Application of siRNA delivery carrier in preparation of medicine for treating osteoarthritis

Abstract

The invention discloses application of a siRNA delivery carrier in preparation of a medicine for treating osteoarthritis, and belongs to the technical field of biological medicine. The siRNA delivery carrier is an articular disc acellular matrix, and is prepared by the following steps: firstly, cleaning an animal temporomandibular articular disc, then carrying out repeated freeze thawing treatment, and then treating the articular disc with a guanidine hydrochloride solution, a pancreatin solution, an SDS (Sodium Dodecyl Sulfate) solution, a DNA (Deoxyribonucleic Acid) enzyme solution and an RNA (Ribonucleic Acid) enzyme solution in sequence to obtain an acellular sample; and after grinding, carrying out digestion treatment in a mixed solution of acetic acid and pepsin, adjusting the pH value, and centrifuging to obtain the product. The preparation method is simple, the prepared delivery carrier has the advantages of being good in biocompatibility, moderate in degradation rate, easy to process and prepare and the like, an effective and continuous treatment strategy can be provided for OA treatment when the delivery carrier is combined with nano particles and siRNA for application, the siRNA can be stably released to target cartilage cells in an osteoarthritis state, and clinical application potential is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of a siRNA delivery carrier in preparing a drug for treating osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a common chronic degenerative disease characterized by synovial inflammation, cartilage degeneration and subchondral bone sclerosis. It is very harmful, causing pain, joint damage and joint dysfunction, reducing the quality of life of patients and bringing a heavy social burden. Osteoarthritis is difficult to treat, and truly effective treatments are very limited.

[0003] Compared with systemic medication, intra-articular injection therapy is a relatively safe and effective treatment for osteoarthritis. It acts directly on the affected joint tissue, reduces the distribution range of the drug in the body, and thus reduces the risk of systemic side effects. In addition, intra-articular injection can achieve high-concentration release of drugs in the joints, improve the therapeutic effect and reduce the dosage of drugs, reducing the burden on systemic organs. However, after the drug is injected into the joint cavity, it will be quickly cleared from the joint space through the subsynovial capillaries and lymphatic vessels. The drug has a short half-life, and multiple, large-dose drug injections are often required to achieve the treatment goal, which may cause systemic toxicity. The intra-articular injection drugs currently used in clinical practice cannot meet the clinical needs of single injection and gradual sustained release. Multiple treatments are also inconvenient and uncomfortable for patients.

[0004] Small interfering RNA (siRNA), as an important component of RNA interference technology, plays an important role in gene silencing therapy with high specificity and effectiveness. However, siRNA is extremely small and can be easily cleared and degraded from the joint cavity and cannot act on the articular cartilage. How to effectively deliver siRNA to the target cells in the cartilage without being degraded is a problem that needs to be solved urgently. Therefore, the development of a degradation-resistant delivery carrier for intra-articular injection to treat osteoarthritis is a research focus in the current field of OA treatment. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an application of a siRNA delivery vector in the preparation of a drug for the treatment of osteoarthritis, so as to solve the problem that the existing siRNA is easily cleared and degraded from the joint cavity during intra-articular injection to treat osteoarthritis and cannot act on the articular cartilage, making it difficult to effectively deliver siRNA to the cartilage.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: A use of a siRNA delivery vector in the preparation of a drug for treating osteoarthritis, wherein the siRNA delivery vector is a decellularized matrix of an articular disc.

[0007] The beneficial effects of the present invention are as follows: the present invention uses the decellularized matrix of the articular disc as a delivery carrier of siRNA, and can realize local injection of the joint cavity through the excellent rheological properties and injectability of the decellularized matrix of the articular disc, and at the same time, through the viscoelastic characteristics of the decellularized matrix of the articular disc itself, the loaded nanoparticle-siRNA can be retained in the joint cavity for a long time, and gradually released in the synovial fluid, thereby achieving effective treatment of arthritis. Compared with sodium hyaluronate, a delivery carrier of traditional siRNA, the decellularized medium of the articular disc prepared by the present invention has better injectability and rheological properties as a loading matrix, and has a significantly better retention effect in the joint cavity after loading siRNA, and can play a role continuously, and still has a certain effect at 42 days after injection, while sodium hyaluronate is used for siRNA loading, and it has been basically completely degraded in just one week.

[0008] Furthermore, the above-mentioned articular disc acellular matrix is ​​prepared by the following method: (1) The temporomandibular joint disc of the animal is first cleaned and then subjected to repeated freeze-thaw treatment. The joint disc after repeated freeze-thaw treatment is then treated with guanidine hydrochloride solution, pancreatic enzyme solution, SDS solution, DNA enzyme solution and RNA enzyme solution in sequence to obtain a decellularized sample; (2) Grinding the decellularized sample obtained in step (1) and digesting it in a mixed solution of acetic acid and pepsin, adjusting the pH, and centrifuging to obtain the product.

[0009] The beneficial effects of adopting the above further technical scheme are as follows: the preparation method of the present invention is simple, the prepared delivery carrier dECM has excellent rheological properties and injectability, can be injected locally into the joint cavity, and can retain the nanoparticle-siRNA in the joint cavity for a long time by utilizing its own viscoelastic characteristics, and gradually release it in the synovial fluid, thereby achieving effective treatment of osteoarthritis. At the same time, the dECM prepared by the present invention is derived from natural cartilage tissue, has good biocompatibility and bioactivity, can provide biomechanical support, and provide structural support and a favorable microenvironment for cartilage in the state of osteoarthritis.

[0010] Furthermore, the animal temporomandibular joint disc in step (1) is a rabbit temporomandibular joint disc.

[0011] Furthermore, the conditions for the repeated freeze-thaw treatment in step (1) are: first standing at -30~-10°C for 10~15 h, then standing at 1~10°C for 10~15 h, and repeating 2~5 times.

[0012] Furthermore, the treatment in the guanidine hydrochloride solution in step (1) is carried out at a temperature of 1-10°C and for a time of 30-40 h. The guanidine hydrochloride solution is prepared by the following method: firstly, guanidine hydrochloride and sodium acetate trihydrate are mixed and dissolved in water, then Tris-EDTA is added to adjust the pH, and finally, a protease inhibitor is added and mixed uniformly to obtain the solution.

[0013] Furthermore, the concentration of guanidine hydrochloride in the guanidine hydrochloride solution is 0.3-0.5 g / mL, the concentration of sodium acetate trihydrate is 5-10 mg / mL, and the volume concentration of the protease inhibitor is 0.5-2%.

[0014] Furthermore, in step (1), the mass concentration of trypsin in the trypsin solution is 0.1-1.0%, the treatment temperature in the trypsin solution is 35-40°C, and the treatment time is 10-15 h; the treatment conditions in the SDS solution are: at room temperature, first treat in an SDS solution with a mass concentration of 0.4-0.6% for 8-12 h, and then treat in an SDS solution with a mass concentration of 0.05-0.2% for 8-12 h; the concentration of the DNA enzyme solution is 80-120 U / mL, the concentration of the RNA enzyme solution is 800-1200 U / mL, and the treatment time in the DNA enzyme solution and the RNA enzyme solution is 3-5 h.

[0015] The beneficial effects of adopting the above further technical scheme are as follows: the present invention uses guanidine hydrochloride solution, pancreatic enzyme solution, SDS solution, DNA enzyme solution and RNA enzyme solution to decellularize the articular disc, which saves 3 days compared with the conventional decellularization process. At the same time, due to the low concentration of SDS used, the addition of DNA enzyme and RNA enzyme can more specifically remove cell components, which is more conducive to the retention of components in natural cartilage tissue. After decellularization by the method of the present invention, the cell components in each area are completely removed, the cell nucleus disappears, and the articular disc tissue does not show obvious loose and sparse changes, and the collagen component in the articular disc dECM is well preserved.

[0016] Furthermore, in step (2), the concentration of acetic acid in the mixed solution of acetic acid and pepsin is 0.3-0.7 mol / L, the mass ratio of pepsin to the decellularized sample is (10-20): (100-200); the digestion temperature is 20-30°C, and the time is 90-100 h.

[0017] Furthermore, in step (2), a NaOH solution with a concentration of 3 to 5 mol / L is used to adjust the pH to neutral.

[0018] The present invention has the following beneficial effects: The present invention provides a method for preparing a siRNA delivery vector for intra-articular injection to treat osteoarthritis. The prepared delivery vector has the advantages of good biocompatibility, moderate degradation rate, no obvious inflammatory effect on the human body after degradation, easy processing and preparation, etc. The prepared dECM combined with nanoparticles and siRNA can provide an effective and sustainable treatment strategy for OA treatment, can stably release siRNA to target chondrocytes in the osteoarthritis state, and has clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a preparation process and research flow chart of the siRNA delivery vector for intra-articular injection to treat osteoarthritis; Figure 2 The results of HE staining of the articular disc before and after decellularization; Figure 3 The results of safranin fast green staining before and after decellularization of the articular disc; Figure 4 The results of immunohistochemical staining of type I collagen before and after decellularization of the articular disc; Figure 5 The diagrams show the general appearance of the dECM prepared in the present invention and the rheological properties evaluation results of clinically used sodium hyaluronate, wherein A shows the general appearance of the prepared articular disc dECM and the general appearance of the currently clinically used sodium hyaluronate, B shows the viscosity curves of articular disc dECM and sodium hyaluronate at different concentrations at different shear rates, and C shows the viscosity of samples at different concentrations and shear rates; Figure 6 This is a test diagram of the effect of dECM prepared by the present invention on the retention of the temporomandibular joint cavity in the OA model; Figure 7 This is a diagram showing the results of HE staining of important organs in an animal model after the dECM prepared by the present invention was injected into the temporomandibular joint cavity; Figure 8 This is a graph showing the test results of dECM+DGL@siRNA transfected normal condylar chondrocytes for 12 hours; Fig. 9 This is a graph showing the test results of OA condylar chondrocytes transfected with dECM+DGL@siRNA prepared by the present invention for 12 hours; Fig.10 This is a result diagram showing the effect of dECM+DGL@siRNA prepared by the present invention on apoptosis of condylar chondrocytes in normal / OA states; Fig.11 Live-dead staining diagram of the effect of dECM+DGL@siRNA on the biological activity of condylar chondrocytes prepared by the present invention; Fig.12 HE staining diagram of the therapeutic effect of dECM prepared by the present invention injected into the joint cavity of OA model for 1.5 months; Fig.13 This is a Safranin Fast Green staining diagram of the therapeutic effect of dECM prepared by the present invention injected into the joint cavity of OA model for 1.5 months; Fig.14 This is a micro-CT reconstruction image of the therapeutic effect of dECM prepared by the present invention injected into the joint cavity of an OA model for 1.5 months. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0021] Embodiment 1: A method for preparing a siRNA delivery vector for intra-articular injection to treat osteoarthritis (preparation and research flow chart as shown in Figure 1 ), including the following steps: (1) Repeated freeze-thaw treatment: The temporomandibular joint discs of 4-month-old New Zealand rabbits were collected intact under sterile conditions and carefully washed with saline to remove blood stains. The rabbit joint discs were first placed at -20°C for 12 h, then at 4°C for 12 h, and the freeze-thaw treatment was repeated three times.

[0022] (2) Prepare guanidine hydrochloride solution: first weigh 38.212 g guanidine hydrochloride and 0.6804 g sodium acetate trihydrate, add 50 mL Milli-Q ultrapure water and stir until completely dissolved, then add 1 mL Tris-EDTA (1×) to adjust the pH to 5.8, finally make up to 100 mL with ultrapure water and add protease inhibitors to make the volume concentration in the solution 1%, and store at 4°C until used.

[0023] (3) Decellularization: First, treat the rabbit articular disc treated in step (1) with the guanidine hydrochloride solution prepared in step (2) at 4°C for 36 h; then treat the rabbit articular disc with a 0.5% trypsin solution at 37°C for 12 h; then treat the rabbit articular disc with a 0.5% SDS solution at room temperature for 10 h, and treat the rabbit articular disc with a 0.1% SDS solution for 10 h; finally, treat the rabbit articular disc in a 100 U / mL DNase solution for 4 h and in a 1000 U / mL RNase solution for 4 h on a shaker at 200 rpm to obtain a decellularized sample.

[0024] (4) dECM digestion: First, weigh 150 mg of the decellularized sample obtained in step (3) and grind it into powder. Add 10 mL of 0.5 mol / L acetic acid and 15 mg of pepsin, and digest it at room temperature for 96 h. Then, slowly add 4 mol / L NaOH solution to adjust the pH to neutral. Finally, centrifuge to obtain an siRNA delivery carrier dECM with injectability and excellent rheological properties for intra-articular injection to treat osteoarthritis.

[0025] Embodiment 2: A method for preparing a siRNA delivery vector for intra-articular injection to treat osteoarthritis comprises the following steps: (1) Repeated freeze-thaw treatment: The temporomandibular joint discs of 4-month-old New Zealand rabbits were collected intact under sterile conditions and carefully washed with saline to remove blood stains. The rabbit joint discs were first placed at -15°C for 15 h, then at 6°C for 15 h, and the freeze-thaw treatment was repeated three times.

[0026] (2) Prepare guanidine hydrochloride solution: first weigh 30 g guanidine hydrochloride and 0.55 g sodium acetate trihydrate, add 50 mL Milli-Q ultrapure water and stir until completely dissolved, then add Tris-EDTA (1×) to adjust the pH to 5.8, finally make up to 100 mL with ultrapure water and add protease inhibitors so that the volume concentration in the solution is 0.8%, and store at 4°C for later use.

[0027] (3) Decellularization: First, treat the rabbit articular disc treated in step (1) with the guanidine hydrochloride solution prepared in step (2) at 4°C for 40 h; then treat the rabbit articular disc with a 0.5% trypsin solution at 37°C for 15 h; then treat the rabbit articular disc with a 0.5% SDS solution at room temperature for 12 h, and then treat the rabbit articular disc with a 0.1% SDS solution for 12 h; finally, treat the rabbit articular disc in a 100 U / mL DNase solution for 5 h and in a 1000 U / mL RNase solution for 5 h on a shaker at 200 rpm to obtain a decellularized sample.

[0028] (4) dECM digestion: First, weigh 150 mg of the decellularized sample obtained in step (3) and grind it into powder. Add 10 mL of 0.5 mol / L acetic acid and 15 mg of pepsin. Digest at room temperature for 100 h. Then slowly add 4 mol / L NaOH solution to adjust the pH to neutral. Finally, centrifuge to obtain an siRNA delivery carrier dECM with injectability and excellent rheological properties for intra-articular injection to treat osteoarthritis.

[0029] Embodiment 3: A method for preparing a siRNA delivery vector for intra-articular injection to treat osteoarthritis comprises the following steps: (1) Repeated freeze-thaw treatment: The temporomandibular joint discs of 4-month-old New Zealand rabbits were collected intact under sterile conditions and carefully washed with saline to remove blood stains. The rabbit joint discs were first placed at -25°C for 10 h, then at 1°C for 10 h, and the freeze-thaw treatment was repeated three times.

[0030] (2) Prepare guanidine hydrochloride solution: first weigh 50 g guanidine hydrochloride and 0.75 g sodium acetate trihydrate, add 50 mL Milli-Q ultrapure water and stir until completely dissolved, then add Tris-EDTA (1×) to adjust the pH to 5.8, finally make up to 100 mL with ultrapure water and add protease inhibitors so that the volume concentration in the solution is 1.2%, and store at 4°C for later use.

[0031] (3) Decellularization: First, treat the rabbit articular disc treated in step (1) with the guanidine hydrochloride solution prepared in step (2) at 4°C for 30 h; then treat the rabbit articular disc with a 0.5% trypsin solution at 37°C for 10 h; then treat the rabbit articular disc with a 0.5% SDS solution at room temperature for 8 h, and treat the rabbit articular disc with a 0.1% SDS solution for 8 h; finally, treat the rabbit articular disc in a 100 U / mL DNase solution for 3 h and in a 1000 U / mL RNase solution for 3 h on a shaker at 200 rpm to obtain a decellularized sample.

[0032] (4) dECM digestion: First, weigh 150 mg of the decellularized sample obtained in step (3) and grind it into powder. Add 10 mL of 0.5 mol / L acetic acid and 15 mg of pepsin, and digest it at room temperature for 90 h. Then, slowly add 4 mol / L NaOH solution to adjust the pH to neutral. Finally, centrifuge to obtain an siRNA delivery carrier dECM with injectability and excellent rheological properties for intra-articular injection to treat osteoarthritis.

[0033] Experimental example: (1) Decellularization effect The decellularized sample obtained in step (3) of Example 1 was immersed in a 4% paraformaldehyde solution for 24 hours. The fixed sample was fully washed with running water and placed in a fully automatic dehydrator for dehydration. The natural articular disc sample that was not decellularized was used as a control group. After being fully infiltrated with paraffin and embedded, the tissue was sliced ​​along the horizontal plane using a paraffin slicer with a thickness of 5 μm. After being dried in an oven at 65°C, HE staining, safranin fast green staining, and type I collagen immunohistochemical staining were performed to verify the decellularization effect.

[0034] The experimental results are as follows Figure 2-Figure 4As shown in the figure, the results show that in the horizontal, longitudinal central and annular sections of the natural articular disc, chondrocytes are evenly distributed in the pits, the cell nuclei are clearly visible in purple-blue, and the extracellular matrix components are arranged tightly and continuously. After decellularization, it can be seen that the cell components in the sections in all directions and regions are removed, the cell nuclei disappear, and the final articular disc tissue does not show obvious loose and sparse changes. Figure 3 and Figure 4 The staining results in the figure proved that the collagen components, especially type I collagen, in the dECM of the articular disc obtained after decellularization were well preserved.

[0035] (2) Rheological properties and joint cavity retention effect The rheological properties and retention effects of sodium hyaluronate, which is widely used for clinical intra-articular injection, and dECM prepared in the present invention in the temporomandibular joint cavity were compared. Sodium hyaluronate and dECM carrying 1.25 nmol cy5-siRNA (Jima) with fluorescent groups were injected into an established rat temporomandibular joint OA model (the OA model was established by injecting 50 μL of physiological saline solution containing 2 mg of sodium iodoacetate into the temporomandibular joint cavity in front of the rat's ear), respectively. The sodium hyaluronate and dECM retained in the joint cavity were observed at multiple continuous time points (1 day, 3 days, 7 days and 42 days) using the PerkinElmer IVIS Lumina III in vivo imaging system.

[0036] The experimental results are as follows Figure 5-Figure 7 shown.

[0037] Figure 5 Figure A in the middle shows the general view of the dECM prepared by the present invention and the sodium hyaluronate used in clinical practice. Figure 5 As shown in Figures B and C, it can be seen from samples of different concentrations and shear rates that the dECM prepared in the present invention has injectability and better rheological properties than sodium hyaluronate.

[0038] Figure 6 The results showed that the degradation rate of Sodium Hyaluronate was extremely fast after injection, and the fluorescence signal decayed rapidly, and it had completely disappeared after 1 week. The dECM prepared by the present invention had a good articular cavity retention effect and could exert its effect continuously, and could continue to play a role in the temporomandibular joint cavity of the OA model.

[0039] The important organs of the experimental group of rats injected with dECM in the joint cavity, including heart, liver, spleen, lung and kidney, were collected for HE staining. The experimental results are as follows: Figure 7As shown, the detection found that compared with the untreated control group, the main organs of the experimental group treated with the dECM of the present invention were normal, the cell morphology in the organs was normal, and no degeneration and necrosis such as atrophy or vacuolization was observed.

[0040] (3) The effect of dECM loaded with cationic nanoparticles-siRNA (dECM+DGL@siRNA) was evaluated by transfecting normal and OA condylar chondrocytes. The cationic nanocarrier DGL in the experimental group was from COLCOM, and the nitrogen-phosphorus ratio of DGL and siRNA was 10:1. 1.5 μL of siRNA at a concentration of 20 μmol / L was added to each well of the six-well plate. In the control group, 1.5 μL of siRNA at a concentration of 20 μmol / L was added to each well of the six-well plate, without dECM and DGL.

[0041] The experimental results are as follows Figure 8 and Fig. 9 As shown, the results show that compared with the control group, the dECM prepared by the present invention can achieve better chondrocyte transfection effect after loading cationic nanoparticles-siRNA.

[0042] (4) Evaluation of the apoptosis and toxicity of dECM+DGL@siRNA articular disc cells Rabbit articular disc cells were resuspended, counted, and inoculated into 24-well plates in different groups at a density of 3×10 4 Each group was set up with 4 replicate wells. After 24 h of cell culture, the culture medium was replaced and cultured in a 37°C constant temperature cell incubator under 5% (volume fraction) carbon dioxide. Five days after the culture medium was replaced, the prepared live / dead staining solution was added to each well, and the live / dead cell staining was observed under a fluorescence microscope and photographed. The control group was pure cell culture without dECM+DGL@siRNA.

[0043] The experimental results are as follows Fig.10 and Fig.11 As shown, it can be observed that the rabbit articular disc cells inoculated in different sample groups are mainly living cells, and basically no dead cells are observed.

[0044] (5) Condylar repair effect test The dECM prepared in the present invention was injected into a rat temporomandibular joint OA model (injection dose of 40 μL) and then observed.

[0045] The experimental results are as follows Figure 12-14 As shown, HE staining ( Fig.12 ) and safranin fast green stain ( Fig.13 ) The results show that the dECM prepared by the present invention can effectively protect the condylar cartilage, making it clear in layers and avoiding degradation and damage. The repair effect of the condylar bone was evaluated by micro-CT ( Fig.14 ), the results showed that dECM can prevent condylar bone resorption and destruction caused by OA.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a siRNA delivery vector in the preparation of a drug for treating osteoarthritis, characterized in that: The siRNA delivery carrier is a decellularized disc matrix.

2. Use of the siRNA delivery vector according to claim 1 in the preparation of a drug for treating osteoarthritis, characterized in that: The decellularized matrix of the articular disc is prepared by the following method: (1) The temporomandibular joint disc of the animal is first cleaned and then subjected to repeated freeze-thaw treatment. The joint disc after repeated freeze-thaw treatment is then treated with guanidine hydrochloride solution, pancreatic enzyme solution, SDS solution, DNA enzyme solution and RNA enzyme solution in sequence to obtain a decellularized sample; (2) Grinding the decellularized sample obtained in step (1) and digesting it in a mixed solution of acetic acid and pepsin, adjusting the pH, and centrifuging to obtain the product.

3. Use of the siRNA delivery vector according to claim 2 in the preparation of a drug for treating osteoarthritis, characterized in that: The conditions for the repeated freeze-thaw treatment in step (1) are: first standing at -30~-10°C for 10~15 h, then standing at 1~10°C for 10~15 h, and repeating 2~5 times.

4. Use of the siRNA delivery vector according to claim 2 in the preparation of a drug for treating osteoarthritis, characterized in that: The temperature for treatment in the guanidine hydrochloride solution in step (1) is 1-10° C. and the time is 30-40 h. The guanidine hydrochloride solution is prepared by the following method: firstly, guanidine hydrochloride and sodium acetate trihydrate are mixed and dissolved in water, then Tris-EDTA is added to adjust the pH, and finally, a protease inhibitor is added and mixed evenly to obtain the solution.

5. Use of the siRNA delivery vector according to claim 4 in the preparation of a drug for treating osteoarthritis, characterized in that: In the guanidine hydrochloride solution, the concentration of guanidine hydrochloride is 0.3-0.5 g / mL, the concentration of sodium acetate trihydrate is 5-10 mg / mL, and the volume concentration of the protease inhibitor is 0.5-2%.

6. Use of the siRNA delivery vector according to claim 2 in the preparation of a drug for treating osteoarthritis, characterized in that: In the step (1), the mass concentration of trypsin in the trypsin solution is 0.1-1.0%, the treatment temperature in the trypsin solution is 35-40°C, and the treatment time is 10-15 h. The conditions for treatment in the SDS solution are: at room temperature, first treat in an SDS solution with a mass concentration of 0.4-0.6% for 8-12 h, and then treat in an SDS solution with a mass concentration of 0.05-0.2% for 8-12 h. The concentration of the DNA enzyme solution is 80-120 U / mL, the concentration of the RNA enzyme solution is 800-1200 U / mL, and the treatment time in the DNA enzyme solution and the RNA enzyme solution is 3-5 h.

7. Use of the siRNA delivery vector according to claim 2 in the preparation of a drug for treating osteoarthritis, characterized in that: In the step (2), the concentration of acetic acid in the mixed solution of acetic acid and pepsin is 0.3-0.7 mol / L, and the mass ratio of pepsin to the decellularized sample is (10-20):(100-200); the digestion temperature is 20-30°C, and the time is 90-100 h.

8. Use of the siRNA delivery vector according to claim 2 in the preparation of a drug for treating osteoarthritis, characterized in that: In the step (2), a NaOH solution with a concentration of 3-5 mol / L is used to adjust the pH to neutral.

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