO.3, and the nucleotide sequence of hsa-mir-324-5p is shown in SEQ ID NO.4.

[0079] Table 1

[0080]

[0081]

[0082] The cell suspension cultured for 36 hours was centrifuged at 1200 rpm for 5 minutes at 4°C, the supernatant was discarded, and the obtained cell pellet was suspended using a transfection kit according to the instructions, the volume of the suspension used was 35 mL, and a mixed solution was obtained.

[0083] Add 210 μL of microRNA stock solution to 35 mL of the mixture, pipette evenly, and the microRNA concentration in the mixture is 0.24 μg / μL. Add the mixture in batches to the Lonza cell nuclear transfection instrument, and electroporate at 220 V for 5 s with a pulse length of 20 ms. Add the electroporated cells to serum-free medium and continue culturing for 24 h.

[0084] To harvest exosomes, use a low-temperature centrifuge to centrifuge the culture solution at 2000 rpm for 15 min at 4°C, and take the supernatant; continue to centrifuge the supernatant at 3000 rpm for 30 min at 4°C, and take the supernatant. Centrifuge the supernatant at 12000 rpm for 50 min at 4°C, discard the supernatant, and obtain the precipitate, which is the prepared exosomes.

[0085] Add 10 mL of PBS solution and shake to dissolve the precipitate to obtain adipose-derived mesenchymal stem cell-derived exosomes carrying microRNA.

[0086] Example 2

[0087] This embodiment provides a method for preparing an exosome composition, and the method for preparing the exosome composition is as follows:

[0088] The digested and separated adipose stem cells were resuspended in 15 mL of PBS solution, and 10 mL of the solution was cultured in 60 mL of serum-free culture medium at 37° C. and 5% carbon dioxide concentration for 36 h to obtain a cell suspension.

[0089] Prepare microRNA master solution: dissolve the synthesized microRNA in TE buffer according to the ratio in Table 1 and prepare 2 mL microRNA master solution, wherein the total RNA concentration of the microRNA master solution is 40 μg / μL (calculated as 1 according to the absorbance value OD at a wavelength of 260 nm).

[0090] 10 mL of the cell suspension cultured for 36 h was taken and centrifuged at 1200 rpm for 5 min at 4°C, the supernatant was discarded, and the obtained cell pellet was suspended using a transfection kit according to the instructions, with a suspension volume of 5 mL to obtain a mixed solution.

[0091] Add 30 μL of microRNA stock solution to 5 mL of the mixture, pipette evenly, and the microRNA concentration in the mixture is 0.24 μg / μL. Add it to the Lonza cell nuclear transfection instrument in batches, and electroporate at 220 V for 5 seconds with a pulse length of 20 ms. Add the electroporated cells to the remaining cell suspension that has not been electroporated, and continue to culture for 24 hours.

[0092] To harvest exosomes, use a low-temperature centrifuge to centrifuge the culture solution at 2000 rpm for 15 min at 4°C, and take the supernatant; continue to centrifuge the supernatant at 3000 rpm for 30 min at 4°C, and take the supernatant. Centrifuge the supernatant at 12000 rpm for 50 min at 4°C, discard the supernatant, and obtain the precipitate, which is the prepared exosomes.

[0093] 10 mL of PBS solution was added and shaken to dissolve the precipitate to obtain the exosome composition.

[0094] Comparative Example 1

[0095] This embodiment provides a method for preparing exosomes, and the method for preparing exosomes is as follows:

[0096] The adipose-derived mesenchymal stem cells after digestion and separation were resuspended in 15 mL of PBS solution, and 10 mL of the solution was cultured in 60 mL of serum-free medium at 37° C. and 5% carbon dioxide concentration for 70 h.

[0097] Take 10 mL of the cell suspension cultured for 70 hours to harvest exosomes. Use a low-temperature centrifuge to centrifuge the culture solution at 2000 rpm for 15 minutes at 4°C and take the supernatant; continue to centrifuge the supernatant at 3000 rpm for 30 minutes at 4°C and take the supernatant. Centrifuge the supernatant at 12000 rpm for 50 minutes at 4°C, discard the supernatant, and obtain the precipitate, which is the prepared exosomes.

[0098] The yields of exosomes obtained by the preparation methods of Examples 1 to 2 and Comparative Example 1 are shown in Table 2.

[0099] Table 2

[0100]

[0101] As can be seen from Table 1, in Example 1, all mesenchymal stem cells were electroporated with microRNA, and in Example 2, some mesenchymal stem cells (10 mL) were electroporated with microRNA. There was little difference between Example 1 and Example 2 in terms of total exosome RNA and total protein reflecting the amount of exosome preparation. Electroporation of all mesenchymal stem cells increased the difficulty of exosome preparation and material requirements. Comparative Example 1 was that all mesenchymal stem cells were not electroporated with microRNA, and the total protein and total RNA content of the prepared exosomes were low.

[0102] Test Example 1

[0103] This test example provides an exosome total RNA content to evaluate the harvest quality. The RNA concentration is detected by spectrophotometry. The total RNA is extracted using an RNA extraction kit according to the instructions. The specific detection steps are as follows:

[0104] 1 mL of the exosome preparation was centrifuged at 15,000 rpm for 60 min at 4°C, the supernatant was discarded, 100 μL of lysis buffer was added to resuspend the exosome pellet, and the mixture was allowed to stand for 5 min.

[0105] Add 50 μL of ethanol to 100 μL of lysate and mix thoroughly. Add the mixed lysate-ethanol solution to the micro filter cartridge assembly and cover it. Centrifuge at 13,000 rpm for 10 seconds and discard the flow-through.

[0106] Add 180 μL Wash Solution 1 to the micro filter cartridge, centrifuge at 13,000 rpm for 10 seconds, and discard the flow-through.

[0107] Add 180 μL Wash Solution 2 / 3 to the micro filter cartridge, centrifuge at 13000 rpm for 10 s, and discard the flow-through. Add 180 μL Wash Solution 2 / 3 to the micro filter cartridge again, centrifuge at 13000 rpm for 10 s, and discard the flow-through.

[0108] Place the micro filter cartridge in a clean collection tube and centrifuge at 13,000 rpm for 60 seconds. Discard the residual liquid and dry the micro filter cartridge for 60 seconds.

[0109] Place the micro filter cartridge into a clean elution tube, add 10 μL of preheated elution buffer, and incubate at room temperature for 60 seconds.

[0110] Centrifuge at 13000 rpm for 30 seconds to obtain RNA. Add 10 μL of preheated elution buffer to the micro filter cartridge again and incubate at room temperature for 60 seconds.

[0111] To determine the total amount of RNA, aspirate the entire RNA solution (about 20 μL), measure the absorbance (OD) at 260 nm without dilution, and calculate the RNA content at about 40 ng / μL per OD.

[0112] The total RNA content detection results are shown in Table 3.

[0113] Table 3

[0114] Group Total RNA content Example 1 Exosome preparation 211.3 μg Example 2 Exosome preparation 203.8 μg Comparative Example 1 Exosome Preparation 54.0 μg

[0115] The test results show that after microRNA is introduced into mesenchymal stem cells and cultured, the secreted exosomes containing microRNA have a higher RNA content.

[0116] Test Example 2

[0117] This test example provides a method for detecting total protein in exosomes, and the total protein reflects the harvest amount of exosomes.

[0118] The exosome preparation was centrifuged at 15000 rpm for 15 min, resuspended with 1 mL of PBS, and 200 μL of RIPA lysis buffer was added. After mixing for 5 min, the mixture was centrifuged at 15000 rpm for 5 min, and the supernatant was collected.

[0119] Prepare gradient concentrations of standard protein by mixing solution A and solution B of the BCA protein concentration assay kit at a ratio of 50:1 to prepare BCA working solution.

[0120] Take 20 μL of each of the standard protein and exosome extracted protein with gradient concentrations and add them to 200 μL of BCA working solution, and incubate them on a 96-well plate at 37°C for 45 min.

[0121] The absorbance at 562 nm was measured on an ELISA instrument, and the exosome protein content was calculated based on the gradient concentration of standard protein. The total protein content test results are shown in Table 4.

[0122] Table 4

[0123] Group Total protein content Example 1 Exosome preparation 92.8 μg Example 2 Exosome preparation 94.3 μg Comparative Example 1 Exosome Preparation 75.2 μg

[0124] The test results show that when microRNA is introduced into mesenchymal stem cells, after culture, the secreted exosomes evaluated by total protein content are higher.

[0125] like Figure 1As shown, the yield of exosomes directly secreted by mesenchymal stem cells in the present invention is higher. The yield of exosomes obtained from 15 mL of adipose tissue is calculated based on protein content. The total protein of exosomes encapsulated by microRNA can reach 95.13 μg (SD = 8.43 μg), while the total protein content of exosomes not encapsulated by the microRNA is only 74.83 μg (SD = 6.31 μg); the total RNA content of exosomes encapsulated by microRNA can reach 201.25 μg (SD = 31.00 μg), while the total RNA content of exosomes not encapsulated by the microRNA is only 51.67 μg (SD = 10.67 μg). The exosomes prepared by this method have more content (in terms of protein content) and a higher total RNA content.

[0126] Test Example 3

[0127] This test example provides a method for detecting exosomes to reduce serum inflammatory factors.

[0128] 7-week-old SD rats were used to collect 300 μL of tail vein blood for the detection of TNFα, IL-6 and IL-1β.

[0129] A rat COPD model was established. On the 1st and 14th days, 100 μL of lipopolysaccharide (containing 200 ng of lipopolysaccharide) was instilled into the trachea. On the 2nd to 13th day and 15th to 42nd day, the rats were fumigated twice a day with 8 cigarettes for 1 hour each time. The interval between fumigation treatments was ≥ 6 hours.

[0130] After the COPD model was established, 300 μL of rat tail vein blood was collected one week later (day 49) for the detection of TNFα, IL-6 and IL-1β.

[0131] On days 50 to 59, the model rats were treated with exosome preparations without microRNA encapsulation and with microRNA encapsulation, respectively. 001-010 (cohort 1) was the non-microRNA encapsulated exosome treatment group, and 011-020 (cohort 2) was the microRNA encapsulated exosome treatment group, 20 μL each time, twice a day, for 10 days.

[0132] On day 59, 300 μL of tail vein blood was collected from rats for detection of TNFα, IL-6, and IL-1β.

[0133] The experimental data are shown in Table 5 and Figure 2 As shown:

[0134] Table 5

[0135]

[0136] Table 5 and Figure 2 The data showed that before induction, the average levels of serum TNFα, IL-6 and IL-1β of 20 SD rats were 45.8 pg / mL (SD = 9.1 pg / mL), 19.9 ng / mL (SD = 2.3 ng / mL), and 27.9 pg / mL (SD = 4.1 pg / mL), respectively; one week after induction, the average levels of serum TNFα, IL-6 and IL-1β of 20 SD rats were 284.9 pg / mL (SD = 34.9 pg / mL), 61.8 ng / mL (SD = 6.4 ng / mL), and 163.8 pg / mL (SD = 7.0 pg / mL), respectively.

[0137] The mice were randomly divided into two groups, receiving exosome preparations without microRNA encapsulation (cohort 1) and after microRNA encapsulation (cohort 2) respectively. After the treatment, the serum TNFα, IL-6 and IL-1β levels were measured. The average levels of TNFα, IL-6 and IL-1β in cohort 1 were 204.3pg / mL (SD = 25.4pg / mL), 53.3ng / mL (SD = 4.7ng / mL), and 117.6pg / mL (SD = 14.4pg / mL), respectively; the average levels of TNFα, IL-6 and IL-1β in cohort 2 were 149pg / mL (SD = 13.1pg / mL), 32.5ng / mL (SD = 3.4ng / mL), and 94.6pg / mL (SD = 8.4pg / mL), respectively.

[0138] Both types of exosome preparations can significantly reduce the level of serum inflammatory factors, but the exosome preparations encapsulating microRNA have a better effect, especially in reducing IL-6. In the present invention, exosomes are good drug delivery carriers on the one hand, loaded with target microRNAs that regulate gene expression, and on the other hand, exosomes themselves also contain macromolecular substances such as proteins, lipids and nucleic acids, which can play a corresponding therapeutic role. The two work together to achieve the technical effect of targeted regulation of gene expression and protein synthesis in tissue cells.

[0139] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention. Sequence Listing <110> Sandai Kangnian (Shanghai) Medical Technology Co., Ltd. <120> A preparation method of exosomes encapsulating microRNA and its products and applications <130> 2021 <160> 4 <170> PatentIn version 3.3 <210> 1 <211> 110 <212> RNA <213> Artificially synthesized sequences <400> 1 gugaaaugu agcgagauuu ucuguugugc uugaucuaac caugugguug cgagguauga 60 guaaaacaug guuccgucaa gcaccaugga acgucacgca gcuuucuaca 110 <210> 2 <211> 87 <212> RNA <213> Artificially synthesized sequences <400> 2 accgcaggga aaaugaggga cuuuuggggg cagauguguu uccauuccac uaucauaaug 60 ccccuaaaaa uccuuauugc ucuugca 87 <210> 3 <211> 64 <212> RNA <213> Artificially synthesized sequences <400> 3 augacugauu ucuuuuggug uucagaguca auauaauuuu cuagcaccau cugaaaucgg 60 uuau 64 <210> 4 <211> twenty two <212> RNA <213> Artificially synthesized sequences <400> 4 cgcauccccu agggcauugg ug 22

Claims

1. A method for preparing an exosome composition, characterized in that: The preparation method comprises the following steps: (1) isolating and culturing mesenchymal stem cells, mixing the mesenchymal stem cells with an electroporation working solution to obtain a mixed solution, and mixing the microRNA mother solution with the mixed solution to obtain a pre-transfection solution; The microRNA mother solution includes hsa-mir-218-1, hsa-mir-365a, hsa-mir-29a and hsa-mir-324-5p; the ratio of hsa-mir-218-1, hsa-mir-365a, hsa-mir-29a and hsa-mir-324-5p in the microRNA mother solution is (4-5):(1-3):(2-3):1; the total RNA concentration of the microRNA mother solution is 3.2-4.8 μg / μL; the volume ratio of the microRNA mother solution to the mixed solution is 3:(400-600); (2) electroporating the pre-transfection solution in step (1); (3) adding the electroporated mesenchymal stem cells obtained in (2) to a cell suspension of mesenchymal stem cells that have not been electroporated, and culturing the mixture to obtain a culture medium; (4) collecting the exosome composition from the culture medium; specifically comprising: subjecting the culture medium to centrifugation once, twice and three times in sequence to obtain exosomes encapsulating microRNA; the temperature of the first centrifugation is 0-8°C, the speed of the first centrifugation is 1800-2200 rpm, and the time of the first centrifugation is 15-30 min; the temperature of the second centrifugation is 0-8°C, the speed of the second centrifugation is 2500-3500 rpm, and the time of the second centrifugation is 30-40 min; the temperature of the third centrifugation is 0-8°C, the speed of the third centrifugation is 11000-15000 rpm, and the time of the third centrifugation is 50-60 min.

2. The method for preparing the exosome composition according to claim 1, characterized in that: Step (1) specifically includes: The tissue is digested and cultured with a digestion solution to obtain a mesenchymal stem cell suspension, and the mesenchymal stem cells are obtained by centrifugation. The mesenchymal stem cells are mixed with an electroporation working solution to obtain a mixed solution, and the microRNA mother solution is mixed with the mixed solution to obtain a pre-transfection solution.

3. The method for preparing the exosome composition according to claim 2, characterized in that: The digestion solution is 0.05-0.2% trypsin digestion solution.

4. The method for preparing the exosome composition according to claim 2, characterized in that: The culture medium used in step (1) is a serum-free culture medium, and the culture time is 30 to 36 hours.

5. The method for preparing the exosome composition according to claim 2, characterized in that: The centrifugal treatment temperature in step (1) is 0-8°C, the centrifugal treatment speed is 1100-1200 rpm, and the centrifugal treatment time is 5-8 min.

6. The method for preparing the exosome composition according to claim 1, characterized in that: The concentration of total RNA in the mixed solution of step (1) is 0.192~2.88 μg / μL.

7. The method for preparing the exosome composition according to claim 1, characterized in that: The mesenchymal stem cells in step (1) are selected from adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells.

8. The method for preparing the exosome composition according to claim 1, characterized in that: The voltage of the electroporation treatment in step (2) is 200-220 V, the shock duration of the electroporation treatment is 4-6 s, and the pulse length of the electroporation treatment is 20-25 ms.

9. The method for preparing the exosome composition according to claim 1, characterized in that: The culture medium used in step (3) is a serum-free culture medium, and the culture time is 20 to 24 hours.

10. Use of the method for preparing the exosome composition according to any one of claims 1 to 9 in preparing a drug for treating lung diseases, characterized in that: The lung disease is chronic obstructive pulmonary disease.

11. The use according to claim 10, characterized in that: The dosage form of the drug is an aerosol dosage form.

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