A hydrogel-state articular cartilage-targeted nano-drug delivery system and preparation method thereof

Abstract

The invention belongs to the technical field of biomedicine, and specifically discloses a hydrogel articular cartilage-targeted nano-drug delivery system and a preparation method thereof. PEG is used to modify G5PAMAM to prepare a 50% PEGylated PAMAM material (PAMAM-PEG-Mal); the PAMAM material is used to carry a synthesized miR-455-3p mimic, and a cysteine-terminated chondrocyte affinity peptide (CAP) and a minimal self peptide (MSP) are connected to synthesize a positively charged cartilage-targeted nano-carrier (CANC); a self-assembled neutral triblock hydrogel (PCEC) is used to package the positively charged cartilage-targeted nano-carrier to obtain a hydrogel articular cartilage-targeted nano-drug delivery system with a solid content of 15%. Compared with the prior art, the hydrogel-state articular cartilage-targeted nanodrug delivery system of the present invention can increase the joint residence time of miR-455-3p mimic, improve the utilization efficiency of miR-455-3p mimic by articular chondrocytes, thereby effectively inhibiting articular cartilage degeneration and OA progression, while having good biosafety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a hydrogel-state articular cartilage-targeted nano drug delivery system and a preparation method thereof. Background Art

[0002] Osteoarthritis (OA) is one of the main causes of long-term pain, loss of working ability and decreased quality of life in the middle-aged and elderly. In modern times, with the changes in lifestyle, especially incorrect exercise, overuse of joints and obesity, the incidence of OA has become younger. Epidemiological data show that there are currently about 140 million OA patients in my country, and the number is increasing year by year with the aging of society. Due to the lack of effective early intervention methods, most OA patients need to undergo artificial joint replacement surgery in the late stage of the disease. Studies have shown that the annual number of artificial joint replacement surgeries in my country is increasing at an average rate of 20% year by year, bringing a heavy burden to society and the economy.

[0003] Currently, the treatments for early osteoarthritis mainly include oral non-inflammatory analgesics (NSAIDs), slow-acting drugs such as glucosamine and chondroitin, anti-anxiety drugs, Chinese patent medicines, intra-articular injections of sodium hyaluronate, glucocorticoids, platelet-rich plasma (PRP), as well as physical therapy and sports rehabilitation. When the disease develops to the middle and late stages and knee joint strangulation occurs, arthroscopic cleaning can be performed, and in the late stages, joint replacement surgery is required.

[0004] Articular cartilage degeneration is the main cause of the development of osteoarthritis. Currently, oral anti-inflammatory analgesics (NSAIDs), slow-acting drugs, anti-anxiety drugs and Chinese patent medicines, as well as intra-articular injections of hyaluronic acid and glucocorticoids can only suppress local inflammation and relieve symptoms for a period of time, but cannot effectively prevent articular cartilage degeneration and OA progression. In recent years, the popular intra-articular platelet-rich plasma therapy does not seem to benefit OA patients significantly. When the damaged cartilage falls off to form loose bodies or the meniscus tears and causes joint ligaments, arthroscopic cleaning can remove the joint ligaments, but it cannot repair the extensively degenerated articular cartilage. Artificial joint replacement surgery performed in the late stage of the disease is traumatic and expensive, and cannot restore the limit and fine movement of the joint. Young patients in particular are also faced with the problem of artificial knee revision.

[0005] Therefore, seeking effective articular cartilage treatment targets and developing a reliable articular cartilage repair method based on this to curb the progression of OA in the early stages of the disease is the key to treatment and is also a problem that urgently needs to be solved.

[0006] Nucleic acid drugs are at the forefront of biopharmaceuticals. Our previous application number CN202010773820.X discloses the use of miR-455-3p as a therapeutic target for osteoarthritis. By constructing bone marrow mesenchymal stem cells that overexpress miR-455-3p and secreting exosomes rich in miR-455-3p, early intervention in osteoarthritis is achieved to treat OA. However, the biological production of exosomes is a natural process. The molecular components of exosomes are complex, the production of miR-455-3p is difficult to stably regulate, the exosomes produced are not molecularly targeted, and the cell culture and exosome separation and purification technology are difficult. Large-scale industrial production cannot be achieved at this stage. In addition, the clinical application of allogeneic exosomes also faces risks such as adverse immune reactions, carcinogenesis, and viral contamination. So far, there has been no unified standard for monitoring the safety and effectiveness of exosomes at home and abroad.

[0007] miRNAmimic can mimic the function of mature miRNA in cells to enhance the regulatory effect of endogenous miRNA. Currently, chemical synthesis has been used to achieve industrial production of miRNAmimic. However, like endogenous miRNA, synthetic miRNAmimic is easily degraded by nucleases in the body, and has poor targeting to articular cartilage, low chondrocyte uptake rate and utilization rate, and cannot be used directly as a drug. Synthetic nano drug delivery systems can be customized according to the characteristics of miRNA, joint environment and cartilage to overcome the above problems. The product quality is stable, easy to achieve industrial production, and has mature quality control and safety evaluation standards. Summary of the invention

[0008] In order to protect miR-455-3p mimic from being easily degraded by nucleases in vivo, and to endow it with the ability to target articular cartilage, penetrate articular cartilage matrix, and be efficiently taken up and utilized by articular chondrocytes, the present invention provides a hydrogel articular cartilage-targeted nano drug delivery system and a preparation method thereof.

[0009] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0010] One of the purposes of the present invention is to provide a hydrogel-state articular cartilage-targeted nano-drug delivery system and a preparation method thereof, comprising the following steps:

[0011] S1, using PEG to modify G5 PAMAM to prepare 50% PEGylated PAMAM material PAMAM-PEG-Mal;

[0012] S2, using PAMAM-PEG-Mal to carry miR-455-3p mimic, and connecting the cysteine-terminated chondrocyte affinity peptide CAP and the minimal self-peptide MSP to synthesize the positively charged cartilage-targeted nanocarrier CANC;

[0013] S3. Use self-assembled neutral triblock hydrogel PCEC to encapsulate the positively charged cartilage-targeted nanocarrier CANC to obtain a hydrogel-state articular cartilage-targeted nanodrug delivery system with a solid content of 15%.

[0014] Furthermore, the step S1 specifically includes:

[0015] S1-1. 100 nmol of G5 PAMAM was dialyzed against NaH2PO4 buffer at pH 6 and mixed with 6.4 μmol of PEG in 0.1 M NaHCO3 buffer at pH 8 overnight. Unattached PEG was removed by dialyzation against PBS buffer at pH 6.5 to prepare 50% PEGylated PAMAM material PAMAM-PEG-Mal.

[0016] Furthermore, the step S2 specifically includes:

[0017] S2-1. Rapidly inject the synthesized miR-455-3pmimic into PAMAM-PEG-Mal at N / P=8:1, mix and incubate at room temperature for 30 minutes to load the miR-455-3p mimic on the PEGylated PAMAM, add the same molar amount of cysteine-terminated chondrocyte affinity peptide CAP and minimal self-peptide MSP at the same time according to 2 times the molar amount of PAMAM, react at 4°C for 2 hours, remove the unconnected polypeptides with ultracentrifugation column, and obtain the dual-peptide modified cartilage-targeted nanoparticle CANC loaded with miR-455-3p mimic, which is freeze-dried in vacuo and stored at -20°C.

[0018] Furthermore, the step S3 specifically includes:

[0019] S3-1, first drying ε-caprolactone ε-CL with calcium hydride, then collecting it in a dry flask by vacuum distillation and storing it in nitrogen, using Sn(Oct)2 as a catalyst, adding PEG and ε-CL in a molar ratio of 91:15 into a polymerization tube, freezing and thawing it three times, heating it to 110°C and reacting it in nitrogen for 2 days; after the reaction, dissolving the product in dichloromethane, purifying it three times by ether precipitation, obtaining a neutral triblock hydrogel PCEC, vacuum freeze-drying it and storing it at -20°C;

[0020] S3-2. Take 182ul phosphate buffer solution (PBS) to dissolve CANC, then add 30.9mg PCEC, shake for a few seconds and let stand overnight at room temperature to obtain a hydrogel articular cartilage-targeted nano-drug delivery system with a solid content of 15%.

[0021] Preferably, in step S1-1, the molecular weight cutoff of the dialysis bag used for dialyzing G5 PAMAM is 3000, and the molecular weight cutoff of the dialysis bag used for dialyzing PBS buffer to remove unattached PEG is 2000.

[0022] Preferably, in step S2-1, the molecular weight cutoff of the ultracentrifugation column is 2000.

[0023] The second purpose of the present invention is to provide a hydrogel articular cartilage-targeted nano-drug delivery system prepared by the above method.

[0024] Compared with the prior art, in the hydrogel-state articular cartilage-targeted nano-drug delivery system of the present invention, CAP can enhance the affinity of nano-carriers to articular cartilage, and MSP can reduce the phagocytosis of nano-carriers by synovial macrophages; finally, the cartilage-targeted nano-carriers are packaged using self-assembled neutral triblock hydrogel (PCEC), which can not only shield the "cation trap" in the body to prolong the joint residence time of the nano-carriers, but also realize the aggregation and stable controlled release of the nano-carriers in the articular cartilage; in addition, the hydrogel-state articular cartilage-targeted nano-drug delivery system prepared by the present invention has injectability, good fluidity and self-healing properties, and can stably release positively charged nano-carriers carrying miR-455-3p mimic at joint temperature; the hydrogel-state articular cartilage-targeted nano-drug delivery system does not affect the biological function of the miR-455-3p mimic carried, and shows good molecular stability in synovial fluid, as well as strong articular cartilage penetration ability and chondrocyte targeting ability; injection of miR-455-3p into the joint cavity of surgically induced OA mice The mimic / hydrogel articular cartilage-targeted nanodrug delivery system increases the joint residence time of miR-455-3p mimic and improves the utilization efficiency of miR-455-3p mimic by articular chondrocytes, thereby effectively inhibiting articular cartilage degeneration and OA progression while having good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention is a flow chart for preparing the hydrogel articular cartilage-targeted nano-drug delivery system.

[0026] Figure 2 The morphology and physical properties of the hydrogel-state articular cartilage-targeted nanodrug delivery system: A is cross-linked into a hydrogel at room temperature, B is injectable, C is self-healing, and D is plastic.

[0027] Figure 3 Rheological mechanics testing of the hydrogel-state articular cartilage-targeted nanodrug delivery system: A is frequency scanning; B is strain scanning; C is temperature scanning; and D is oscillation test.

[0028] Figure 4 The scanning electron micrograph of the hydrogel-state articular cartilage-targeted nanodrug delivery system, the transmission electron micrograph of the released nanocarriers, and the release rate graph: A is the scanning electron micrograph; B is the transmission electron micrograph of the sustained-release microparticles in the chamber; C is the sustained-release rate.

[0029] Figure 5 This is an in vitro biological test of the hydrogel-state articular cartilage-targeted nanodrug delivery system: A is the stability of miR-455-3p mimic in synovial fluid; B is the transfection ability; C is the ability to penetrate the articular cartilage matrix; D is chondrocyte targeting.

[0030] Figure 6 The effectiveness test of the hydrogel-state articular cartilage-targeted nanodrug delivery system in the in vivo treatment of OA: A is the flowchart of mouse OA joint modeling and treatment; B is the safranin-fast green staining and type II collagen immunohistochemistry of the modeled joint tissue section; C is the CT image of the modeled joint; D is the detection of articular cartilage homeostasis indicators of the modeled joint.

[0031] Figure 7 This is a safety test of the hydrogel-state articular cartilage-targeted nanodrug delivery system for the in vivo treatment of OA: A shows that there were no gross differences in the heart, liver, spleen, lung and kidney organs of mice; B shows that there were no differences in the tissue sections of the heart, liver, spleen, lung and kidney organs of mice; C shows that there were no differences in the routine blood test results of mice; D shows that there were no differences in the liver, kidney and heart function indexes of mice. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0033] Unless otherwise specified, the raw materials and reagents used in the following examples were commercially available.

[0034] Reference Figure 1 This embodiment exemplifies a hydrogel-state articular cartilage-targeted nano-drug delivery system and a preparation method thereof, comprising the following steps:

[0035] S1, using PEG to modify G5 PAMAM to prepare 50% PEGylated PAMAM material (PAMAM-PEG-Mal);

[0036] S1-1, 100 nmol of G5 PAMAM (purchased from Dendritech) was dialyzed against a pH = 6 NaH2PO4 buffer (molecular weight cutoff of 3000) and mixed with 6.4 μmol of SM (PEG) 12 (purchased from Thermo Fisher Scientific) in a 0.1 M NaHCO3 buffer at pH = 8 overnight, and dialyzed against a pH = 6.5 PBS buffer (molecular weight cutoff of 2000) to remove unconnected PEG, thereby preparing a 50% PEGylated PAMAM material (PAMAM-PEG-Mal);

[0037] S2, using 50% PEGylated PAMAM material to carry miR-455-3p mimic, and connecting cysteine-terminated chondrocyte affinity peptide CAP and minimal self-peptide MSP to synthesize positively charged cartilage-targeted nanocarrier CANC;

[0038] S2-1, the synthesized miR-455-3p mimic (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) was rapidly injected into the PEGylated PAMAM material (PAMAM-PEG-Mal) according to N / P=8:1, the mixture was mixed and incubated at room temperature for 30 minutes to load the miR-455-3pmimic on the PEGylated PAMAM, and the same molar amount of the cysteine-terminated chondrocyte affinity peptide CAP (amino acid sequence: Cys-DWRVIIPPRPS, purchased from Suzhou Qiangyao Biotechnology Co., Ltd.) and the minimal self-peptide MSP (amino acid sequence: Cys-GNYTCEVTELTREGETIIELK, purchased from Suzhou Qiangyao Biotechnology Co., Ltd.) were added at the same time according to 2 times the molar amount of PAMAM, and the reaction was carried out at 4°C for 2 hours. The unconnected polypeptides were removed by ultracentrifugation column (molecular weight cutoff of 2000) to obtain the double-peptide modified cartilage-targeted nanocarrier CANC carrying miR-455-3p mimic, which was freeze-dried in vacuo and stored at -20°C;

[0039] S3, using self-assembled neutral triblock hydrogel PCEC to package the positively charged cartilage-targeted nanocarrier CANC to obtain a hydrogel-state articular cartilage-targeted nanodrug delivery system with a solid content of 15%;

[0040] S3-1, first drying ε-caprolactone ε-CL (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) with calcium hydride, then collecting it in a dry flask by vacuum distillation, and storing it in nitrogen, using Sn(Oct)2 as a catalyst, adding PEG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and ε-CL in a molar ratio of 91:15 into a polymerization tube, freezing and thawing it three times, heating it to 110°C and reacting it in nitrogen for 2 days; after the reaction, dissolving the product in dichloromethane, purifying it three times by ether precipitation, obtaining a neutral triblock hydrogel PCEC, vacuum freeze-drying it and storing it at -20°C;

[0041] S3-2. Take 182ul phosphate buffer solution (PBS) to dissolve CANC, then add 30.9mg PCEC, shake for a few seconds and let stand overnight at room temperature to obtain a hydrogel-state articular cartilage-targeted nanodrug delivery system PCEC@CANC with a solid content of 15%.

[0042] See also Figure 2 A, B, and D in the figure, at room temperature, the hydrogel-state articular cartilage-targeted nano-drug delivery system prepared in this example can self-assemble to form a hydrogel, and the formed hydrogel can be smoothly injected through a 31G needle and can be shaped at will; see Figure 2 In Figure C, the hydrogel-state articular cartilage-targeted nanodrug delivery systems dyed red and blue were placed in contact under humid conditions at 37°C. After 2 hours, the contact surfaces blended with each other, indicating good self-healing properties.

[0043] The neutral triblock hydrogel PCEC and the hydrogel articular cartilage targeted nano-drug delivery system PCEC@CANC prepared in this example were subjected to rheological tests using a rheometer, and commercial hyaluronic acid (HA) - Alge (Seikagaku Kogyo Co., Ltd.) was used as a control. The test results are shown in FIG. Figure 3 As shown, Figure 3 As shown in Figure A, the frequency sweep of the rheometer confirmed the elastic properties of PCEC and PCEC@CANC hydrogels. Figure 3 As shown in B in Figure 1, the strain scan further shows the yield property of the material. After being subjected to a certain stress, the storage modulus and loss modulus of both materials drop rapidly and transform into viscous properties. The lower yield stress of PCEC@CANC indicates that it is easier to inject. Figure 3 As shown in D in Figure 1, the oscillation test also confirmed that the rapid change of the storage modulus of PCEC and PCEC@CANC under high and low strains supports the rapid self-healing characteristics of the material. Figure 3 As shown in Figure 3C, PCEC@CANC also exhibits temperature sensitivity. When the temperature exceeds 36°C, the storage modulus decreases relative to the loss modulus and the viscosity of the material increases.

[0044] Furthermore, the scanning electron microscopy results of the hydrogel-based articular cartilage-targeted nanodrug delivery system PCEC@CANC are shown in Figure 2. Figure 4 The cartilage-targeted nanocarrier CANC shown in A is encapsulated in the voids of the neutral triblock hydrogel PCEC. Further, we placed the hydrogel-state articular cartilage-targeted nanodrug delivery system PCEC@CANC loaded with FAM-miR-455-3p mimic into a Transwell chamber (0.4 μm pore size), collected the culture medium in the culture well at 37°C for transmission electron microscopy detection, and measured the fluorescence value every 24 hours to calculate its release rate. Figure 4 B and Figure 4 As shown in Figure C, at 37°C, the hydrogel-state articular cartilage-targeted nanodrug delivery system PCEC@CANC can slowly release CANC loaded with miR-455-3p mimic at a stable rate.

[0045] In vitro experiments:

[0046] The synovial fluid of KOA patients was obtained (the synovial fluid was obtained from patients who underwent joint replacement surgery in the Department of Joint Surgery of the First Affiliated Hospital of Sun Yat-sen University, with ethical approval IRB no. 2013C-110, and the patients had signed informed consent). 1 nmol of free miR-455-3p mimic or PCEC@CANC loaded with an equal amount of miR-455-3p mimic was incubated with the synovial fluid at 37°C, and synovial fluid samples were collected regularly. The content of miR-455-3p mimic in the synovial fluid of the two groups was detected by PCR gel electrophoresis. Figure 5 PCEC@CANC can significantly enhance the stability of miR-455-3p mimic in synovial fluid. Similarly, primary articular chondrocytes were stimulated with 50 nM free miR-455-3p mimic or CANC loaded with an equal amount of miR-455-3p mimic, and q-PCR was used to detect changes in cartilage-related genes and cartilage degeneration-related genes. Figure 5 The CANC shown in B in the figure not only does not affect the biological function of the miR-455-3p mimic it carries, but also enhances its cellular utilization efficiency. Furthermore, we used articular cartilage discs of equal size (including complete articular cartilage and subchondral bone), and contacted the articular cartilage surface of the articular cartilage disc with 1 nmol free FAM-miR-455-3p mimic solution or PCEC@CANC loaded with an equal amount of FAM-miR-455-3p mimic. After 72 hours, the articular cartilage disc was cut open from the center, sliced, and then the penetration of FAM-miR-455-3p mimic was observed under a fluorescence microscope. Figure 5As shown in Figure C, PCEC@CANC loaded FAM-miR-455-3p mimic showed stronger penetration ability in articular cartilage. Finally, we tested the targeting ability of CANC to articular chondrocytes and macrophages. We added 50nM free FAM-miR-455-3p mimic or CANC loaded with an equal amount of FAM-miR-455-3p mimic to articular chondrocytes and macrophages, respectively. After 24 hours, the uptake of miR-455-3pmimic by the two cells was detected by fluorescence microscopy. Figure 5 As shown in D, articular chondrocytes have a stronger uptake ability for miR-455-3p mimic loaded by CANC, while macrophages have reduced uptake and consumption of miR-455-3p mimic. The articular chondrocyte targeting of CANC is conducive to improving its therapeutic efficiency.

[0047] In vivo experiments:

[0048] First, a mouse OA model of medial meniscus instability (DMM) was constructed. 12-week-old C57 / BL6 male mice (from the Institute of Model Animals, Nanjing University) were anesthetized by intraperitoneal injection of 1% pentobarbital (8 μL / g). The right knee joint was prepared and disinfected with iodine. The skin, subcutaneous tissue and knee joint capsule were cut in sequence, and the medial meniscus tibial ligament was cut to free the medial meniscus to cause knee instability. The joint capsule and skin wounds were sutured with 5-0 silk sutures. Ampicillin (20 mg / kg) was injected subcutaneously every day for 3 days after surgery to prevent infection, and buprenorphine (0.05 mg / kg) was injected subcutaneously every day for analgesia.

[0049] In the mouse OA model of medial meniscus instability (DMM), PBS solution or 1 nmol miR-455-3p mimic was injected into the joint cavity weekly for 4 consecutive weeks starting from 2 weeks after modeling, or a single injection of the hydrogel-state articular cartilage-targeted nanodrug delivery system PCEC@CANC containing 4 nmol miR-455-3p mimic was given, and the mice were killed at the 10th week of modeling (see Figure 6 A in Figure 1). Knee joint tissue section staining showed that PCEC@CANC, a hydrogel-based articular cartilage-targeted nanodrug delivery system loaded with 4 nmol of miR-455-3p mimic, was injected into the joint cavity once, resulting in the least degree of articular cartilage degeneration and the least osteophyte formation on CT (see Figure 6 B and C in the figure). PCR detection of cartilage tissue further confirmed that it can better promote the high expression of cartilage-related molecules and inhibit the expression of cartilage degradation-related molecules (see Figure 6 D), indicating that the hydrogel-state articular cartilage-targeted nanodrug delivery system PCEC@CANC can significantly inhibit the progression of OA through a single administration into the joint cavity.

[0050] Then, the important organs (heart, liver, spleen, lung, kidney) of mice were taken for tissue sectioning and blood test. Figure 7 As shown by Figure 7 As shown in A, there was no significant difference in the heart, liver, spleen, lung and kidney of mice. Figure 7 As shown in B, there was no difference in the tissue sections of the mouse heart, liver, spleen, lung and kidney. Figure 7 As shown in C, there was no difference in the blood routine test results of mice. Figure 7 As shown in D, there was no difference in the liver, kidney and heart function indexes of mice. In summary, the hydrogel-based articular cartilage-targeted nano-drug delivery system has good biosafety.

[0051] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel-state articular cartilage-targeted nano-drug delivery system, characterized in that: The following steps are involved: S1, using PEG to modify G5 PAMAM to prepare 50% PEGylated PAMAM material PAMAM-PEG-Mal; S2, using PAMAM-PEG-Mal to carry miR-455-3p mimic, and connecting the cysteine-terminated chondrocyte affinity peptide CAP and the minimal self-peptide MSP to synthesize the positively charged cartilage-targeted nanocarrier CANC; S3. Use self-assembled neutral triblock hydrogel PCEC to package the positively charged cartilage-targeted nanocarrier CANC to obtain a hydrogel-state articular cartilage-targeted nanodrug delivery system with a solid content of 15%.

2. The method for preparing the hydrogel articular cartilage-targeted nano-drug delivery system according to claim 1, characterized in that: The step S1 specifically includes: S1-1. 100 nmol of G5 PAMAM was dialyzed against NaH2PO4 buffer at pH 6 and mixed with 6.4 μmol of PEG in 0.1 M NaHCO3 buffer at pH 8 overnight. Unattached PEG was removed by dialyzation against PBS buffer at pH 6.5 to prepare 50% PEGylated PAMAM material PAMAM-PEG-Mal.

3. The method for preparing the hydrogel articular cartilage-targeted nano-drug delivery system according to claim 2, characterized in that: The step S2 specifically includes: S2-1. Rapidly inject miR-455-3p mimic into PAMAM-PEG-Mal at N / P=8:1, mix and incubate at room temperature for 30 minutes to load miR-455-3p mimic on PEGylated PAMAM, add chondrocyte affinity peptide CAP and minimal self-peptide MSP in the same molar amount as 2 times the molar amount of PAMAM at the same time, react at 4°C for 2 hours, remove unconnected polypeptides with ultracentrifugation column, and obtain dual-peptide modified cartilage-targeted nanocarrier CANC loaded with miR-455-3p mimic, which is freeze-dried in vacuo and stored at -20°C.

4. The method for preparing the hydrogel articular cartilage-targeted nano-drug delivery system according to claim 3, characterized in that: The step S3 specifically includes: S3-1, first drying ε-caprolactone ε-CL with calcium hydride, then collecting it in a dry flask by vacuum distillation and storing it in nitrogen, using Sn(Oct)2 as a catalyst, adding PEG and ε-CL in a molar ratio of 91:15 into a polymerization tube, freezing and thawing it three times, heating it to 110°C and reacting it in nitrogen for 2 days; after the reaction, dissolving the product in dichloromethane, purifying it three times by ether precipitation, obtaining a neutral triblock hydrogel PCEC, vacuum freeze-drying it and storing it at -20°C; S3-2. Take 182 ul of phosphate buffer solution (PBS) to dissolve CANC, then add 30.9 mg of PCEC, shake for a few seconds and let stand overnight at room temperature to obtain a hydrogel-state articular cartilage-targeted nanodrug delivery system with a solid content of 15%.

5. The method for preparing the hydrogel articular cartilage-targeted nano-drug delivery system according to claim 2, characterized in that: In the step S1-1, the molecular weight cutoff of the dialysis bag used for dialyzing G5 PAMAM is 3000, and the molecular weight cutoff of the dialysis bag used for dialyzing PBS buffer to remove unattached PEG is 2000.

6. The method for preparing the hydrogel articular cartilage-targeted nano-drug delivery system according to claim 3, characterized in that: In step S2-1, the molecular weight cutoff of the ultracentrifugation column is 2000.

7. A hydrogel articular cartilage-targeted nano-drug delivery system prepared by the method according to any one of claims 1 to 6.

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