Application of an siRNA delivery vector in the preparation of a medicament for treating osteoarthritis

Abstract

The present invention discloses the application of an siRNA delivery vector in the preparation of a drug for treating osteoarthritis, belonging to the technical field of biomedicine. The above siRNA delivery vector is an acellular matrix of the articular disc, which is prepared by the following method: first, the animal temporomandibular joint disc is washed and then subjected to repeated freeze-thaw treatment, and then the joint disc is successively treated with guanidine hydrochloride solution, trypsin solution, SDS solution, DNase solution and RNase solution to obtain an acellular sample; after grinding, it is digested in a mixed solution of acetic acid and pepsin, the pH is adjusted, and centrifuged to prepare. The preparation method of the present invention has the advantages of simple preparation method, good biocompatibility of the prepared delivery vector, moderate degradation rate, easy processing and preparation, etc. The combined application with nanoparticles and siRNA can provide an effective and continuous treatment strategy for OA treatment, can stably release siRNA targeting chondrocytes in the state of osteoarthritis, and has the potential for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to the application of an siRNA delivery vector in the preparation of 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 highly harmful, causing pain, joint damage, and joint dysfunction, reducing the quality of life of patients and bringing a heavy social burden. The treatment of osteoarthritis is difficult, and truly effective treatment means are very limited.

[0003] Compared with the systemic drug administration route, intra-articular injection treatment is a relatively safe and effective treatment method for osteoarthritis. It directly acts on the affected joint tissues, reducing the distribution range of the drug in the body, thereby reducing the risk of systemic side effects. In addition, intra-articular injection can achieve high-concentration release of the drug in the joint, improving the treatment effect and reducing the drug dosage, and reducing the burden on the whole body organs. However, after the drug is injected into the joint cavity, it will be rapidly cleared from the joint space through the sub-synovial capillaries and lymphatic vessels, and the drug half-life is short. To achieve the treatment purpose, multiple and large-dose drug injections are often required, which may cause systemic toxicity. Currently, the intra-articular injection drugs 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 part of RNA interference technology, plays an important role in gene silencing therapy. It has high specificity and effectiveness. However, siRNA is extremely small and is easily cleared and degraded from the joint cavity and cannot act on articular cartilage. How to effectively deliver siRNA to the target cells in cartilage without being degraded is an urgent problem to be solved. Therefore, the development of a degradation-resistant delivery vector for intra-articular injection treatment of osteoarthritis is a research focus in the current field of OA treatment. Summary of the Invention

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

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] Application of an siRNA delivery vector in the preparation of a drug for treating osteoarthritis, wherein the siRNA delivery vector is an acellular matrix of the articular disc.

[0008] The beneficial effects of the present invention are as follows: The present invention uses the acellular matrix of the articular disc as the delivery vector of siRNA. Through the excellent rheological properties and injectability of the acellular matrix of the articular disc, local injection into the joint cavity can be achieved. At the same time, due to the viscoelastic characteristics of the acellular 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 the traditional siRNA delivery vector sodium hyaluronate, the acellular matrix of the articular disc prepared in the present invention has better injectability and rheological properties as a loading matrix. After loading siRNA, it has a significantly better retention effect in the joint cavity, can continuously play a role, and still has a certain effect 42 days after injection, while when using sodium hyaluronate to load siRNA, it has basically been completely degraded in only one week.

[0009] Furthermore, the above acellular matrix of the articular disc is prepared by the following method:

[0010] (1) First, wash the animal temporomandibular joint disc and then perform repeated freeze-thaw treatment. Then, treat the joint disc after repeated freeze-thaw treatment successively with guanidine hydrochloride solution, trypsin solution, SDS solution, DNase solution, and RNase solution to obtain a decellularized sample.

[0011] (2) Grind the decellularized sample obtained in step (1) and then perform digestion treatment in a mixed solution of acetic acid and pepsin, adjust the pH, and centrifuge to prepare it.

[0012] The beneficial effects of adopting the above further technical solution are as follows: The preparation method of the present invention is simple. The prepared delivery vector dECM has excellent rheological properties and injectability, can achieve local injection into the joint cavity, and can make nanoparticle-siRNA retain in the joint cavity for a long time by using its own viscoelastic characteristics and gradually release in the synovial fluid, thereby achieving effective treatment of osteoarthritis. At the same time, the dECM prepared in the present invention is derived from natural cartilage tissue, has good biocompatibility and biological activity, can provide biomechanical support, and provides structural support and a favorable microenvironment for cartilage in the state of osteoarthritis.

[0013] Furthermore, in step (1), the animal temporomandibular joint disc is a rabbit temporomandibular joint disc.

[0014] Furthermore, the conditions for the repeated freeze-thaw treatment in step (1) are: first, stand at -30~-10°C for 10~15 h, then stand at 1~10°C for 10~15 h, and repeat 2~5 times.

[0015] Further, in step (1), the treatment is carried out in a guanidine hydrochloride solution at a temperature of 1 - 10 °C for 30 - 40 h; the guanidine hydrochloride solution is prepared by the following method: first, guanidine hydrochloride and sodium acetate trihydrate are mixed and dissolved in water, then Tris-EDTA is added to adjust the pH, and finally protease inhibitors are added and mixed evenly to obtain it.

[0016] Further, 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 protease inhibitors is 0.5 - 2%.

[0017] Further, 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 DNase solution is 80 - 120 U / mL, the concentration of RNase solution is 800 - 1200 U / mL, and the treatment time in both the DNase solution and the RNase solution is 3 - 5 h.

[0018] The beneficial effects of adopting the above further technical solutions are as follows: The present invention uses guanidine hydrochloride solution, trypsin solution, SDS solution, DNase solution and RNase solution to perform decellularization treatment on the articular disc, saving 3 days compared with the conventional decellularization treatment process. At the same time, due to the lower concentration of SDS used, adding DNase and RNase can more specifically remove cell components, which is more conducive to the retention of components in natural cartilage tissue. After the decellularization treatment by the method of the present invention, the cell components in each region are completely removed, the cell nuclei disappear, and there is no obvious loose and sparse change in the articular disc tissue, and the collagen components in the dECM of the articular disc are well preserved.

[0019] Further, in 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 treatment temperature is 20 - 30 °C, and the time is 90 - 100 h.

[0020] Further, in step (2), a NaOH solution with a concentration of 3 - 5 mol / L is used to adjust the pH to neutral.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a preparation method of an siRNA delivery vector for intra-articular injection in the treatment of osteoarthritis. The prepared delivery vector has the advantages of good biocompatibility, moderate degradation rate, no obvious inflammatory effect on the human body after degradation, and easy processing and preparation. The combined application of the prepared dECM with nanoparticles and siRNA can provide an effective and sustained treatment strategy for OA treatment, can stably release siRNA targeting chondrocytes in the state of osteoarthritis, and has the potential for clinical application. Brief Description of the Drawings

[0023] Figure 1 It is a process flow chart and a research flow chart of the siRNA delivery vector for intra-articular injection in the treatment of osteoarthritis according to the present invention;

[0024] Figure 2 It is a graph of the HE staining results before and after decellularization of the articular disc;

[0025] Figure 3 It is a graph of the safranin-fast green staining results before and after decellularization of the articular disc;

[0026] Figure 4 It is a graph of the immunohistochemical staining results of type I collagen before and after decellularization of the articular disc;

[0027] Figure 5 It is a graph of the gross view of the dECM prepared according to the present invention and the rheological property evaluation results of sodium hyaluronate for clinical use. Among them, A is the gross view of the articular disc dECM after preparation and the gross view of sodium hyaluronate currently used clinically, B is the viscosity curve graph of the articular disc dECM and sodium hyaluronate with different concentrations at different shear rates, and C is the viscosity graph of the samples at different concentrations and shear rates;

[0028] Figure 6 It is a graph for detecting the retention effect of the dECM prepared according to the present invention in the temporomandibular joint cavity in the OA model;

[0029] Figure 7 It is a graph of the HE staining detection results of important organs in the animal model after injection of the dECM prepared according to the present invention into the temporomandibular joint cavity;

[0030] Figure 8 It is a graph of the detection results after the dECM + DGL@siRNA prepared according to the present invention transfected normal condylar chondrocytes for 12 hours;

[0031] Figure 9 It is a graph of the detection results after the dECM + DGL@siRNA prepared according to the present invention transfected OA condylar chondrocytes for 12 hours;

[0032] Figure 10 It is a graph of the influence results of the dECM + DGL@siRNA prepared according to the present invention on the apoptosis of normal / OA state condylar chondrocytes;

[0033] Figure 11 This is a live / dead staining image of the biological activity of dECM+DGL@siRNA prepared by the present invention on condylar chondrocytes;

[0034] Figure 12 This is a HE staining image of the treatment effect of dECM prepared by the present invention after 1.5 months of intra-articular injection in an OA model;

[0035] Figure 13 This is a safranin-fast green staining image of the treatment effect of dECM prepared by the present invention after 1.5 months of intra-articular injection in an OA model;

[0036] Figure 14 This is a micro-CT reconstruction image of the treatment effect of dECM prepared by the present invention after 1.5 months of intra-articular injection in an OA model. Detailed implementation mode

[0037] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained by purchasing in the market.

[0038] Example 1:

[0039] A preparation method of an siRNA delivery vector for intra-articular injection in the treatment of osteoarthritis (the preparation and research flow chart is as Figure 1 shown), including the following steps:

[0040] (1) Repeated freeze-thaw treatment: Under sterile conditions, the temporomandibular joint disc of 4-month-old New Zealand rabbits was collected intact and carefully washed with physiological saline to remove blood. The rabbit joint disc was first placed at -20 °C for 12 h, then at 4 °C for 12 h, and repeated 3 times for repeated freeze-thaw treatment.

[0041] (2) Preparation of guanidine hydrochloride solution: First, weigh 38.212 g of guanidine hydrochloride and 0.6804 g of sodium acetate trihydrate, add 50 mL of Milli-Q ultrapure water and stir until completely dissolved, then add 1 mL of Tris-EDTA (1×) to adjust the pH to 5.8, and finally make up the volume to 100 mL with ultrapure water and add a protease inhibitor so that its volume concentration in the solution is 1%, and store it at 4 °C for later use.

[0042] (3)Decellularization treatment: First, treat the rabbit articular disc after the treatment 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% (mass concentration) trypsin solution at 37 °C for 12 h; then treat the rabbit articular disc with a 0.5% (mass concentration) SDS solution at room temperature for 10 h and with a 0.1% (mass concentration) SDS solution for 10 h; finally, place it on a shaker at 200 rpm and treat it in a 100 U / mL DNase solution for 4 h and in a 1000 U / mL RNase solution for 4 h to obtain a decellularized sample.

[0043] (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 96 h, then slowly add 4 mol / L NaOH solution to adjust the pH to neutral, and finally centrifuge to obtain dECM, an injectable siRNA delivery carrier with excellent rheological properties for intra-articular injection treatment of osteoarthritis.

[0044] Example 2:

[0045] A preparation method of an siRNA delivery carrier for intra-articular injection treatment of osteoarthritis, comprising the following steps:

[0046] (1)Repeated freeze-thaw treatment: Under aseptic conditions, collect the temporomandibular joint discs of 4-month-old New Zealand rabbits intact and carefully wash away the blood with physiological saline. First, place the rabbit articular disc at -15 °C for 15 h, then at 6 °C for 15 h, and repeat 3 times for repeated freeze-thaw treatment.

[0047] (2)Prepare guanidine hydrochloride solution: First, weigh 30 g of guanidine hydrochloride and 0.55 g of sodium acetate trihydrate, add 50 mL of Milli-Q ultrapure water and stir until completely dissolved, then add Tris-EDTA (1×) to adjust the pH to 5.8, and finally make up the volume to 100 mL with ultrapure water and add a protease inhibitor so that its volume concentration in the solution is 0.8%, and store it at 4 °C for standby.

[0048] (3)Decellularization treatment: First, treat the rabbit articular disc after the treatment 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% (mass concentration) trypsin solution at 37 °C for 15 h; then treat the rabbit articular disc with a 0.5% (mass concentration) SDS solution at room temperature for 12 h and with a 0.1% (mass concentration) SDS solution for 12 h; finally, place it on a shaker at 200 rpm and treat it in a 100 U / mL DNase solution for 5 h and in a 1000 U / mL RNase solution for 5 h to obtain a decellularized sample.

[0049] (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, and finally centrifuge to obtain dECM, an injectable siRNA delivery carrier with excellent rheological properties for intra-articular injection treatment of osteoarthritis.

[0050] Example 3:

[0051] A preparation method of an siRNA delivery carrier for intra-articular injection treatment of osteoarthritis, comprising the following steps:

[0052] (1)Repeated freeze-thaw treatment: Under aseptic conditions, collect the temporomandibular joint discs of 4-month-old New Zealand rabbits intact and carefully wash away the blood with physiological saline. First, place the rabbit articular disc at -25 °C for 10 h, then at 1 °C for 10 h, and repeat 3 times for repeated freeze-thaw treatment.

[0053] (2)Prepare guanidine hydrochloride solution: First, weigh 50 g of guanidine hydrochloride and 0.75 g of sodium acetate trihydrate, add 50 mL of Milli-Q ultrapure water and stir until completely dissolved, then add Tris-EDTA (1×) to adjust the pH to 5.8, and finally make up the volume to 100 mL with ultrapure water and add a protease inhibitor so that its volume concentration in the solution is 1.2%, and store it at 4 °C for standby.

[0054] (3)Decellularization treatment: First, treat the rabbit articular disc after the treatment 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% (mass concentration) trypsin solution at 37 °C for 10 h; then treat the rabbit articular disc with a 0.5% (mass concentration) SDS solution at room temperature for 8 h and with a 0.1% (mass concentration) SDS solution at room temperature for 8 h; finally, place it on a shaker at 200 rpm and treat it in a 100 U / mL DNase solution for 3 h and in a 1000 U / mL RNase solution for 3 h to obtain a decellularized sample.

[0055] (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 90 h, then slowly add 4 mol / L NaOH solution to adjust the pH to neutral, and finally centrifuge to obtain an injectable dECM with excellent rheological properties as an siRNA delivery vector for intra-articular injection treatment of osteoarthritis.

[0056] Experimental examples:

[0057] (1)Decellularization effect

[0058] Take the decellularized sample obtained in step (3) of Example 1, immerse it in a 4% (mass fraction) paraformaldehyde solution for fixation for 24 h, wash the fixed sample thoroughly with running water, place it in a fully automatic dehydrator for dehydration, and use the natural articular disc sample without decellularization treatment as a control group. After fully infiltrating with paraffin and embedding, use a paraffin slicer to slice the tissue along the horizontal plane, with a slice thickness of 5 μm. After baking the slices in an oven at 65 °C, perform HE staining, safranin-fast green staining, and type I collagen immunohistochemical staining to verify the decellularization effect.

[0059] The experimental results are as Figures 2 - 4 shown. The results show that in the horizontal, longitudinal central, and annular region slices of the natural articular disc, chondrocytes are evenly distributed in the lacunae, and the cell nuclei are clearly visible in purple-blue. The extracellular matrix components are arranged tightly and continuously. After decellularization treatment, it can be seen that the cell components in the slices in all directions and regions are removed, the cell nuclei disappear, and at the same time, there is no obvious loose and sparse change in the final articular disc tissue. Figure 3 and Figure 4 the staining results in prove that the collagen components, especially type I collagen components, in the articular disc dECM obtained after decellularization are well preserved.

[0060] (2)Rheological properties and intra-articular retention effect

[0061] Compare the rheological properties and the retention effect in the temporomandibular joint cavity of sodium hyaluronate, which is widely used in clinical intra-articular injection, and the dECM prepared by the present invention. In the established rat temporomandibular joint OA model (an 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 ear), sodium hyaluronate and dECM carrying 1.25 nmol of cy5-siRNA (from GenePharma) with a fluorescent group were respectively injected. The retention of sodium hyaluronate and dECM in the joint cavity was observed at multiple consecutive time points (1 day, 3 days, 7 days, and 42 days) through a PerkinElmer IVIS Lumina III in vivo imaging system.

[0062] The experimental results are as Figures 5 - 7 shown.

[0063] Figure 5 Figure A in shows the general view of the dECM prepared by the present invention and the clinically used sodium hyaluronate. According to the results detected by the rheometer, as shown in Figures B and C in Figure 5 , it can be seen from the samples with different concentrations and shear rates that the dECM prepared by the present invention has injectability and better rheological properties compared to sodium hyaluronate.

[0064] Figure 6 It shows that the degradation rate of sodium hyaluronate after injection is extremely fast, the fluorescence signal decays rapidly, and it has completely disappeared at 1 week, while the dECM prepared by the present invention has a good retention effect in the joint cavity, can continuously exert its effect, and can continuously play a role in the temporomandibular joint cavity of the OA model.

[0065] The main organs of the heart, liver, spleen, lungs, and kidneys of the experimental group of rats injected with dECM into the joint cavity were collected for HE staining. The experimental results are as Figure 7 shown. It was detected that compared with the untreated control group, there were no abnormalities in the main organs of the experimental group treated with the dECM of the present invention, the cell morphology in the organs was normal, and no manifestations of degeneration and necrosis such as atrophy or vacuolization were seen.

[0066] (3) Detect the transfection effect of normal and OA state condylar chondrocytes after dECM is loaded with cationic nanoparticles-siRNA (dECM+DGL@siRNA). The cationic nanocarrier DGL in the experimental group is from COLCOM Company. The nitrogen-phosphorus ratio of DGL and siRNA is 10:1. 1.5 μL of siRNA with a concentration of 20 μmol / L is added to each well of a six-well plate. In the control group, 1.5 μL of siRNA with a concentration of 20 μmol / L is added to each well of a six-well plate, without adding dECM and DGL.

[0067] The experimental results are as follows Figure 8 and Figure 9 shown. The results indicate that, compared with the control group, after the dECM loaded with cationic nanoparticles-siRNA prepared by the present invention, better transfection effects on chondrocytes can be achieved.

[0068] (4)Evaluate the apoptotic and toxic effects of dECM+DGL@siRNA on articular disc cells

[0069] After resuspending and counting rabbit articular disc cells, they were seeded into 24-well plates of different groups at an inoculation density of 3×10 4 cells / well, with 4 replicates in each group. After inoculation, the cells were cultured for 24 h and then the medium was changed, and they were cultured in a constant temperature cell incubator at 37℃ under the condition of 5% (volume fraction) carbon dioxide. On the 5th day after changing the medium, the prepared live / dead staining solution was added to each well, and the staining conditions of live or dead cells were observed under a fluorescence microscope and photographed. The control group was pure cell culture without adding dECM+DGL@siRNA.

[0070] The experimental results are as follows Figure 10 and Figure 11 shown. It can be observed that the rabbit articular disc cells inoculated in different sample groups are mainly live cells, and basically no dead cells are seen.

[0071] (5)Examination of the condylar repair effect

[0072] The dECM prepared by the present invention was injected into a rat temporomandibular joint OA model (the injection dose was 40 μL) and then observed.

[0073] The experimental results are as follows Figures 12 - 14 shown. The results of HE staining ( Figure 12 ), and safranin-fast green staining ( Figure 13 ), prove that the dECM prepared by the present invention can effectively protect the condylar cartilage, making its layers clear and avoiding degradation and damage. The repair effect of condylar bone was evaluated by micro-CT ( Figure 14 ), and the results showed that dECM can avoid the absorption and destruction of condylar bone caused by OA.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of acellular disc matrix as a siRNA delivery carrier 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), digesting it in a mixed solution of acetic acid and pepsin, adjusting the pH, and centrifuging to obtain; The temperature of the 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 uniformly to obtain the solution; 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 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; 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 hours.

2. The use of the decellularized disc matrix according to claim 1 as a siRNA delivery carrier 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.

3. Use of the acellular matrix of articular disc according to claim 1 as a siRNA delivery carrier 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%.

4. Use of the decellularized disc matrix according to claim 1 as a siRNA delivery carrier 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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