Cyclic IL-10mRNA nano preparation as well as preparation method and application thereof

Abstract

The invention discloses a cyclic IL-10mRNA nano preparation as well as a preparation method and application thereof, and belongs to the technical field of biology. The annular IL-10mRNA nano preparation is prepared by the following method: 1, synthesizing chemically modified annular IL-10mRNA in vitro, and 2, preparing the annular IL-10mRNA nano preparation capable of being delivered in vivo by a self-assembly method. The annular IL-10mRNA can regulate immune cells in synovial membranes of articular cavities and inhibit articular cartilage injury, so that the effect of treating joint inflammatory diseases is achieved. The application has the advantages that the annular IL-10mRNA is successfully delivered into the joint cavity with the joint inflammatory disease through the nano system, and the inflammation environment in the joint cavity with the joint inflammatory disease is adjusted, so that the development of inflammation is inhibited; the cyclic IL-10 promotes cartilage repair so as to improve the disease progress of joint inflammatory diseases.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a cyclic IL-10 mRNA nano preparation and a preparation method and application technology thereof. Background Art

[0002] Common inflammatory arthritis (IA) diseases include osteoarthritis (OA), rheumatoid arthritis (RA), spondyloarthritis, psoriatic arthritis (PsA), etc. In the past nearly three decades, the incidence of IA has been increasing rapidly both at home and abroad. According to the data of the World Health Organization (WHO), there are currently about 18 million people suffering from rheumatoid arthritis, 528 million people suffering from osteoarthritis, and the number of osteoarthritis patients in my country has reached nearly 140 million. Inflammatory joint disease and osteoarthritis cause pain, limited mobility, joint destruction and even deformity or disability in patients, which brings a heavy burden to patients and their families. At present, the treatment methods for inflammatory joint diseases mainly include disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, chondroprotective agents and surgical treatment, but the current treatment methods can only play a role in delaying the course of the disease.

[0003] Interleukin 10 (IL-10) is a multi-effect cytokine with important immunomodulatory functions, which is mainly secreted by antigen-presenting cells, such as activated T cells, monocytes, B cells, and macrophages. IL10 can affect the activities of many cell types in the immune system. High levels of IL-10 inhibit their antigen presentation ability, resulting in inefficient T cell activation. The presence of chronic antigens further exhausts T cells and induces the production of IL-10. As a result, T cells develop "tolerance" to antigens, and high levels of IL10 can promote the proliferation of regulatory T cells (Treg) and helper T cells 2 (Th2), thereby achieving the effect of inhibiting inflammatory responses. In addition, in healthy joints, IL-10 and its receptor IL-10R are present in multiple joint tissues. In osteoarthritis, chondrocytes express more IL-10 and its receptors. IL-10 can affect the proliferation of osteoclasts and osteoblasts through the receptor activator of nuclear factor-kappa B (RANK) / RANK ligand (RANKL) / osteoprotegerin (OPG) axis, and participate in maintaining bone metabolism balance. IL-10 can also significantly antagonize the expression of IL-6, MMP-1 and MMP-3 induced by TNF-α, increase the expression of chondrocyte formation markers Col II, SOX9 and glycosaminoglycans, thereby inhibiting chondrocyte apoptosis and regulating the inflammatory response of chondrocytes. Therefore, IL-10 has great application potential in inflammatory joint diseases. However, current clinical trials related to IL-10 (such as recombinant cytokines, fusion proteins, etc.) have problems such as blood toxicity and short half-life.

[0004] Therefore, there is still an urgent need to develop a new IL-10 preparation or method with a long half-life and low blood toxicity so that IL-10 related diseases can be better treated and the application and development of IL-10 technology can be promoted. Summary of the invention

[0005] The first object of the present invention is to disclose a cyclic IL-10 mRNA nanoformulation.

[0006] The second object of the present invention is to disclose a method for preparing the above-mentioned cyclic IL-10 mRNA nanoformulation.

[0007] The third object of the present invention is to disclose the application of the above-mentioned cyclic IL-10 mRNA nano preparation.

[0008] The technical solution of the present invention involves the following three aspects:

[0009] In a first aspect, the present invention provides a nucleic acid sequence of IL-10 mRNA and a method for synthesizing the same. The IL-10 mRNA of the present invention can theoretically be any mRNA sequence that can produce IL-10, including but not limited to self-reporter gene IL-10 mRNA, self-amplifying IL-10 mRNA, and circular IL-10 mRNA.

[0010] In a second aspect, the present invention provides a delivery system for delivering IL-10 mRNA. The delivery system of the present invention can theoretically be any delivery system capable of delivering mRNA, including but not limited to cationic lipids, lipid nanoformulations, polymer nanoformulations, and the like.

[0011] In a third aspect, the present invention provides a new strategy for treating joint inflammatory diseases using IL-10 mRNA nanoformulations, which can significantly improve the progression of such diseases. The joint inflammatory diseases described in the present invention include, but are not limited to, osteoarthritis (OA), rheumatoid arthritis (RA), spondyloarthritis, psoriatic arthritis (PsA), etc.

[0012] The objective of the present invention is achieved through the following technical solutions:

[0013] A method for preparing a cyclic IL-10 mRNA nanoformulation, wherein the method comprises the following steps:

[0014] (1) PCR amplify the human IL-10 gene open reading frame plasmid carrying the T7 promoter and the T7 terminator, and purify the PCR product to form a linearized DNA;

[0015] In vitro transcription: The Novagen T7 transcription kit was used with 1-2 μg of in vitro transcription template, 7.5 mM guanosine triphosphate, 7.5 mM 5-methyl-cytidine triphosphate, 7.5 mM adenosine triphosphate, and 7.5 mM pseudouridine-5'-triphosphate. The reaction was carried out at 37°C for 16 hours, followed by deoxyribonuclease treatment to obtain IL-10 mRNA;

[0016] IL-10 mRNA was purified by high performance liquid chromatography (HPLC), and the salts in the buffer were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa); after washing with enzyme-free water three times, the IL-10 mRNA was collected in enzyme-free water for further use or stored in a -80°C refrigerator; further purified IL-10 mRNA was obtained;

[0017] The further purified IL-10 mRNA was digested with Biyuntian RNase R to enrich the circular IL-10 mRNA: 20 μg IL-10 mRNA, 20 U RNase R, the reaction was carried out at 37°C for 30 minutes, 85°C for 15 minutes, and then purified by high performance liquid chromatography, washed three times using an ultrafiltration device, and the circular IL-10 mRNA was collected in enzyme-free water for further use or stored in a -80°C refrigerator; the desired circular IL-10 mRNA was obtained;

[0018] (2) dissolving SM-102, distearoylphosphatidylcholine (DSPC), cholesterol (Choi), dimyristoylglycerol-polyethylene glycol (DMG-PEG) and distearoylphosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG) in anhydrous ethanol to form uniform solutions with concentrations of 10 mg / ml, 20 mg / ml, 20 mg / ml, 10 mg / ml and 20 mg / ml, respectively;

[0019] SM-102, DSPC, Choi, DMG-PEG, and DSPE-PEG were prepared into solution A with a total liposome concentration of 7.5 mg / mL according to a molar ratio of 50:10:38.5:0.75:0.75;

[0020] 10 μg of cyclic IL-10 mRNA was mixed with pH 4 citrate buffer, 25.7 μl of the solution was quickly added to the mixture of mRNA and citrate buffer on an oscillator at 800 rpm, shaken for 30 s, and then 180 μl of PBS was added dropwise, wherein the PBS was of molecular biology grade.

[0021] The organic solvent and free compounds in the formed nanoformulation dispersion were removed by centrifugation using an EMD Millipore MWCO 100 kDa ultrafiltration device; after washing three times with high-pressure water, the cyclic IL-10 mRNA nanoformulation was collected and dispersed in pH 7.4-PBS buffer for further use or stored in a 4°C refrigerator.

[0022] The method for preparing a circular IL-10 mRNA nanoformulation described in the above technical solution, wherein: the IL-10 mRNA is any mRNA sequence that can produce IL-10.

[0023] The method for preparing a circular IL-10 mRNA nanoformulation described in the above technical solution, wherein: the IL-10 mRNA is a self-reporter gene IL-10 mRNA, a self-amplifying IL-10 mRNA or a circular IL-10 mRNA.

[0024] The method for preparing a circular IL-10 mRNA nanoformulation described in the above technical solution, wherein: the delivery product containing the IL-10 mRNA nanoformulation is any delivery system capable of delivering mRNA.

[0025] The method for preparing a cyclic IL-10 mRNA nanoformulation described in the above technical solution, wherein: the delivery system capable of delivering mRNA is a cationic lipid, a lipid nanoformulation or a polymer nanoformulation.

[0026] A circular IL-10 mRNA nano preparation, wherein: the nano preparation is prepared by the preparation method described in any of the technical solutions in the above technical solutions.

[0027] The application of the cyclic IL-10 mRNA nano preparation described in the above technical solution in the preparation of treatment for inflammatory diseases of joints.

[0028] In the application described in the above technical solution, the joint inflammatory disease is osteoarthritis (OA), rheumatoid arthritis (RA), spondyloarthritis or psoriatic arthritis (PsA).

[0029] The application described in the above technical solution, wherein the application of the cyclic IL-10 mRNA nano preparation in the preparation of a method for treating inflammatory joint diseases refers to the application in the preparation of anti-inflammatory functional drugs.

[0030] In the application described in the above technical solution, the anti-inflammatory means can significantly reduce the inflammatory response induced by LPS or can significantly improve the progression of joint inflammatory diseases.

[0031] The present invention has the following beneficial effects:

[0032] 1. Circular IL-10 can regulate the inflammatory environment in the joint cavity of inflammatory joint diseases, thereby inhibiting the development of inflammation. At the same time, IL-10 can promote cartilage repair and improve the disease process of inflammatory joint diseases.

[0033] 2. Annular IL-10 mRNA nanoformulations can efficiently deliver IL-10 into cells, translate IL-10 protein, and exhibit a longer half-life, enabling better treatment of IL-10-related diseases and promoting the application and development of IL-10 technology.

[0034] 3. Local injection into the joint cavity can also avoid systemic blood toxicity caused by recombinant cytokines and systemic immunosuppressive reactions caused by IL-10. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 1. Figure 1 Characterization of cyclic IL-10 mRNA; wherein Figure 1 A is the total ion current of the in vitro transcription product by high performance liquid chromatography. Figure 1 B is the 2% gel electrophoresis diagram, Figure 1 C is the Sanger sequencing spectrum of the splicing site.

[0036] 2. Figure 2 The invention is to characterize the size and morphology of the circular IL-10mRNA nanoformulation (IL-10mRNA / LNPs); Figure 2 A is the DLS size distribution; Figure 2 B shows the size change of LNPs within 5 days; Figure 2 C is TEM imaging.

[0037] 3. Figure 3 Figure 2 is a graph showing the delivery efficiency and biocompatibility of cyclic IL-10 mRNA nanoformulations; wherein, Figure 3 A is the expression time of EGFP nanoformulation in cells; Figure 3 B shows lysosomal escape of cy5-IL 10 nanoformulation; Figure 3 C is the expression time of IL-10 in cells; Figure 3 D~ Figure 3 E is the immunogenicity and cytotoxicity of IL-10 nanoformulation.

[0038] 4. Figure 4 The therapeutic effect of cyclic IL-10mRNA nanoformulation in inflammatory diseases of joints; Figure 4 A is a diagram of the treatment cycle for rheumatoid arthritis; Figure 4 B shows that IL-10 nanoformulation reduces IL-6 secretion in vitro; Figure 4 C is the thickness change curve of rat toes; Figure 4 D is the change curve of clinical scores of rats; Figure 4E is a comparison of the toes of rats in different groups. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the technical solution of the present invention, the following specific embodiments are provided. Obviously, the embodiments described in this case are part of the embodiments of the present invention, but 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.

[0040] Unless otherwise specified, the experimental materials used in the following examples of the patent application of the present invention can be purchased from conventional biochemical reagent companies.

[0041] 1. Materials:

[0042] SM-102 and LPS were purchased from MCE. Distearoylphosphatidylcholine (DSPC), cholesterol (Choi), dimyristoylglycerol-polyethylene glycol and (DMG-PEG) distearoylphosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG) were from Sigma-Aldrich.

[0043] Immunization Grade Porcine Type II Collagen and Incomplete Freund's Adjuvant were purchased from Chondrex, Inc. (Washington State, USA).

[0044] Lipofectamine 2000 (Lip2k) and TE buffer (20X) were purchased from Invitrogen, Carlsbad, California, USA.

[0045] IL-10 plasmid, EGFP plasmid and circular RNA vector sequences were from Miaoling plasmid platform. T7 High Yield RNA Transcription Kit was from Novozymes Biotech Co., Ltd. The relevant primers were from Qingke Biotech Co., Ltd.

[0046] 2. Cell lines:

[0047] Mouse mononuclear macrophage leukemia cells (RAW264.7) and human renal epithelial cells (293T) were purchased from Meisen Biotechnology Co., Ltd. and cultured in DMEM medium. 1% penicillin / streptomycin antibiotics (Thermo-Fisher Scientific) and 10% fetal bovine serum (FBS; AusGeneX) were added to the cell culture medium.

[0048] Embodiment 1:Preparation of cyclic IL-10 and EGFP mRNA lipid nanoformulations:

[0049] 1. In vitro synthesis of chemically modified circular IL-10 mRNA and EGFP mRNA.

[0050] Circular enhanced green fluorescent protein (EGFP) and circular IL-10 mRNA (mRNA) were synthesized using in vitro transcription technology (IVT). A non-translated region, intron splicing region, non-translated region and ribosome entry site region were designed at the 5' end of the circular RNA to enhance translation initiation, and a non-translated region and intron splicing region were designed at the 3' end of the RNA to form circular IL-10 mRNA during in vitro transcription. In order to avoid immune stimulation caused by mRNA, pseudouridine-5'-triphosphate (Pseudo-UTP) was used to replace conventional uridine triphosphate. The specific steps are:

[0051] 1. The human IL-10 and EGFP gene open reading frame plasmids carrying T7 promoter and T7 terminator were used as polymerase chain reaction (PCR) amplification templates (plasmids were purchased from Zhongyuan Biotechnology and amplified in DH5α), and the T7 promoter sequence was used as the forward primer and the T7 terminator sequence was used as the reverse primer for PCR amplification. The Novagen PCR kit was used, and the reaction was performed at 98°C for 10s, 60°C for 5s, and 72°C for 15s for a total of 34 cycles to amplify the template. The amplified products were separated by 1% gel electrophoresis and purified using the Novagen gel recovery kit to form linearized DNA as a template for in vitro transcription;

[0052] 2. Perform in vitro transcription (IVT) of IL-10 mRNA, EGFP-mRNA and cy5 IL-10 mRNA respectively:

[0053] 2.1. In vitro transcription of IL-10 mRNA: Use the Novagen T7 transcription kit with 1-2 μg in vitro transcription template, 7.5 mM guanosine triphosphate, 7.5 mM 5-methyl-cytidine triphosphate, 7.5 mM adenosine triphosphate, and 7.5 mM pseudouridine-5'-triphosphate, and react at 37°C for 16 hours, followed by deoxyribonuclease (DNase) treatment to obtain IL-10 mRNA;

[0054] 2.2. In vitro transcription of EGFP-mRNA: the steps are the same as those in 2.1, except that the in vitro transcription template is the in vitro transcription template of EGFP, to obtain EGFP mRNA;

[0055] 2.3. In vitro transcription of cy5 IL-10 mRNA: The steps are the same as 2.1, except that 2.5 mM cy5-labeled uridine triphosphate is added to the reaction system; cy5 IL-10 mRNA is obtained.

[0056] 3. The IL-10 mRNA, EGFP mRNA and cy5 IL-10 mRNA obtained in step 2 were purified by semi-preparative high performance liquid chromatography (HPLC) to obtain a high performance liquid phase total ion current diagram of the in vitro transcription product, and the salts in the buffer were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO100 kDa); after washing 3 times with enzyme-free water, the IL-10 mRNA and EGFP mRNA were collected in enzyme-free water for further use or stored in a -80°C refrigerator; further purified IL-10 mRNA, EGFP mRNA and cy5 IL-10 mRNA were obtained.

[0057] 4. The IL-10mRNA, EGFP mRNA and cy5 IL-10mRNA further purified in step 3 were digested with Biyuntian RNase R to enrich circular IL-10mRNA, circular EGFP mRNA and circular cy5 IL-10mRNA: 20 μg IL-10mRNA, 20U RNase R, the reaction was carried out at 37°C for 30 min and 85°C for 15 min, then purified by semi-preparative high performance liquid chromatography (HPLC), washed 3 times using an ultrafiltration device, and the circular IL-10mRNA, circular EGFP mRNA and circular cy5IL-10mRNA were collected in enzyme-free water for further use or stored in a -80°C refrigerator to obtain the desired circular IL-10mRNA, circular EGFP mRNA and circular cy5 IL-10mRNA.

[0058] In order to evaluate whether the product of step 4 is a circular IL-10 mRNA, the IL-10 mRNA further purified from the in vitro transcription product in step 3 and the circular IL-10 mRNA digested with Bio-Tech RNase R in step 4 were subjected to electrophoresis analysis in a 2% agarose gel; electrophoresis conditions: 100V, 30min. Using 4ug of circular IL-10 mRNA in step 4 as a reverse transcription template, the template was reverse transcribed using the Novozymes reverse transcription kit, the reaction was carried out at 37°C for 15min and 85°C for 5s, and the obtained cDNA product was subjected to Sanger sequencing by Qingke Biotechnology Co., Ltd. The results are as follows Figure 1 As shown, Figure 1 A is the total ion current of the HPLC product of in vitro transcription in step 3. Figure 1 B is the 2% gel electrophoresis diagram, Figure 1 C is the reverse transcription Sanger sequencing spectrum.

[0059] 2. Preparation and characterization of nanoparticles:

[0060] Circular IL-10 mRNA, cyclic EGFP mRNA and cyclic cy5 IL-10 mRNA nanoformulations (lipid polymer composite nanoformulations encapsulating mRNA) were prepared by a self-assembly method. The method specifically includes the following steps:

[0061] 1. SM-102, distearoylphosphatidylcholine (DSPC), cholesterol (Choi), dimyristoylglycerol-polyethylene glycol (DMG-PEG) and distearoylphosphatidylethanolamine-methoxypolyethylene glycol (DSPE-PEG) were dissolved in anhydrous ethanol to form uniform solutions with concentrations of 10 mg / ml, 20 mg / ml, 20 mg / ml, 10 mg / ml and 20 mg / ml respectively. ;

[0062] 2. SM-102, DSPC, Choi, DMG-PEG and DSPE-PEG were prepared into solution A with a total liposome concentration of 7.5 mg / mL according to a molar ratio of 50:10:38.5:0.75:0.75;

[0063] 3. 10 μg of cyclic IL-10 mRNA was mixed with a pH 4 citrate buffer, 25.7 μl of solution A was quickly added to the mixture of mRNA and citrate buffer on an oscillator at 800 rpm, shaken for 30 s, and then 180 μl of PBS was added dropwise, wherein the PBS was of molecular biology grade;

[0064] 4. Use ultrafiltration device (EMD Millipore, MWCO 100kDa) to remove organic solvents and free compounds in the formed nanoformulation dispersion by centrifugation; after washing with high-pressure water three times, the cyclic IL-10mRNA nanoformulation was collected and dispersed in pH7.4-PBS buffer for further use or stored in a 4°C refrigerator, and the prepared cyclic IL-10mRNA nanoformulation was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Dynamic light scattering (DLS, Brookhaven Instruments, USA) determined the size and stability of cyclic IL-10mRNA nanoformulations in phosphate buffer at 4°C for 5 days (the results are shown in Figure 2). Figure 2 A, 2B). The morphology of the mRNA nanoformulation was observed under transmission electron microscopy (JEOL-2100) at 200KV and 101μA (as shown in Figure 2 C).

[0065] According to the method of steps 1-4, circular EGFP mRNA and circular cy5 IL-10 mRNA nanoformulations were prepared respectively, wherein in step 3, 10 μg of circular EGFP mRNA and circular cy5 IL-10 mRNA were mixed with citric acid buffer at pH=4.

[0066] 5. Detection of cytotoxicity of cyclic IL-10 mRNA nanoformulations: 293T cells were evenly plated in 96-well plates at a density of 5000 cells per well. After 24 hours of cell attachment, the cells were transfected with different concentrations (0.5, 1, 2 or 3 μg / ml) of cyclic IL-10 mRNA nanoformulations for 12 hours, and then 0.1 ml of fresh complete medium was added and further cultured for 24 hours to evaluate cell viability and transfection efficiency. Lip2k was used as a positive control for transfection efficiency. Cell viability was tested by cck-8, a non-toxic cell viability detection method, and real-time cell proliferation can be checked by a microplate reader (TECAN, Infinite M200 Pro). The absorbance was detected at 450 nm and 630 nm using a 96-well Spectramax plate reader (Molecular Devices, Sunnyvale, California); the results are shown in Figure 2. Figure 3 As shown in D, the cyclic IL-10 mRNA nanoformulation is not cytotoxic to cells.

[0067] 6. To further verify the effectiveness of in vitro transfection, we selected cyclic EGFP-mRNA and cyclic cy5 IL-10 mRNA nanoformulations as model mRNAs. 293T cells were evenly plated in a 96-well plate at a density of 5000 cells per well. After 24 hours of cell attachment, the cells were transfected with 1 μg / ml cyclic EGFP-mRNA nanoformulation for 12 hours, and then 0.1 ml of fresh complete medium was added and further cultured. Pictures were taken under an inverted fluorescence microscope every 12 hours in the first 48 hours, and every 24 hours after 48 hours. The percentage of transfected enhanced green fluorescent protein-positive cells was calculated and analyzed using imaj-j to determine the half-life of green fluorescent protein. A large amount of green fluorescence was detected in 293T cells transfected with cyclic EGFP-mRNA nanoformulations and the commercial transfection reagent Lipofectamine 2000 (Lip2k), and green fluorescence expression was still present 200 hours after transfection ( Figure 3 A). Circular cy5 IL-10 mRNA nanoformulation can effectively deliver cy5-labeled mRNA into the cytoplasm. After 6 hours of culture, a large amount of cy5-labeled mRNA escapes from lysosomes and diffuses into the cytoplasm. In contrast, naked mRNA cannot enter cells after 6 hours of culture ( Figure 3 B).

[0068] 7.293T cells were evenly plated in a 24-well plate at a density of 20,000 cells per well. After 24 hours of cell attachment, the cells were transfected with different concentrations (0.5, 1, 2 or 3 μg / ml) of cyclic IL-10 mRNA nanoformulations and the cell supernatant was collected at 6h, 12h, 24h, 36h, 48h, 72h, 96h, 120h and 144h of transfection and 500μl of fresh culture medium was replaced. Compared with the cyclic IL-10 mRNA nanoformulation, Lip2k was used as a positive control for transfection efficiency. IL-10 secretion was tested by Elisa. IL-10 secretion can be checked by a microplate reader (TECAN, Infinite M200 Pro). The absorbance was detected at 450nm using a 96-well Spectramax plate reader (Molecular Devices, Sunnyvale, California), and IL-10 secretion was still detectable at 144h ( Figure 3 C).

[0069] 8. Detection of the immunogenicity of cyclic IL-10 mRNA nanoformulations: RAW264.7 cells were evenly plated in a 96-well plate at a density of 5000 cells per well. After 24 hours of cell attachment, the cells were transfected with cyclic IL-10 mRNA nanoparticles at different concentrations (0.5, 1, 2 or 3 μg / ml) for 12 hours, and then 0.1 ml of fresh complete culture medium was added and further cultured for 24 hours, and the cell supernatant was collected for the immunogenicity determination of the nanoformulation. The immunogenicity was assessed by Elisa testing of IL-6 secretion, which can be checked by a microplate reader (TECAN, Infinite M200 Pro). The absorbance was detected at 450 nm using a 96-well Spectramax plate reader (Molecular Devices, Sunnyvale, California); the results are shown in the table below. Figure 3 As shown in E, the cyclic IL-10 mRNA nanoformulation is immunostimulatory.

[0070] 3. IL-10mRNA nanoformulation is delivered to the joint cavity of RAW264.7 and joint inflammatory disease models to exert therapeutic functions:

[0071] (I) Experimental methods and steps:

[0072] 1. In vitro anti-inflammatory experiment: RAW264.7 cells were evenly plated in 12-well plates at a density of 50,000 cells per well. After 24 hours of cell attachment, the cells were induced with 100 ng / mL of LPS for 6 hours, and the cells were transfected with different concentrations (0.5, 1, 2 or 3 μg / ml) of cyclic IL-10 mRNA nanoformulations for 12 hours, and then 1 ml of fresh complete medium was added and further cultured for 48 hours. The cell supernatant was collected and the in vitro anti-inflammatory activity of the cyclic IL-10 mRNA nanoformulation was evaluated by Elisa test of IL-6 secretion. IL-6 secretion can be checked by microplate reader (TECAN, Infinite M200 Pro). The absorbance was detected at 450 nm using a 96-well Spectramax plate reader (Molecular Devices, Sunnyvale, California). Adherent cells were collected, total RNA was extracted from the cells using the Trizol method, and total RNA was reverse transcribed using HiScript II 1st Strand cDNA Synthesis Kit (Norvastatin) to obtain cDNA. The changes in IL-6 at the mRNA level were detected using a fluorescent quantitative qPCR instrument (CFX96 Touch).

[0073] 2. IL-10mRNA nanoformulation for the treatment of rheumatoid arthritis: Select 7-8 week-old immune mature SD rats and emulsify IFA with collagen solution. Insert the bevel of the needle upward and parallel to the tail, 2 cm from the base of the tail, until the needle tip is 0.5 cm from the base, and inject 0.2ml (200μg collagen) emulsion subcutaneously at the base of the tail. A booster injection was performed on the 7th day after the initial immunization. The collagen-IFA emulsion was prepared as described above and 0.1ml was injected subcutaneously into the tail. For the booster injection, the needle was inserted 3 cm from the base of the tail until the tip reached 1.5 cm from the base. The booster injection was injected at a different location from the initial injection. The disease was assessed by qualitative clinical scoring and determination of paw thickness using a thickness gauge. After modeling, the IL-10mRNA treatment group was injected with cyclic IL-10mRNA nanoformulation into the hind leg ankle joint cavity every week, with a dosage of 5μg / rat. The blank group was given the same dose of PBS, and the same dose of nanoformulation without cyclic IL-10mRNA was used to exclude the effect of nanoformulation on the disease process. Figure 4 As shown in A.

[0074] 3. IL-10mRNA nanoformulation for the treatment of osteoarthritis: SD rats weighing 200-250g were selected. After the rats were anesthetized with tribromoethanol, they were fixed and the right hind limbs were shaved. The skin of the legs was cut to expose the knee joint. A scalpel was used to make a T-shaped opening at the knee joint. The knee joint was flexed as much as possible to expose the anterior cruciate ligament and fully expose the knee joint. The posterior extension of the lateral meniscus was removed and the anterior cruciate ligament was cut. After hemostasis, the wound was sutured and the joint cavity was closed layer by layer. Penicillin was injected intramuscularly every day for 3 days after surgery to prevent wound infection. Two weeks after surgery, the IL-10mRNA treatment group of rats was injected with cyclic IL-10mRNA nanoformulation into the knee joint cavity of the hind legs of the rats, with a dose of 5μg / rat. The blank group was given the same dose of PBS, and the same dose of nanoformulation without cyclic IL-10mRNA was used to exclude the effect of nanoformulation on the disease process. The disease was evaluated by osteophyte formation in the rat joint cavity, the degree of joint movement limitation, X-ray, small animal CT and immunohistochemistry.

[0075] (II) Experimental results:

[0076] 1. For the LPS-induced in vitro inflammation model of RAW264.7, cells were transfected with IL-10 protein mRNA at different concentrations (0.5, 1, 2 or 3 μg / ml) for 12 hours, and the same dose of PBS was used as a blank control. The same dose of nanoformulations without cyclic IL-10 mRNA was used to exclude the effect of nanoformulations on inflammation. After 48 hours of culture, the secretion of inflammatory factor IL-6 and the mRNA expression level of IL-6 showed that different doses of cyclic IL-10 mRNA had different degrees of anti-inflammatory activity, among which 3 μg / ml had the best anti-inflammatory effect. The secretion of inflammatory factor IL-6 was not significantly different from that of the blank control (such as Figure 4 B).

[0077] 2. In the treatment of rheumatoid arthritis model, the thickness of the hind paws of rats in the model group and the group without cyclic IL-10 mRNA nanoformulation increased continuously, affecting the normal walking of the rats. After administration of cyclic IL-10 mRNA nanoformulation, the thickness of the hind paws decreased, and the walking gait was normal. There was no significant difference compared with the blank control. The thickness of the hind paws and the clinical scores were Figure 4 C, as shown in 4D.

[0078] 3. In the osteoarthritis model, rats treated with cyclic IL-10mRNA nanoformulations had fewer osteophytes and normal walking gait.

[0079] We designed a nanosystem to deliver IL-10mRNA to the joint cavity for the treatment of inflammatory joint diseases. The results showed that IL-10mRNA was successfully delivered to LPS-induced mouse mononuclear macrophage leukemia cells (RAW264.7) through the nanosystem, which could significantly reduce the inflammatory response induced by LPS. At the same time, the successful delivery of IL-10mRNA to the joint cavity of inflammatory joint diseases through the nanosystem can significantly improve the progression of inflammatory joint diseases.

[0080] The above description is only a preferred embodiment of the present invention and does not impose any formal or substantial limitation on the present invention. Any technician familiar with the profession can make use of the technical contents disclosed above without departing from the scope of the technical solution of the present invention, and any slight changes, modifications and evolutions made by equivalent changes are all equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a cyclic IL-10 mRNA nanoformulation, characterized in that: The method comprises the following steps: (1) PCR amplify the human IL-10 gene open reading frame plasmid carrying the T7 promoter and T7 terminator, and purify the PCR product to form a linearized DNA as an in vitro transcription template; In vitro transcription: The Novagen T7 transcription kit was used with 1-2 μg of in vitro transcription template, 7.5 mM guanosine triphosphate, 7.5 mM 5-methyl-cytidine triphosphate, 7.5 mM adenosine triphosphate, and 7.5 mM pseudouridine-5'-triphosphate. The reaction was carried out at 37°C for 16 hours, followed by deoxyribonuclease treatment to obtain IL-10 mRNA; IL-10 mRNA was purified by high performance liquid chromatography, and the salts in the buffer were removed by centrifugation using an EMD Millipore MWCO 100 kDa ultrafiltration device; after washing three times with enzyme-free water, the IL-10 mRNA was collected in enzyme-free water for further use or stored in a -80°C refrigerator; further purified IL-10 mRNA was obtained; The further purified IL-10 mRNA was digested with Biyuntian RNase R to enrich the circular IL-10 mRNA: 20 μg IL-10 mRNA, 20 U RNase R, the reaction was carried out at 37°C for 30 minutes, 85°C for 15 minutes, and then purified by high performance liquid chromatography, washed three times using an ultrafiltration device, and the circular IL-10 mRNA was collected in enzyme-free water for further use or stored in a -80°C refrigerator; the desired circular IL-10 mRNA was obtained; (2) dissolving SM-102, distearoylphosphatidylcholine, cholesterol, dimyristoylglycerol-polyethylene glycol and distearoylphosphatidylethanolamine-methoxypolyethylene glycol in anhydrous ethanol to form uniform solutions with concentrations of 10 mg / ml, 20 mg / ml, 20 mg / ml, 10 mg / ml and 20 mg / ml, respectively; SM-102, DSPC, Choi, DMG-PEG, and DSPE-PEG were prepared into solution A with a total liposome concentration of 7.5 mg / mL according to a molar ratio of 50:10:38.5:0.75:0.75; 10 μg of cyclic IL-10 mRNA was mixed with pH 4 citrate buffer, 25.7 μl of solution A was quickly added to the mixture of mRNA and citrate buffer on an oscillator at 800 rpm, shaken for 30 s, and then 180 μl of PBS was added dropwise, where PBS was molecular biology grade; The organic solvent and free compounds in the formed nanoformulation dispersion were removed by centrifugation using an EMD Millipore MWCO 100 kDa ultrafiltration device; after washing three times with high-pressure water, the cyclic IL-10 mRNA nanoformulation was collected and dispersed in pH 7.4-PBS buffer for further use or stored in a 4°C refrigerator.

2. The method for preparing a cyclic IL-10 mRNA nanoformulation according to claim 1, characterized in that: The IL-10 mRNA is any mRNA sequence that can produce IL-10.

3. The method for preparing a cyclic IL-10 mRNA nanoformulation according to claim 2, characterized in that: The IL-10 mRNA is a self-reporter gene IL-10 mRNA, a self-amplified IL-10 mRNA or a circular IL-10 mRNA.

4. The method for preparing a cyclic IL-10 mRNA nanoformulation according to claim 1, characterized in that: The delivery preparation containing the IL-10 mRNA nanoformulation is any delivery system capable of delivering mRNA.

5. The method for preparing a cyclic IL-10 mRNA nanoformulation according to claim 4, characterized in that: Delivery systems capable of delivering mRNA are cationic lipids, lipid nanoformulations or polymer nanoformulations.

6. A cyclic IL-10 mRNA nanoformulation, characterized in that: The nano preparation is prepared by the preparation method described in any one of claims 1 to 5.

7. Use of the cyclic IL-10 mRNA nanoformulation according to claim 6 in the preparation of a drug for treating inflammatory joint diseases.

8. The use according to claim 7, characterized in that: The joint inflammatory disease is osteoarthritis, rheumatoid arthritis, spondyloarthritis or psoriatic arthritis.

9. The use according to claim 7, characterized in that: The application of the cyclic IL-10 mRNA nano preparation in the preparation of a treatment for joint inflammatory diseases refers to the application in the preparation of anti-inflammatory functional drugs.

10. The use according to claim 9, characterized in that: The anti-inflammatory effect refers to the ability to significantly reduce the inflammatory response induced by LPS or to significantly improve the course of joint inflammatory diseases.

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