Circular il-10 mRNA nano-preparation, and preparation method therefor and use thereof

Abstract

The present invention belongs to the field of biotechnology. Provided are a circular IL-10 mRNA nano-preparation, and a preparation method therefor and the use thereof. The circular IL-10 mRNA nano-preparation is prepared by means of the following method: 1, synthesizing a chemically modified circular IL-10 mRNA in vitro; and 2, preparing, by means of self-assembly, a circular IL-10 mRNA nano-preparation that can be delivered in vivo. The circular IL-10 mRNA inhibits injury of articular cartilage by means of regulating immune cells in the synovial membrane of an articular cavity, thereby achieving the effect of treating inflammatory joint diseases.

Description

A cyclic IL-10 mRNA nanoformulation, its preparation method and application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a cyclic IL-10 mRNA nanoparticle formulation, its preparation method, and its application technology. Background Technology

[0002] Common inflammatory joint diseases (IA) include osteoarthritis (OA), rheumatoid arthritis (RA), spondyloarthritis, and psoriatic arthritis (PsA). Over the past three decades, the incidence of IA has been rapidly increasing both domestically and internationally. According to the World Health Organization (WHO), approximately 18 million people currently suffer from rheumatoid arthritis, and 528 million from osteoarthritis. In my country alone, the number of osteoarthritis patients is approaching 140 million. Inflammatory joint diseases and osteoarthritis cause pain, limited mobility, joint destruction, and even deformities or disabilities, placing a heavy burden on patients and their families. Current treatments for inflammatory joint diseases mainly include disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, chondroprotective agents, and surgery. However, current treatments can only slow the progression of the disease.

[0003] Interleukin-10 (IL-10) is a pleiotropic cytokine with important immunomodulatory functions, primarily secreted by antigen-presenting cells such as activated T cells, monocytes, B cells, and macrophages. IL-10 can influence the activity of many cell types in the immune system. High levels of IL-10 inhibit their antigen-presenting capacity, leading to inefficient T cell activation. The presence of chronic antigens further depletes T cells and induces IL-10 production. Therefore, T cells develop "tolerance" to antigens, and high levels of IL-10 can promote the proliferation of regulatory T cells (Tregs) and helper T cells 2 (Th2), thereby suppressing the inflammatory response. 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 receptor. IL-10 can influence osteoclast and osteoblast proliferation through nuclear factor-kappa B (RANK) / RANK ligand (RANKL) / osteoplastin (OPG) axis receptor activators, participating in maintaining bone metabolic homeostasis. IL-10 can also significantly antagonize the expression of TNF-α-induced IL-6, MMP-1, and MMP-3, and 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 of IL-10 (such as recombinant cytokines and fusion proteins) have problems such as hematologic toxicity and short half-life.

[0004] Therefore, there is still an urgent need to develop a new type of IL-10 formulation or method with a long half-life and low blood toxicity, so as to better treat IL-10-related diseases and promote the application and development of IL-10 technology. Technical issues

[0005] The first objective of this invention is to disclose a cyclic IL-10 mRNA nanoformulation.

[0006] The second objective of this invention is to disclose a method for preparing the above-mentioned cyclic IL-10 mRNA nanoparticles.

[0007] The third objective of this invention is to disclose the application of the above-mentioned cyclic IL-10 mRNA nanoformulation. Technical solutions

[0008] The technical solution of this invention relates to 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 described in this invention can theoretically be any mRNA sequence capable of producing IL-10, including but not limited to self-reporter gene IL-10 mRNA, self-amplified IL-10 mRNA, and circular IL-10 mRNA.

[0010] Secondly, the present invention provides a delivery system for delivering IL-10 mRNA. The delivery system described in this invention can theoretically be any delivery system capable of delivering mRNA, including but not limited to cationic lipids, lipid nanoparticles, polymer nanoparticles, etc.

[0011] Thirdly, this invention provides a novel strategy for treating inflammatory joint diseases using IL-10 mRNA nanoparticles, which can significantly improve the progression of these diseases. The inflammatory joint diseases described in this invention include, but are not limited to, osteoarthritis (OA), rheumatoid arthritis (RA), spondyloarthritis, and psoriatic arthritis (PsA).

[0012] The objective of this invention is achieved through the following technical solution:

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

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

[0015] In vitro transcription: The Novizan 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 salts in the buffer were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa). After washing three times with enzyme-free water, the IL-10 mRNA was collected in enzyme-free water for further use or stored at -80°C to obtain further purified IL-10 mRNA.

[0017] The further purified IL-10 mRNA was enriched into circular IL-10 mRNA by digestion with Beyotime RNase R: 20 μg IL-10 mRNA, 20 U RNase R, the reaction was carried out at 37℃ for 30 min and at 85℃ for 15 min, then purified by high performance liquid chromatography, washed three times with an ultrafiltration device, and the circular IL-10 mRNA was collected in enzyme-free water for further use or stored at -80℃; the desired circular IL-10 mRNA was obtained.

[0018] (2) SM-102, distearate phosphatidylcholine (DSPC), cholesterol (Choi), dimyristoyl glycerol-polyethylene glycol (DMG-PEG) and distearate phosphatidyl ethanolamine-methoxy polyethylene glycol (DSPE-PEG) were dissolved in anhydrous ethanol to form homogeneous 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 a solution A with a total liposome concentration of 7.5 mg / mL by mixing SM-102, DSPC, Choi, DMG-PEG, and DSPE-PEG in a molar ratio of 50:10:38.5:0.75:0.75.

[0020] Mix 10 μg of circular IL-10 mRNA with citrate buffer at pH 4. Quickly add 25.7 μl of the solution to the mRNA and citrate buffer mixture on an 800 rpm shaker for 30 s. Then add 180 μl of molecular biology grade PBS dropwise.

[0021] Organic solvents and free compounds in the formed nano-formulation 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 nano-formulation was collected and dispersed in pH 7.4-PBS buffer for further use or storage at 4°C.

[0022] The method for preparing a circular IL-10 mRNA nanoparticle formulation described in the above technical solution, wherein: the IL-10 mRNA is any mRNA sequence capable of producing IL-10.

[0023] The above technical solution describes a method for preparing a circular IL-10 mRNA nanoparticle formulation, wherein the IL-10 mRNA is a self-reporter gene IL-10 mRNA, a self-amplified IL-10 mRNA, or a circular IL-10 mRNA.

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

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

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

[0027] The application of the cyclic IL-10 mRNA nanoparticles described in the above technical solution in the preparation of treatments for inflammatory joint diseases.

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

[0029] The application of the above-described technical solution, wherein the application of the cyclic IL-10 mRNA nanoparticles in the preparation of treatments for inflammatory joint diseases refers to its application in the preparation of anti-inflammatory drugs.

[0030] In the application described above, the anti-inflammatory effect refers to the ability to significantly reduce LPS-induced inflammatory responses or significantly improve the progression of inflammatory joint diseases. Beneficial effects

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

[0032] 1. Circulating 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, thereby improving the disease progression of inflammatory joint diseases.

[0033] 2. Circular IL-10 mRNA nanoparticles can efficiently deliver IL-10 into cells, translate it into 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 hematologic toxicity caused by recombinant cytokines and systemic immunosuppression caused by IL-10. Attached Figure Description

[0035] Figure 1 shows the characterization of circular IL-10 mRNA; Figure 1A is the total ion chromatogram of the in vitro transcription product in high performance liquid chromatography, Figure 1B is the 2% gel electrophoresis image, and Figure 1C is the Sanger sequencing spectrum of the splicing site.

[0036] 2. Figure 2 shows the size and morphology characterization of the circular IL-10 mRNA nanoparticles (IL-10 mRNA / LNPs); Figure 2A shows the size distribution of DLS; Figure 2B shows the size change of LNPs over 5 days; Figure 2C shows TEM imaging.

[0037] 3. Figure 3 shows the delivery efficiency and biocompatibility of the circular IL-10 mRNA nanoformulation; Figure 3A shows the expression time of the EGFP nanoformulation in cells; Figure 3B shows the lysosomal escape of the cy5-IL 10 nanoformulation; Figure 3C shows the expression time of IL-10 in cells; Figures 3D-3E show the immunogenicity and cytotoxicity of the IL-10 nanoformulation.

[0038] 4. Figure 4 shows the therapeutic effect of cyclic IL-10 mRNA nanoparticles in joint inflammatory diseases; Figure 4A shows the treatment cycle of rheumatoid arthritis; Figure 4B shows the reduction of IL-6 secretion in vitro by IL-10 nanoparticles; Figure 4C shows the curve of rat toe thickness change; Figure 4D shows the curve of rat clinical score change; Figure 4E shows the comparison of rat toes in different groups. The best embodiment of the present invention

[0039] To facilitate understanding of the technical solutions of the present invention, the following specific embodiments are provided. Obviously, the embodiments described herein are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

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

[0041] I. Materials:

[0042] SM-102 and LPS were purchased from MCE. Disteazylophosphatidylcholine (DSPC), cholesterol (Choi), dimyristoylglycerol-polyethylene glycol, and (DMG-PEG) disteazylophosphatidylethanolamine-methoxy polyethylene 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), TE buffer (20X) was purchased from Invitrogen, Carlsbad, California, USA.

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

[0046] II. Cell lines:

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

[0048] Example 1: Preparation of cyclic IL-10 and EGFP mRNA lipid nanoparticles:

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

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

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

[0052] 2. In vitro transcription (IVT) was performed on IL-10 mRNA, EGFP-mRNA, and cy5 IL-10 mRNA, respectively:

[0053] 2.1 In vitro transcription of IL-10 mRNA: The Novizan T7 transcription kit was used together with 1-2 μg of in vitro transcription template, 7.5 mM guanosine triphosphate, 7.5 mM 5-methylcytidine 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 (DNase) treatment to obtain IL-10 mRNA.

[0054] 2.2. In vitro transcription of EGFP-mRNA: The steps are the same as in 2.1, except that the in vitro transcription template is an EGFP in vitro transcription template, and EGFP mRNA is obtained.

[0055] 2.3. In vitro transcription of cy5 IL-10 mRNA: The steps are the same as in 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 the total ion chromatogram of the in vitro transcription products. Salts in the buffer were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa). After washing three times with enzyme-free water, the IL-10 mRNA and EGFP mRNA were collected in enzyme-free water for further use or stored at -80°C. Further purified IL-10 mRNA, EGFP mRNA, and cy5 IL-10 mRNA were obtained.

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

[0058] To assess whether the product of step 4 was circular IL-10 mRNA, the further purified IL-10 mRNA from the in vitro transcription product of step 3 and the circular IL-10 mRNA digested with Beyotime RNase R in step 4 were analyzed by electrophoresis on a 2% agarose gel; electrophoresis conditions: 100 V, 30 min. Using 4 μg of the circular IL-10 mRNA from step 4 as a reverse transcription template, the template was reverse transcribed using the Novizumab reverse transcription kit at 37℃ for 15 min followed by 85℃ for 5 s. The resulting cDNA product was sequenced by Sanger sequencing using Qingke Biotechnology Co., Ltd. The results are shown in Figure 1, where Figure 1A is the high-performance liquid chromatography total ion chromatogram of the in vitro transcription product from step 3, Figure 1B is the 2% gel electrophoresis image, and Figure 1C is the reverse transcription Sanger sequencing spectrum.

[0059] II. Preparation and Characterization of Nanoparticles:

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

[0061] 1. Distearate 102, distearate phosphatidylcholine (DSPC), cholesterol (Choi), dimyristoyl glycerol-polyethylene glycol (DMG-PEG), and distearate phosphatidylethanolamine-methoxy polyethylene glycol (DSPE-PEG) were dissolved in anhydrous ethanol to form homogeneous solutions with concentrations of 10 mg / ml, 20 mg / ml, 20 mg / ml, 10 mg / ml, and 20 mg / ml, respectively.

[0062] 2. Prepare solution A with a total liposome concentration of 7.5 mg / mL by mixing SM-102, DSPC, Choi, DMG-PEG, and DSPE-PEG in a molar ratio of 50:10:38.5:0.75:0.75;

[0063] 3. Mix 10 μg of circular IL-10 mRNA with citrate buffer at pH 4. Quickly add 25.7 μl of solution A to the mixture of mRNA and citrate buffer on a shaker at 800 rpm, shake for 30 s, and then add 180 μl of molecular biology grade PBS dropwise.

[0064] 4. Organic solvents and free compounds in the formed nanoparticle dispersion were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa). After washing three times with high-pressure water, the cyclic IL-10 mRNA nanoparticles were collected and dispersed in pH 7.4-PBS buffer for further use or storage at 4°C. The prepared cyclic IL-10 mRNA nanoparticles were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Dynamic light scattering (DLS, Brookhaven Instruments, USA) determined the size and stability of the cyclic IL-10 mRNA nanoparticles in phosphate buffer at 4°C over 5 days (results are shown in Figures 2A and 2B). Transmission electron microscopy (JEOL-2100) was used to observe the morphology of the mRNA nanoparticles at 200 kV and 101 µA (as shown in Figure 2C).

[0065] Circular EGFP mRNA and circular cy5 IL-10 mRNA nanoparticles were prepared according to steps 1-4, wherein in step 3, 10 μg of circular EGFP mRNA and circular cy5 IL-10 mRNA were mixed with citrate buffer at pH = 4.

[0066] 5. Detection of Cytotoxicity of Circular IL-10 mRNA Nanoparticles: 293T cells were uniformly seeded in 96-well plates at a density of 5000 cells per well. After 24 hours of cell adhesion, cells were transfected with different concentrations (0.5, 1, 2, or 3 μg / ml) of circular IL-10 mRNA nanoparticles for 12 hours, followed by incubation with 0.1 ml of fresh complete culture medium for another 24 hours to assess cell viability and transfection efficiency. Lip2k was used as a positive control for transfection efficiency. Cell viability was assessed using CCK-8, a non-toxic cell viability assay that allows real-time cell proliferation to be observed using a microplate reader (TECAN, Infinite M200 Pro). Absorbance was measured at 450 nm and 630 nm using a 96-well Spectramax plate reader (Molecular Device, Sunnyvale, CA). The results are shown in Figure 3D; the circular IL-10 mRNA nanoparticles did not exhibit cytotoxicity to the cells.

[0067] 6. To further validate the effectiveness of in vitro transfection, we selected circular EGFP-mRNA and circular cy5 IL-10 mRNA nanoparticles as model mRNAs. 293T cells were evenly seeded in 96-well plates at a density of 5000 cells per well. After 24 hours of cell adhesion, cells were transfected with 1 μg / ml of circular EGFP-mRNA nanoparticles for 12 hours, followed by the addition of 0.1 ml of fresh complete culture medium and further culturing. Images were taken every 12 hours under an inverted fluorescence microscope for the first 48 hours, and every 24 hours thereafter. The percentage of cells transfected with enhanced green fluorescent protein (GFP) was calculated using imaj-j to determine the half-life of GFP. Abundant green fluorescence was detected in 293T cells transfected with both the circular EGFP-mRNA nanoparticles and the commercially available transfection reagent Lipofectamine 2000 (Lip2k), and green fluorescence expression remained even 200 hours after transfection (Figure 3A). Circular cy5 IL-10 mRNA nanoparticles effectively delivered cy5-labeled mRNA into the cytoplasm. After 6 hours of culture, a large amount of cy5-labeled mRNA escaped from lysosomes and diffused into the cytoplasm. In contrast, naked mRNA could not enter the cells after 6 hours of culture (Figure 3B).

[0068] 7.293T cells were evenly seeded in 24-well plates at a density of 20,000 cells per well. After 24 hours of cell adhesion, cells were transfected with different concentrations (0.5, 1, 2, or 3 μg / ml) of circular IL-10 mRNA nanoparticles. Cell supernatant was collected at 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, and 144 h after transfection, and 500 μl of fresh culture medium was added. Lip2k was used as a positive control for transfection efficiency compared to the circular IL-10 mRNA nanoparticles. IL-10 secretion was assessed using an ELISA reader (TECAN, Infinite M200 Pro). IL-10 secretion was detected at 450 nm using a 96-well Spectramax plate reader (Molecular Device, Sunnyvale, CA), and IL-10 secretion was still detectable at 144 h (Figure 3C).

[0069] 8. Detection of Immunogenicity of Circular IL-10 mRNA Nanoparticles: RAW264.7 cells were uniformly seeded in 96-well plates at a density of 5000 cells per well. After 24 hours of cell adhesion, cells were transfected with different concentrations (0.5, 1, 2, or 3 μg / ml) of circular IL-10 mRNA nanoparticles for 12 hours, followed by incubation with 0.1 ml of fresh complete culture medium for another 24 hours. The cell supernatant was collected for the immunogenicity assay of the nanoparticles. Immunogenicity was assessed by ELISA testing of IL-6 secretion, which was examined using a microplate reader (TECAN, Infinite M200 Pro). Absorbance was measured at 450 nm using a 96-well Spectramax plate reader (Molecular Device, Sunnyvale, California); the results are shown in Figure 3E, indicating that the circular IL-10 mRNA nanoparticles were immunostimulatory.

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

[0071] (I) Experimental methods and procedures:

[0072] 1. In vitro anti-inflammatory assay: RAW264.7 cells were evenly seeded in 12-well plates at a density of 50,000 cells per well. After 24 hours of cell adhesion, cells were induced with 100 ng / mL LPS for 6 hours. Cells were then transfected with different concentrations (0.5, 1, 2, or 3 μg / ml) of circular IL-10 mRNA nanoparticles for 12 hours, followed by incubation with 1 ml of fresh complete culture medium for 48 hours. Cell supernatant was collected, and the in vitro anti-inflammatory activity of the circular IL-10 mRNA nanoparticles was evaluated by ELISA testing of IL-6 secretion. IL-6 secretion was examined using a microplate reader (TECAN, Infinite M200 Pro). Absorbance was measured at 450 nm using a 96-well Spectramax plate reader (Molecular Device, Sunnyvale, California). Adherent cells were collected, and total RNA was extracted from the cells using the trizol method. The total RNA was reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (Novizan). Changes in IL-6 at the mRNA level could be detected using a quantitative real-time qPCR instrument (CFX96 Touch).

[0073] 2. IL-10 mRNA Nanoparticles for Rheumatoid Arthritis: Immunoadapted SD rats aged 7-8 weeks were selected. IFA was emulsified with collagen solution. A needle was inserted bevel-up, parallel to the tail, 2 cm from the base of the tail, until the needle tip was 0.5 cm from the base. 0.2 ml (200 µg collagen) of the emulsion was injected subcutaneously at the base of the tail. A booster injection was given 7 days after the initial immunization. The collagen-IFA emulsion was prepared as described above, and 0.1 ml was injected subcutaneously at 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 performed at a different location than the initial injection. Disease was assessed using qualitative clinical scoring and paw thickness determination using a thrombectomy. After model establishment, circulated IL-10 mRNA nanoparticles were injected into the ankle joint cavity of rats in the IL-10 mRNA treatment group every week at a dose of 5 µg / rat. The control group received the same dose of PBS. The influence of the nanoparticles on disease progression was eliminated by administering the same dose of nanoparticles without circulated IL-10 mRNA. The model establishment and treatment time points are shown in Figure 4A.

[0074] 3. IL-10 mRNA Nanoparticles for Osteoarthritis Treatment: SD rats weighing 200-250 g were selected. After anesthesia with tribromoethanol, the rats were fixed, and the hair on their right hind limb was shaved. The skin of the leg was cut open to expose the knee joint. A T-shaped incision was made at the knee joint using a scalpel. The knee joint was flexed as much as possible to expose the anterior cruciate ligament and fully expose the knee joint. The lateral meniscus was removed, and the anterior cruciate ligament was cut along the suture. After hemostasis, the wound was sutured, and the joint cavity was closed layer by layer. For 3 days postoperatively, each rat was injected intramuscularly with penicillin daily to prevent wound infection. Two weeks postoperatively, rats in the IL-10 mRNA treatment group received an intra-articular injection of circulated IL-10 mRNA nanoparticles in their hind leg at a dose of 5 µg / rat. The control group received the same dose of PBS. The effect of the nanoparticles on disease progression was eliminated by administering the same dose of nanoparticles without circulated IL-10 mRNA. Disease was evaluated by osteophyte formation in the rat joint cavity, the degree of joint movement restriction, 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, cells were transfected with different concentrations (0.5, 1, 2 or 3 μg / ml) of IL-10 protein mRNA for 12 hours. PBS was used as a blank control, and the same dose of nanoparticles without circulated IL-10 mRNA was used to exclude the influence of nanoparticles on inflammation. After 48 hours of culture, the secretion of the inflammatory factor IL-6 and the expression level of IL-6 mRNA showed that different doses of circulated IL-10 mRNA had varying degrees of anti-inflammatory activity, with 3 μg / ml showing the best anti-inflammatory effect. The secretion of the inflammatory factor IL-6 was not significantly different from the blank control (as shown in Figure 4B).

[0077] 2. In the treatment of a rheumatoid arthritis model, the hind paw thickness of rats in both the model group and the group receiving nanoparticles without cyclic IL-10 mRNA continuously increased, affecting normal walking. After administration of the cyclic IL-10 mRNA nanoparticles, hind paw thickness decreased, and gait returned to normal, with no significant difference compared to the blank control. Hind paw thickness and clinical scores are shown in Figures 4C and 4D.

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

[0079] We designed a nanosystem to deliver IL-10 mRNA into the joint cavity for the treatment of inflammatory joint diseases. Results showed that IL-10 mRNA was successfully delivered via nanosystem to LPS-induced mouse mononuclear macrophage leukemia cells (RAW264.7), significantly reducing the LPS-induced inflammatory response. Simultaneously, successful delivery of IL-10 mRNA into the joint cavity of inflammatory joint diseases via nanosystem significantly improved the progression of these diseases.

[0080] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. Any modifications, alterations, and variations made by those skilled in the art without departing from the scope of the present invention using the disclosed technical content are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and variations made to the above embodiments based on the essential technology of the present invention are still within the scope of the present invention. Embodiments of the present invention

[0081] Type the description paragraph of embodiments of the present invention here. Industrial applicability

[0082] Type the industrial utility description paragraph here. Sequence List Free Content

[0083] Type the free content description paragraph for the sequence list here.

Claims

1. A method for preparing a cyclic IL-10 mRNA nanoparticle formulation, characterized in that, The method includes the following steps: (1) PCR amplification of the open reading frame plasmid of human IL-10 gene carrying T7 promoter and T7 terminator, and purification of PCR product to form linearized DNA as in vitro transcription template. In vitro transcription: The Novizan 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. 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 at -80°C to obtain further purified IL-10 mRNA. The further purified IL-10 mRNA was enriched into circular IL-10 mRNA by digestion with Beyotime RNase R: 20 μg IL-10 mRNA, 20 U RNase R, the reaction was carried out at 37℃ for 30 min and at 85℃ for 15 min, then purified by high performance liquid chromatography, washed three times with an ultrafiltration device, and the circular IL-10 mRNA was collected in enzyme-free water for further use or stored at -80℃; the desired circular IL-10 mRNA was obtained. (2) SM-102, distearylphosphatidylcholine, cholesterol, dimyristoylglycerol-polyethylene glycol and distearylphosphatidylethanolamine-methoxypolyethylene glycol were dissolved in anhydrous ethanol to form homogeneous 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 a solution A with a total liposome concentration of 7.5 mg / mL by mixing SM-102, DSPC, Choi, DMG-PEG, and DSPE-PEG in a molar ratio of 50:10:38.5:0.75:0.

75. Mix 10 μg of circular IL-10 mRNA with citrate buffer at pH 4. Quickly add 25.7 μl of solution A to the mixture of mRNA and citrate buffer on a shaker at 800 rpm and shake for 30 s. Then add 180 μl of molecular biology grade PBS dropwise. Organic solvents and free compounds in the formed nano-formulation 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 nano-formulation was collected and dispersed in pH 7.4-PBS buffer for further use or storage at 4°C.

2. The method for preparing a cyclic IL-10 mRNA nanoparticle formulation according to claim 1, characterized in that: The IL-10 mRNA is any mRNA sequence capable of producing IL-10.

3. The method for preparing a cyclic IL-10 mRNA nanoparticle formulation 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 nanoparticle formulation according to claim 1, characterized in that: The delivery product containing IL-10 mRNA nanoformulation can be any delivery system capable of delivering mRNA.

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

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

7. The application of the cyclic IL-10 mRNA nanoformulation according to claim 6 in the preparation of treatments for inflammatory joint diseases.

8. The application according to claim 7, characterized in that: The inflammatory joint diseases mentioned are osteoarthritis, rheumatoid arthritis, spondyloarthritis, or psoriatic arthritis.

9. The application according to claim 7, characterized in that: The application of the cyclic IL-10 mRNA nanoparticles in the preparation of drugs for treating inflammatory joint diseases refers to their application in the preparation of anti-inflammatory drugs.

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

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