Microenvironment response type in-situ injection hydrogel microsphere as well as preparation method and application thereof

Abstract

The invention provides a microenvironment response type in-situ injection hydrogel microsphere as well as a preparation method and application thereof, which are used for solving the problem of poor cartilage repair and bone joint bioactive substance treatment effect, and the main problem of poor effect is that the treatment effect is influenced due to the absence of prevention and early treatment measures. The poor secondary problem is the problem of insufficient delivery efficiency and retention time caused by the carrying method of the bioactive substances; the invention relates to a microenvironment response type in-situ injection hydrogel microsphere, a preparation method and application thereof, the injection gel microsphere is prepared from cationic liposome and methacrylic acid gelatin, the cationic liposome contains bioactive substances and hydrazone bonds, the injection gel microsphere is injected in situ in a bone joint cavity, and the hydrogel microsphere has the advantages that the bioactive substances and the hydrazone bonds are separated; and when the pH is reduced, bioactive substances are released and are used for preventing and repairing cartilage deformation.

Description

Technical Field

[0001] The present invention relates to the field of medical device technology, specifically to the field of functional hydrogel preparation technology, and in particular to a microenvironment responsive in situ injectable hydrogel microsphere, a preparation method and an application thereof. Background Art

[0002] Osteoarthritis is a chronic degenerative joint disease that mainly occurs in middle-aged and elderly people, seriously affecting their quality of life. Cartilage degeneration is a key change in osteoarthritis, and cartilage repair is also a method for treating osteoarthritis in the middle and late stages.

[0003] KGN is a small molecule that can promote the differentiation of chondrocytes. As one of the active ingredients in life, it plays a key role in promoting cartilage repair. In addition, cartilage-targeted peptides can provide targeted treatment and facilitate cartilage repair.

[0004] In addition to considering the effects of bioactive substances on cartilage repair and bone and joint treatment, it is also necessary to pay attention to the delivery system of the active substances. Because the structure of the cartilage matrix is ​​dense and has a high concentration of negative charges, conventional delivery systems cannot achieve good results. This is also an important aspect of the optimization of the present invention.

[0005] When we talk about repair and treatment, we do not consider prevention. If we can intervene in time before the cartilage undergoes morphological changes, we can achieve the dual effects of prevention and treatment.

[0006] Currently, there is a method for preparing GelMA microspheres carrying KGN through microfluidic technology, but the following problems still limit its clinical effectiveness: First, the active substances are currently released by rupturing the microspheres through friction, which does not take into account the importance of previous microenvironmental changes and cannot fully utilize the information of the microenvironment to construct preventive treatment measures and increase the clinical scope of applicability; the second is the problem of drug delivery efficiency. The structure of the cartilage matrix is ​​dense and has a high concentration of negative charges, which prevents the penetration of drugs; the third is how to further increase the retention time of active substances and improve the therapeutic effect. Summary of the invention

[0007] The present invention provides a microenvironment-responsive in situ injectable hydrogel microsphere, a preparation method and an application thereof, which are used to solve the problem of poor treatment effects of cartilage repair and bone and joint bioactive substances. The main problem is the lack of prevention and early treatment measures, which affects the treatment effect. The secondary problem is the delivery efficiency and residence time that are insufficient due to the method of carrying the bioactive substances.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions: A microenvironment-responsive in situ injectable hydrogel microsphere, a preparation method and an application thereof. The injectable gel microsphere is prepared from cationic liposomes and methacrylate gelatin. The cationic liposomes contain bioactive substances and hydrazone bonds. The injectable gel microsphere is injected in situ into a bone joint cavity and releases bioactive substances when the pH is lowered, so as to prevent and repair cartilage deformation.

[0009] Furthermore, the bioactive substances are KGN and WYRGRL peptides.

[0010] Furthermore, the preparation method of the cationic liposome is: S121: 6.5 mg of DOTAP, 3 g of soybean lecithin, 1 g of cholesterol, 2.5 mg of DSPE-HyD-PEG-COOH, 2.5 mg of DSPE-PEG-WYRGRL and 3 mg of KGN were dissolved in 30 mL of dichloromethane and 10 mL of ethyl acetate and evaporated at 50 °C for 30 min using a rotary evaporator to form a lipid film; S122: 6 mL of PBS was added to dissolve the lipid film, and the film was sonicated in an ultrasonic cleaner for 5 min, then hydrated at 37° C. for 1 h, cooled to 25° C., and then sonicated in a water bath for 2 min to obtain cationic liposome WDLK.

[0011] The ultrasonic parameters in step S22 are: 600w, working for 3s / pausing for 1s.

[0012] Furthermore, the preparation method of the DSPE-PEG-WYRGRL is: S111: Dissolve 200 mg of DSPE-PEG-NHS in 10 mL of PBS buffer solution; S112: Add 1 mL of PBS buffer solution containing WYRGRL peptide and stir the mixture for 8-12 h; S113: The mixture was placed in a dialysis bag, dialyzed with physiological saline, and lyophilized to obtain DSPE-PEG-WYRGRL.

[0013] Furthermore, the preparation method of the methacrylate gelatin is: S211: 10 g of porcine gelatin was completely dissolved in 250 mL of PBS solution at 60 °C under magnetic stirring; S212: Add 6 mL of methacrylic anhydride dropwise to the S211 solution under stirring at 50 °C and continue stirring for 8-12 h; S213: The mixture was placed in a 14000 kDa dialysis bag, dialyzed against pure water at 40°C for 3-5 days, and the reaction product was freeze-dried to obtain methacrylate gelatin GM.

[0014] Furthermore, the preparation method of the injectable gel microspheres is: S221: aqueous phase, GM, WDLK, and photoinitiator are ultrasonically treated in a PBS solution until the solution becomes clear, and this solution is used as the aqueous phase; S222: oil phase, a mineral oil mixture containing 2% by weight of Span 80 is used as the oil phase; S223: The water phase and the oil phase were connected to a microfluidic device, WDLKG droplets were formed through the microfluidic device, the droplets were photocrosslinked for 5 min, placed in a microcentrifuge tube containing 1 mL of isopropanol, suspended for 15 min, and then centrifuged at 4000 r / min to obtain WYRGRL hydrogel microspheres.

[0015] Wherein, according to weight ratio, the final concentration of GM in the water phase is 10%, the final concentration of WDLK is 2.5%, and the final concentration of the photoinitiator is 0.5%.

[0016] Furthermore, in step S221, the photoinitiator selected is one of the following: 2-hydroxy-2-methyl-1-phenyl-1-propanone, 1-hydroxy-cyclohexyl-phenyl-ketone, 2,2-dimethoxy-2-phenylacetophenone.

[0017] Furthermore, the WDLKG hydrogel microspheres have a round, honeycomb-shaped porous structure, and the size of the microspheres is 100 μm.

[0018] Furthermore, the WDLKG hydrogel microspheres are released faster at pH 5.0 than at pH 7.4.

[0019] Furthermore, the degradation rate of the WDLKG hydrogel microspheres after 28 days was 81.23±5.97%.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The WDLK and WDLKG prepared by the present invention have good pH responsiveness. When the pH decreases, the nanoparticles show The shrunken and degraded state indicates that the liposomes disintegrate and release the bioactive substance KGN; By forming WDLK into hydrogel microspheres, the hydrogel microspheres are made pH-responsive, but their release rate is slowed down, which has a better Long-lasting sustained-release effect and longer degradation time; Since WDLK is surface-modified by WYRGRL peptide, WDLK and WDLKG have cartilage targeting properties. Promote cartilage differentiation; experiments have shown that WDLKG promotes the expression of type II collagen and aggrecan protein, thereby maintaining cartilage morphology and having a good protective effect; From the experimental results, we can see that WDLKG can inhibit synovitis in osteoarthritis and has a good therapeutic effect on cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the synthesis of WDLK and WDLKG in Example 1; Figure 2 is a TEM image of WDLK treated with a PBS buffer solution of pH=7.0 in Example 2; Figure 3 is a TEM image of WDLK treated with a PBS buffer solution of pH=5.0 in Example 2; Figure 4 is a particle size distribution diagram of WDLK treated with PBS buffer solution of pH=7.0 in Example 2; Figure 5 is a particle size distribution diagram of WDLK treated with a PBS buffer solution of pH=5.0 in Example 2; Figure 6 is the Zeta potential diagram of WDLK particles in Example 2; Figure 7 is a microscopic image of WDLKG hydrogel microspheres of Example 2; Figure 8 is the particle size distribution diagram of WDLKG hydrogel microspheres in Example 2; Fig. 9 is a scanning electron microscope SEM image of WDLKG hydrogel microspheres in Example 2; Fig.10 This is a fluorescence microscope image of WDLKG hydrogel microspheres in Example 2; Fig.11 is an in vitro KGN release curve of WDLKG in a PBS buffer solution of pH=5.0 and WDLK in a PBS buffer solution of pH=7.4 and pH=5.0 according to Example 2; Fig.12 is a schematic diagram of Example 2 showing the release of KGN from WDLK and the promotion of chondrocyte anabolism; Fig.13 is the degradation curve diagram of GM and WDLKG in Example 2; Fig.14 This is a photo of the hemolytic appearance of WDLKG in Example 2 after incubation for 6 h and 12 h; Fig.15 This is the fluorescence microscopic image of primary mouse chondrocytes treated with WDLK liposomes for 8 h, 24 h, and 48 h in Example 3; Fig.16 This is the fluorescence intensity graph of primary mouse chondrocytes treated with WDLK liposomes in Example 3 for 8h, 24h, and 48h; Fig.17 This is a fluorescence microscopic picture of human cartilage tissue treated with WDLK liposomes for 48 hours in Example 3; Fig.18 This is a fluorescence intensity graph of human cartilage tissue treated with WDLK liposomes for 48 hours in Example 3; Fig.19 This is a comparison diagram of Example 3, in which CCK-8 assay was used to evaluate the effect of WDLKG hydrogel microspheres on the activity of primary mouse chondrocytes; Fig. 20 This is a red and blue staining comparison diagram of Example 3 WDLKG hydrogel microspheres after treating high-density cell culture; Fig.21 This is a comparison chart of average optical density values ​​after high-density cell culture treated with WDLKG hydrogel microspheres in Example 3; Fig. 22 This is a qPCR comparison diagram of protein expression of primary mouse chondrocytes stimulated by WDLKG hydrogel microspheres in Example 3; Fig.23 This is a protein immunoblot image and quantitative analysis image of protein expression of primary mouse chondrocytes stimulated by WDLKG hydrogel microspheres in Example 3; Fig.24 This is the immunofluorescence image and quantitative analysis image of protein expression of primary mouse chondrocytes stimulated by WDLKG hydrogel microspheres in Example 3; Fig.25 The immunofluorescence image and quantitative analysis image of the expression of glycoprotein in primary mouse chondrocytes stimulated by WDLKG hydrogel microspheres in Example 3; Fig.26 This is a comparison chart of fluorescence intensity and intensity statistical analysis chart of in vivo detection at 0, 3, 7, and 10 days after WDLKG was injected into the left and right knee joint cavities of mice in Example 3; Fig. 27 This is the staining image of the main organs of mice after WDLKG injection in Example 3; Fig.28 This is a statistical result diagram of blood biochemical markers of WDLKG-injected mice after death in Example 3; Fig.29 This is a HE staining result of knee joint sections and synovium around the joint after WDLKG was injected into a mouse model in Example 3; Fig.30 This is a graph showing the synovitis score of the synovial membrane around the joints of mice after WDLKG injection treatment in Example 3; Fig.31 This is a picture of safranin O and fast green staining of a mouse knee joint section after WDLKG injection in Example 3; Fig.32 This is a graph of the knee osteoarthritis score (OARSI) of mice after WDLKG injection treatment in Example 3; Fig.33 This is the immunofluorescence staining and quantitative analysis of type II collagen in the knee joint sections of mice after WDLKG injection treatment in Example 3; Fig.34 This is an immunofluorescence staining and quantitative analysis diagram of aggrecan protein in the knee joint sections of mice after WDLKG injection treatment in Example 3. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0023] Cartilage repair is a difficult and time-consuming process. If we can shift the focus of cartilage repair (osteoarthritis treatment) forward and start treatment before the cartilage is deformed, the difficulty of treatment will be greatly reduced and the effect will be better. To realize this idea, we cannot do without the study of the microenvironment. Studies have shown that with the progression of osteoarthritis, the degeneration of blood vessels around the joints will intensify. As the main way to transport oxygen and glucose, the deterioration of blood vessels will cause chondrocytes to be in a low glucose and hypoxic state. The increase in anaerobic glycolysis will lead to acidification of the cartilage microenvironment, that is, a decrease in pH. Therefore, we will consider adjusting the release mechanism of bioactive materials from mechanical friction rupture release to pH stimulation release.

[0024] References: Hu Y, Wu H, Xu T, Wang Y, Qin H, Yao Z, et al. Defactinib attenuatesosteoarthritis by inhibiting positive feedback loop between H-type vessels and MSCs in subchondral bone. J Orthop Transl. 2020;24:12–22. Dong L, Zhao Y, Sun C, Ou Yang Z, Chen F, Hu W, et al. ASIC1a-CMPK2-mediated M1 macrophage polarization exacerbates chondrocyte senescence inosteoarthritis through IL-18. Int Immunopharmacol. 2023;124:110878. According to the background technology, the bioactive substances we selected are Kartogenin (KGN) and cartilage targeting peptide WYRGRL peptide. In order to prolong the treatment time and reduce the rapid loss of bioactive substances, a microsphere sustained-release system is usually used. However, due to the particularity of cartilage structure, the delivery efficiency of the current sustained-release system is poor. Therefore, one of the important aspects of the present invention is to develop an efficient drug delivery system.

[0025] The reagent used in the present invention is as follows: Porcine gelatin (300 Bloom), 1,2-distearoyl-sn-glycero-3-hydrazone-polyethylene glycol acid (DSPE-HyD-PEG-COOH) (purity 97%), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) (molecular weight 20 kDa), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-sodium polyethylene glycol (DSPE-PEG-NHS) (purity 98%), WYRGRL peptide (purity 98%), pure soybean lecithin (98%), pharmaceutical grade cholesterol, analytical grade kartogenin (KGN), dichloromethane, ethyl acetate, analytical grade methacrylic anhydride, phosphate buffer solution (PBS).

[0026] The terms or abbreviations used in this invention are: The injectable hydrogel microspheres provided in this embodiment that can be used for in situ injection are prepared by microfluidics and photopolymerization methods. The hydrogel microspheres are formed by embedding liposomes into methacrylate gelatin using liposomes as carriers of bioactive substances, cationic lipids and hydrazone bonds (pH response).

[0027] The preparation method of hydrogel microspheres is as follows: Cationic liposome preparation Synthesis of DSPE-PEG-WYRGRL This step is to perform WYRGRL surface modification on the liposome. Specifically: S111: Dissolve 200 mg of DSPE-PEG-NHS in 10 mL of PBS buffer solution; S112: Add 1 mL of PBS buffer solution containing WYRGRL peptide and stir the mixture for 8-12 h; S113: The mixture was placed in a dialysis bag, dialyzed with physiological saline, and lyophilized to obtain DSPE-PEG-WYRGRL.

[0028] Preparation of cationic liposome WDLK This step is to prepare the liposomes into cationic liposomes, which are beneficial to overcoming the problem of high concentration of negative charges in the cartilage matrix and improving the efficiency of drug delivery.

[0029] S121: 6.5 mg of DOTAP, 3 g of soybean lecithin, 1 g of cholesterol, 2.5 mg of DSPE-HyD-PEG-COOH, 2.5 mg of DSPE-PEG-WYRGRL and 3 mg of KGN were dissolved in 30 mL of dichloromethane and 10 mL of ethyl acetate and evaporated at 50 °C for 30 min using a rotary evaporator to form a lipid film; S122: 6 mL of PBS was added to dissolve the lipid film, and the film was sonicated in an ultrasonic cleaner for 5 min, then hydrated at 37° C. for 1 h, cooled to 25° C., and then sonicated in a water bath for 2 min to obtain cationic liposome WDLK.

[0030] The ultrasonic parameters in step S22 are: 600w, working 3s / pausing 1s.

[0031] Preparation of WYRGRL hydrogel microspheres Preparation of methacrylated gelatin GM This step is a method for preparing methacrylate gelatin from porcine gelatin.

[0032] S211: 10 g of porcine gelatin was completely dissolved in 250 mL of PBS solution at 60 °C under magnetic stirring; S212: Add 6 mL of methacrylic anhydride dropwise to the S211 solution under stirring at 50 °C and continue stirring for 8-12 h; S213: The mixture was placed in a 14000 kDa dialysis bag, dialyzed against pure water at 40°C for 3-5 days, and the reaction product was freeze-dried to obtain methacrylate gelatin GM.

[0033] Preparation of WYRGRL hydrogel microspheres This step is to prepare injectable hydrogel microspheres, such as Figure 1 shown.

[0034] S221: aqueous phase, GM, WDLK, and photoinitiator are ultrasonically treated in a PBS solution until the solution becomes clear, and this solution is used as the aqueous phase; S222: oil phase, a mineral oil mixture containing 2% (by weight) Span 80 is used as the oil phase; S223: The water phase and the oil phase were connected to a microfluidic device, WDLKG droplets were formed through the microfluidic device, the droplets were photocrosslinked for 5 min, placed in a microcentrifuge tube containing 1 mL of isopropanol, suspended for 15 min, and then centrifuged at 4000 r / min to obtain WYRGRL hydrogel microspheres.

[0035] Among them, the final concentration of GM in the water phase is 10% (weight ratio), the final concentration of WDLK is 2.5% (weight ratio), and the final concentration of the photoinitiator is 0.5% (weight ratio). The photoinitiators selected are: 2-hydroxy-2-methyl-1-phenyl-1-propanone (Darocur 1173), 1-hydroxy-cyclohexyl-phenyl-ketone (Irgacure 184), and 2,2-dimethoxy-2-phenylacetophenone (Irgacure 651).

[0036] This example is based on Example 1 and characterizes and tests the performance of injectable hydrogel microspheres.

[0037] WDLK Characterization The WDLK liposomes were characterized by transmission electron microscopy (TEM) and treated with PBS buffer solutions at pH = 7.0 and pH = 5.0, respectively. Figure 2 and Figure 3 As shown, it can be seen that when the pH decreases, the nanoparticles show a shrunken and degraded state, which indicates that the WDLK liposomes disintegrate and show good pH responsiveness.

[0038] like Figure 4 and Figure 5 As shown in the figure, the results of dynamic light scattering (DLS) analysis showed that the hydrodynamic diameter of WDLK liposomes was 181.2 nm and the polydispersity index (PDI) was 0.192, indicating that the WDLK liposomes were uniform in size and well dispersed; after being treated with PBS buffer solution at pH 5.0, the hydrodynamic diameter was reduced to 84.4 nm and the PDI increased to 0.62. These changes indicated that the structure and size distribution of WDLK liposomes had changed, and also proved the responsiveness to pH.

[0039] like Figure 6 As shown, from the Zeta potential diagram, it can be seen that the potential of WDLK is 31.9 mV, which is higher than 30 mV, indicating that WDLK liposomes have high stability.

[0040] WDLKG Characterization like Figure 7 , Figure 8 , Fig. 9 As shown, the WDLKG hydrogel microspheres have a round, honeycomb-like porous structure with high connectivity between the pores. Tests show that the size of the microspheres is about 100 μm.

[0041] WDLK liposomes were labeled with fluorescein isothiocyanate. Fig.10As shown, under a fluorescence microscope, green fluorescence was evenly distributed in the WDLKG hydrogel microspheres, and there was no fluorescence outside the microspheres, indicating that the WDKL liposomes were well encapsulated in the hydrogel.

[0042] From the characterization of WDLK in the first part above, it can be seen that WDLK has good pH responsiveness. Since WDLKG is encapsulated with WDLK liposomes, it is also pH responsive. When the pH decreases, KGN will be released. The results are shown in Figure 11. In WDLK liposomes, at pH 5.0, due to structural dissociation, its release is faster than at pH 7.4; however, under the same pH=5.0 condition, KGN is released slower from WDLKG than from WDLK, with a more sustained sustained release effect. Fig.12 The results show that KGN is released from WDLK liposomes and has an anabolism-promoting effect on chondrocytes.

[0043] Both GM and WDLKG can be degraded, and the results are as follows Fig.13 As shown in the figure, after 28 days, the degradation rate of WDLKG was 81.23±5.97%, which was lower than 95.00±2.43% of GM. It can be seen that the degradation time of WDLKG will be longer.

[0044] WDLKG Biosafety Assessment WDLKG is a hydrogel microsphere used for in situ injection. Although it is less likely to enter the blood, its biosafety still needs to be evaluated. Blood compatibility is assessed using a hemolysis test, with results such as Fig.14 As shown, at 6h and 12h, the hemolysis rates of WDLKG were 1.16±0.16% and 3.03±0.25%, respectively, both lower than 5%, indicating good blood compatibility.

[0045] This example is based on Example 1 and Example 2, and discusses the application of injectable hydrogel microspheres in cartilage repair and osteoarthritis treatment.

[0046] Cartilage targeting specificity of WDLK Mouse chondrocytes Primary mouse chondrocytes were treated with WDLK for 8 h, 24 h, and 48 h, and the location of WDLK in chondrocytes was determined by fluorescence microscopy. Fig.15 As shown, WDLK has good fluorescence intensity and strong targeting specificity for chondrocytes. In the experiment, the control sample used was liposomes that were not modified with WYRGRL peptide. Further statistical analysis showed that Fig.16 , WDLK has a higher fluorescence intensity.

[0047] Human cartilage tissue This experiment is a further experiment on cartilage targeting ability based on the mouse chondrocyte experiment. Human cartilage tissue collected from total knee replacement was cultured under WDLK conditions for 48 hours. Fig.17 The fluorescence microscopy images showed that WDLK exhibited excellent cartilage targeting ability; after further statistical analysis, WDLK had a higher fluorescence intensity, such as Fig.18 shown.

[0048] WDLKG promotes chondrocyte differentiation Primary mouse chondrocytes were co-cultured with WDLKG for 24 and 48 h, and CCK-8 assay revealed that Fig.19 As shown in Figure 2, the cell activity was not significantly affected compared to WDLK. The cells were stained with alcian blue and safranin O. The staining results are shown in Figure 2. Fig. 20 As shown in the results, WDLKG has the strongest staining, indicating that its cartilage differentiation level is the highest; through further statistical analysis, such as Fig.21 As shown, the average optical density of alcian blue and safranin O after WDLKG treatment was higher than that of the control group (*P < 0.05).

[0049] The results of qPCR and western blot analysis were as follows Fig. 22 and Fig.23 As shown, WDLKG promoted the expression of type II collagen and aggrecan proteins. Fig.24 and Fig.25 The results of immunofluorescence images also showed that WDLKG stimulation significantly increased the expression of type II collagen and aggrecan proteins in primary mouse chondrocytes.

[0050] In vivo sustained release and biosafety of WDLKG hydrogel microspheres Animal experiments were conducted on mice to verify the sustained release effect and biosafety of WDLKG hydrogel microspheres after in situ injection in vivo. The red fluorescent dye Cy5.5 was used to label the WDLKG hydrogel microspheres, and the WDLKG hydrogel microspheres were injected into the right joint cavity of the mice. The fluorescence intensity was recorded using a live imaging system. The results are shown in Figure 2. Fig.26 As shown, the fluorescence intensity of the knee joint injected with WDLKG was significantly higher than that of the knee joint injected with WDLK, indicating that WDLKG has a better sustained-release effect.

[0051] Fig. 27 and Fig.28 As relevant results of the biosafety of WDLKG, HE staining of heart, liver, spleen, lung and kidney sections showed that WDLKG was generally normal in the above organs, and no statistically significant differences were observed in blood parameters and liver function data, indicating good biosafety.

[0052] WDLKG hydrogel microsphere effectiveness experiment This experiment verified the therapeutic effect of WDLKG hydrogel microspheres on osteoarthritis cartilage degeneration, and mice with unstable medial meniscus (DMM) were injected intra-articularly every 10 days for 8 weeks. Fig.29 As shown in the results, WDLKG can maintain cartilage morphology and has a good protective effect. Further observation of the synovium around the knee joint showed that WDLKG could inhibit synovitis in osteoarthritis compared with the DMM group. Fig.30 The scoring results can also confirm this conclusion.

[0053] Fig.31 The staining results of the mouse knee joints further revealed the effect of WDLKG on cartilage degeneration. The observation of the range and saturation of Safranin O staining showed that WDLKG had a good therapeutic effect on cartilage. Fig.32 The OARSI scores shown are consistent with the above results. Fig.33 and Fig.34 The results showed that WDLKG promoted the expression of type II collagen and aggrecan protein.

[0054] The above are only preferred embodiments of the present invention. Based on the inventive concept of the present invention and the above embodiments, any optimization performed by ordinary technicians in the field based on their knowledge should be included in the protection scope of the present invention.

Claims

1. A microenvironment-responsive in situ injectable hydrogel microsphere, a preparation method and its application, characterized in that: The injectable gel microspheres are prepared from cationic liposomes and methacrylate gelatin. The cationic liposomes contain bioactive substances and hydrazone bonds. The injectable gel microspheres are injected in situ into the bone joint cavity and release the bioactive substances when the pH is reduced, so as to prevent and repair cartilage deformation.

2. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 1, characterized in that: The biologically active substances are KGN and WYRGRL peptides.

3. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 1, characterized in that: The preparation method of the cationic liposome is: S121: 6.5 mg of DOTAP, 3 g of soybean lecithin, 1 g of cholesterol, 2.5 mg of DSPE-HyD-PEG-COOH, 2.5 mg of DSPE-PEG-WYRGRL and 3 mg of KGN were dissolved in 30 mL of dichloromethane and 10 mL of ethyl acetate and evaporated at 50 °C for 30 min using a rotary evaporator to form a lipid film; S122: 6 mL of PBS was added to dissolve the lipid film, and after ultrasonic treatment in an ultrasonic cleaner for 5 min, the mixture was hydrated at 37 °C for 1 h, cooled to 25 °C, and then ultrasonicated in a water bath for 2 min to obtain cationic liposome WDLK; The ultrasonic parameters in step S22 are: 600w, working for 3s / pausing for 1s.

4. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 3, characterized in that: The preparation method of the DSPE-PEG-WYRGRL is: S111: Dissolve 200 mg of DSPE-PEG-NHS in 10 mL of PBS buffer solution; S112: Add 1 mL of PBS buffer solution containing WYRGRL peptide and stir the mixture for 8-12 h; S113: The mixture was placed in a dialysis bag, dialyzed with physiological saline, and lyophilized to obtain DSPE-PEG-WYRGRL.

5. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 4, characterized in that: The preparation method of the methacrylate gelatin is: S211: 10 g of porcine gelatin was completely dissolved in 250 mL of PBS solution at 60 °C under magnetic stirring; S212: Add 6 mL of methacrylic anhydride dropwise to the S211 solution under stirring at 50 °C and continue stirring for 8-12 h; S213: The mixture was placed in a 14000 kDa dialysis bag, dialyzed against pure water at 40°C for 3-5 days, and the reaction product was freeze-dried to obtain methacrylate gelatin GM.

6. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 5, characterized in that: The preparation method of the injectable gel microspheres is as follows: S221: aqueous phase, GM, WDLK, and photoinitiator are ultrasonically treated in a PBS solution until the solution becomes clear, and this solution is used as the aqueous phase; S222: oil phase, a mineral oil mixture containing 2% by weight of Span 80 is used as the oil phase; S223: connecting the water phase and the oil phase to a microfluidic device, forming WDLKG droplets through the microfluidic device, photocrosslinking the droplets for 5 min, placing them in a microcentrifuge tube containing 1 mL of isopropanol, suspending them for 15 min, and centrifuging them at 4000 r / min to obtain WYRGRL hydrogel microspheres; Wherein, according to weight ratio, the final concentration of GM in the water phase is 10%, the final concentration of WDLK is 2.5%, and the final concentration of the photoinitiator is 0.5%.

7. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 6, characterized in that: In step S221, the photoinitiator selected is one of the following: 2-hydroxy-2-methyl-1-phenyl-1-propanone, 1-hydroxy-cyclohexyl-phenyl-ketone, 2,2-dimethoxy-2-phenylacetophenone.

8. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 1, characterized in that: The WDLKG hydrogel microspheres are round, honeycomb-shaped, porous structures, and the size of the microspheres is 100 μm.

9. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 1, characterized in that: The WDLKG hydrogel microspheres are released faster under the condition of pH 5.0 than under the condition of pH 7.

4.

10. The microenvironment-responsive in situ injectable hydrogel microspheres, preparation method and application thereof according to claim 1, characterized in that: The degradation rate of the WDLKG hydrogel microspheres after 28 days was 81.23±5.97%.

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