Injectable hydrogel microspheres containing organic diselenide bonds as well as preparation method and application of injectable hydrogel microspheres

Abstract

The invention belongs to the field of biomedical materials, and relates to injectable hydrogel microspheres containing an organic diselenide bond and a preparation method and application thereof.The preparation method comprises the steps that alkynyl-containing hyaluronic acid is subjected to thiol-ene click chemical grafting to activate selenocysteine, HS containing the diselenide bond is obtained after oxidation, and the injectable hydrogel microspheres containing the organic diselenide bond are obtained; the preparation method comprises the following steps: preparing an injectable hydrogel microsphere HSPHR, crosslinking the injectable hydrogel microsphere with MMP13 responsive peptide to obtain MMP13 responsive hyaluronic acid HSR, blending methylacryloylated hyaluronic acid, methylacryloyl RGD peptide and HSR to obtain a water phase, and preparing the injectable hydrogel microsphere HSPHR through a microfluidic device. The injectable hydrogel microsphere has better injectability and stability, can respond to a pathological microenvironment of osteoarthritis OA, provides continuous release of microelement organic selenium, recovers oxidation-reduction balance of cartilage, subchondral bone and synovial cells, realizes repair of cartilage matrix, and has the advantages of simple preparation process and low cost. The composition can be well applied to the injury and repair process of osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to an injectable hydrogel microsphere containing an organic diselenide bond, and a preparation method and application thereof. Background Art

[0002] Osteoarthritis (OA) is one of the most common degenerative joint diseases, with increasing incidence and prevalence worldwide, particularly among the elderly population. The 2021 Global Burden of Disease Study showed that the global burden of OA increased significantly from 1990 to 2021, with approximately 466.3 million new cases reported in 2021 and a prevalence of approximately 606.9 million cases. Its pathological process involves changes in multiple joint structures. Destruction of the cartilage matrix is ​​a prominent feature of OA, often characterized by abnormal chondrocyte metabolism and overexpression of matrix-degrading enzymes, including matrix metalloproteinases (MMPs) and members of the ADAMTS family. In addition, proliferation and sclerosis of the subchondral bone are key aspects of the OA disease process. Studies have shown that alterations in the subchondral bone microenvironment, such as bone marrow edema and angiogenesis, may precede cartilage degeneration and are closely associated with pain and OA progression. Synovial hyperplasia is another key pathological feature of OA, often accompanied by low-grade inflammation. Cell proliferation and fibrosis in synovial tissue may be mediated by a variety of inflammatory factors and signaling pathways. For example, the role of CEMIP in synovial inflammation and fibrosis has been demonstrated. Furthermore, chronic synovial inflammation may also affect the health of cartilage and subchondral bone through the release of inflammatory mediators. In summary, the pathogenesis of OA is complex and diverse, involving the interaction of multiple joint structures, including cartilage, subchondral bone, and synovium. A deeper understanding of these mechanisms is crucial for developing effective treatment strategies.

[0003] Selenoproteins, as important antioxidants, play a key role in maintaining the body's redox balance. The biosynthesis of selenoproteins requires the incorporation of selenocysteine, a unique amino acid that is incorporated into proteins through translational recoding of the UGA stop codon. Selenoproteins are crucial in regulating oxidative stress and inflammatory responses. For example, glutathione peroxidase and thioredoxin reductase are two important selenoproteins that play a key role in scavenging hydrogen peroxide and lipid peroxides, thereby protecting cells from oxidative damage. In recent years, research on selenoproteins in the fields of orthopedics and osteoarthritis has gradually increased. Through their antioxidant properties, selenoproteins can effectively mitigate the damage of oxidative stress to the skeletal system, thus playing an important role in the prevention and treatment of orthopedic diseases such as osteoarthritis.

[0004] In recent years, nanoselenium, as a new form of selenium supplementation, has attracted widespread attention due to its unique properties and potential biological effects. Studies have shown that compared with traditional selenium compounds, nanoselenium has significant antioxidant and anti-inflammatory properties, while also displaying lower toxicity. However, selenium nanoparticles have problems such as unclear metabolic pathways and rapid metabolism in the body, which limits their application in the treatment of OA. Precisely targeting lesions provides new ideas for the precise treatment of degenerative joint diseases.

[0005] Therefore, providing a bioactive material that can simultaneously achieve multiple regulation of the pathological environment in the joint and restore the metabolic level of selenoproteins has better clinical application prospects. Summary of the Invention

[0006] In response to the shortcomings of the existing technology, the present invention provides an injectable hydrogel microsphere containing organic diselenide bonds, a preparation method and application thereof, which can achieve multiple regulation of OA pathology, have excellent injectability and stability, effectively restore the level of cellular selenoprotein metabolism, and restore the redox balance in cartilage cells. It has good application prospects in the treatment of early OA and late cartilage defects.

[0007] The technical solution provided by the present invention is as follows:

[0008] The present invention provides a method for preparing injectable hydrogel microspheres containing organic diselenide bonds, comprising the following steps: Hyaluronic acid HA is reacted with an alkyne-containing reagent to generate alkyne-containing HA; After methylation and reduction activation, selenocysteine ​​Sec was grafted onto alkyne-containing HA via a thiol-alkyne click reaction, and then oxidized to form diselenide-crosslinked HS. The carboxyl groups of HS were activated with EDC / NHS, and the MMP13-responsive peptide KCGPQG was added for amidation reaction to obtain MMP13-responsive hyaluronic acid HSR; Methacryl-modified hyaluronic acid HAMA is prepared by reacting hyaluronic acid HA with methacrylic anhydride; HSR, HAMA and methacryloyl RGD peptide are mixed as an aqueous phase, and hydrogel microspheres are prepared using a microfluidic device. The hydrogel microspheres are then cured by ultraviolet light, washed and freeze-dried to obtain the injectable hydrogel microspheres HSPHR.

[0009] Furthermore, the alkyne-containing reagent is an alkyne isocyanate Alkyne-NCO, the molar ratio of HA to Alkyne-NCO is 1:1.5-3, and the preparation method of the alkyne-containing HA comprises: HA was dissolved in PBS at pH 7.0-8.0, and after deoxygenation by nitrogen, the pH was adjusted to 8.0-9.0. Alkyne-NCO was added and reacted at 0-4°C for 2-6 h. The pH was adjusted to 6.5-7.5, and the HA containing alkyne groups was obtained by dialyzing and freeze-drying.

[0010] Furthermore, the method for methylating and reducing selenocysteine ​​Sec comprises: Sec was dissolved in PBS at pH 7.0-8.0, triethylamine (TEA) was added first, and then CH3I was slowly added. The reaction was carried out in the dark for 4-6 hours to generate a thiol methylation product. Subsequently, TCEP was added and the pH was adjusted to 9.0-9.5. The product was stirred at room temperature for 1-2 hours to generate the thiolate Sec-S. - The molar ratio of Sec to TEA is 1:1~3, the molar ratio of Sec to CH3I is 1:1~3, and the final concentration of TCEP is 0.1~1.0 M.

[0011] Furthermore, the preparation method of the diselenide bond cross-linked HS includes: The HA containing alkyne groups was combined with Sec-S - Dissolve in PBS at pH 7.0-8.0, deoxygenate with nitrogen, add CuSO4 and sodium ascorbate, and react in the dark for 16-24 hours. After dialysis to remove free Sec, lyophilize to obtain the monoselenium-modified product HA-Sec. HA-Sec was dissolved in PBS at pH 7.0-8.0, H2O2 was added and reacted at room temperature for 2-4 h, the reaction was terminated with Na2S2O3, the unreacted H2O2 was removed by dialysis, and then freeze-dried to obtain HS cross-linked with diselenide bonds; The molar ratio of the alkyne-containing HA to Sec-S- is 1:1-5, the concentrations of CuSO4 and sodium ascorbate are 50-100 μM and 5-10 mM, respectively, and the concentration of H2O2 is 1-3% w / v.

[0012] Furthermore, in the preparation process of the MMP13-responsive hyaluronic acid HSR, the molar ratio of HS, EDC and NHS is 1-2:5:5, the concentration of the MMP13-responsive peptide KCGPQG is 0.5-1.0 mg / mL, the amidation reaction temperature is 60° C., and the time is 6-8 h.

[0013] Furthermore, in the process of preparing hydrogel microspheres using a microfluidic device, HSR, HAMA and methacryloyl RGD peptide were mixed in a mass ratio of 1-6:2-5:1 to form the aqueous phase, and Span 80 and sorbitol isopropyl ester were mixed in a mass ratio of 1:9 to form the oil phase. The flow rate ratio of the aqueous phase to the oil phase was controlled to be 500:15.

[0014] The present invention also provides an injectable hydrogel microsphere containing an organic diselenide bond, which is prepared according to the above-mentioned preparation method.

[0015] The present invention also provides the use of the above-mentioned injectable hydrogel microspheres containing organic diselenide bonds in the preparation of medicines for treating early osteoarthritis and late cartilage defects.

[0016] Furthermore, the hydrogel microspheres are used to treat early osteoarthritis and late cartilage defects by removing reactive oxygen species in and outside the body and correcting selenoprotein metabolic disorders.

[0017] The present invention also provides a pharmaceutical composition comprising the above-mentioned injectable hydrogel microspheres containing organic diselenide bonds.

[0018] Beneficial effects

[0019] (1) The present invention can prepare hydrogel microspheres responsive to the pathological environment of osteoarthritis. The hydrogel microspheres have good biocompatibility. The hydrogel can also release organic selenium element HSe in response to ROS and MMP13 protein, thereby restoring intracellular selenoprotein metabolism and depleting excessive ROS generated in the joints, thereby forming a virtuous cycle, effectively antagonizing the spread of inflammation in the inflammatory area of ​​the joint, and having application value in reversing the pathology of osteoarthritis.

[0020] (2) The experimental results showed that the hydrogel microspheres prepared by the present invention were injected into the joint cavity to treat early arthritis and repair late cartilage damage. After a period of time, the samples were taken for observation and it was found that the repair effect of the hydrogel group was much better than that of the control group. It can be seen that the hydrogel microspheres can be used as an effective treatment measure for arthritis and provide a new reference for the treatment of OA throughout the entire course of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The macroscopic morphology of the organic diselenide hydrogel microspheres of the present invention is shown in FIG.

[0022] Figure 2 The microscopic morphology of the organic diselenide hydrogel microspheres of the present invention;

[0023] Figure 3 The organic diselenide hydrogel of the present invention responds to the environment in vivo and in vitro;

[0024] Figure 4 The in vitro anti-osteoarthritis effect of the organic diselenide hydrogel microspheres of the present invention;

[0025] Figure 5 The therapeutic effect of the organic diselenide hydrogel microspheres of the present invention on early osteoarthritis;

[0026] Figure 6The therapeutic effect of the organic diselenide hydrogel microspheres of the present invention on late-stage cartilage defects;

[0027] Figure 7 For the present invention, a micro single camera was used to perform macroscopic evaluation of the femoral defect of the specimen. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0029] Main reagents: Hyaluronic acid (HA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), methacrylic anhydride (MA), NaOH, and selenocysteine ​​(Sec) were provided by Shanghai MacLean Biochemical Technology Co., Ltd., alkynyl isocyanate (Alkyne-NCO) was provided by Hefei Tianjian Chemical Co., Ltd., methacryloyl RGD peptide was provided by Engineering for Life, and MMP13-responsive peptide was synthesized by Hefei Sener Biotechnology Co., Ltd. Example 1

[0030] The embodiment of the present invention provides a method for preparing injectable hydrogel microspheres containing organic diselenide bonds, comprising the following steps:

[0031] S1. Preparation of alkynylated HA:

[0032] S11, introducing terminal alkynes into the HA backbone to provide active sites for subsequent click reactions;

[0033] S111. HA (1 g) was dissolved in PBS (100 mL, pH 8.0), and deoxygenated with nitrogen for 30 min. NaHCO3 (0.5 g, 6 mmol) was then added to adjust the pH to 8.5.

[0034] S112, slowly add Alkyne-NCO (0.2 g, 1.5 mmol) dropwise, stir in an ice bath (4°C) and react for 2 h;

[0035] S113. Adjust the pH to 7.0 with 1 M HCl, dialyze to remove unreacted Alkyne-NCO, and lyophilize to obtain alkynylated HA.

[0036] S2. Preparation of hyaluronic acid (HS) containing diselenide cross-linking:

[0037] S21 converts the sulfhydryl group (-SH) of selenocysteine ​​(Sec) into a highly reactive thiolate;

[0038] S211. Dissolve selenocysteine ​​Sec (0.5 g, 1.5 mmol) in PBS (50 mL, pH 8.0) and add TEA (molar ratio Sec:TEA = 1:1.5).

[0039] S212, slowly add CH3I (molar ratio Sec:CH3I = 1:2), and react in the dark for 4 h to generate a thiol methylation product (Sec-SCH3);

[0040] S213, TCEP (1 mL, 0.5 M) was added to reduce the disulfide bonds, and then NaOH was added dropwise to adjust the pH to 9.0. The mixture was stirred at room temperature for 1 h to obtain Sec-S - , dialyzed and freeze-dried for later use.

[0041] S22, linking activated selenocysteine ​​to alkyned HA via a thiol-alkyne reaction;

[0042] S221, HA containing alkyne groups (1 mM) was mixed with Sec-S - (5 mM) was dissolved in PBS (100 mL, pH 8.0) and deoxygenated by nitrogen gas;

[0043] S222, then add CuSO4 (50 μM) and sodium ascorbate (5 mM), and stir in the dark for 24 h;

[0044] S223. Remove free Sec by dialysis, stir at room temperature (25°C) in the dark overnight, and lyophilize to obtain the monoselenium-modified product HA-Sec.

[0045] S23, forming Se-Se bonds between hyaluronic acid chains through oxidation reaction, enhancing structural stability;

[0046] HA-Sec was dissolved in PBS (20 mL, pH 7.4), H2O2 (1 mL, 3% w / v) was added, and the mixture was stirred at room temperature for 2 h. Na2S2O3 (0.5 g) was added to terminate the oxidation, and the unreacted H2O2 was removed by dialysis. The mixture was then freeze-dried to obtain the Se-Se-HA hydrogel precursor (HS). At this point, a stable Se-Se structure already existed in the hydrogel.

[0047] S3. Grafting MMP13-responsive peptide onto HS to synthesize MMP13-responsive HA (HSR):

[0048] S31, activate the carboxyl groups of HS by EDC / NHS, and incubate at 60°C for 6 h;

[0049] S32 and MMP13 responsive peptide (the sequence of the MMP13 responsive short peptide is KCGPQG, and the concentration of the MMP13 responsive peptide in the solution needs to reach 0.5 mg / ml) are subjected to amidation reaction with HS, and the HSR powder is obtained by dialyzing and freeze-drying.

[0050] S4. Preparation of methacrylated hyaluronic acid (HAMA):

[0051] HA was dissolved in Tris buffer containing acetic anhydride at low temperature (4°C) for 2 h. HA was then dissolved in MES buffer (pH 6.0) and deoxygenated with nitrogen. Methacrylic anhydride was slowly added at a molar ratio (HA hydroxyl group:MA ≈ 1:2) while maintaining a controlled temperature at 4°C. After 8 h, EDC / NHS (molar ratio HA:EDC:NHS = 1:5:5) was added to activate the carboxylic acid groups and promote the reaction. Acetyl groups were hydrolyzed with 0.1 M NaOH solution, and the pH was adjusted to neutral. Byproducts were removed by dialysis using a 3000 Da molecular weight cutoff (MWCO) dialysis bag with deionized water for 3 days, with the water changed every 12 h. The purified MeHA was concentrated by ultrafiltration to remove small molecular weight impurities. The purified MeHA was freeze-dried to obtain a white powder.

[0052] S5. HSR, HAMA and methacryloyl-RGD (HR) peptide were mixed and multi-network hydrogel microspheres (HSPHR) were prepared using microfluidic technology:

[0053] S51, HSR, HAMA, and methacryloyl RGD (HR) peptide were mixed at a mass ratio of 5:5:1 to prepare the aqueous solution for the microfluidic device;

[0054] S52, using Span 80 and sorbitol isopropyl ester in a mass ratio of 1:9 to prepare the oil phase solution for the microfluidic device;

[0055] S53, the microfluidic instrument parameters are adjusted to make the water phase: oil phase = 500:15 to uniformly flow out the above two-phase solution;

[0056] S54, outflow channel is photocured using 305nm UV light;

[0057] S55. The solidified hydrogel microspheres are washed with ethanol and DEPC water respectively, and freeze-dried for storage.

[0058] Take 50 mg of freeze-dried hydrogel microsphere powder and soak it in PBS buffer. Use a glass bottle to observe the macroscopic structure of the HSPHR microspheres, such as morphology, color, size, etc. Figure 1 As shown, HSPHR exhibits a pink macroscopic appearance that is unique to selenium, indicating that the diselenide bonds are successfully cross-linked into the hydrogel microspheres.

[0059] The freeze-dried hydrogel microsphere powder was directly subjected to scanning electron microscopy to evaluate the microstructure of the microspheres. Figure 2 As shown in the figure, the composite microspheres cross-linked by hyaluronic acid containing diselenide bonds have a more obvious pore structure, and the increased porosity is more conducive to the material exchange between cells and microspheres.

[0060] Example 2

[0061] Compared with Example 1, the difference is that the mixing ratio of HSR, HAMA, and methacryloyl RGD (HR) peptide in S5 is 6:3:1. The freeze-dried hydrogel microsphere powder was directly subjected to scanning electron microscopy to evaluate the microstructure of the microspheres. Figure 2 As shown in the figure, with the increase of HSR concentration, the pore size of the hydrogel microspheres gradually becomes larger and the rigidity decreases, which is manifested as an increase in the volume of the microspheres.

[0062] Example 3

[0063] Compared with Example 1, the difference is that the mixing ratio of HSR, HAMA, and methacryloyl RGD (HR) peptide in S5 is 1:2:1. The freeze-dried hydrogel microsphere powder was directly subjected to scanning electron microscopy to evaluate the microstructure of the microspheres. Figure 2 As shown in the figure, the pore size of the hydrogel microspheres gradually becomes smaller and the volume of the microspheres decreases with the decrease of HSR concentration.

[0064] Comparative Example 1

[0065] Compared with Example 1, the difference is that only HAMA and methacryloyl RGD (HR) peptide are added to S5 to prepare hydrogel microspheres.

[0066] Take 50 mg of freeze-dried hydrogel microsphere powder and soak it in PBS buffer. Use a glass bottle to observe the morphology, color, size and other macroscopic structures of the HAMA microspheres. Figure 1 As shown, HAMA microspheres appear colorless or white, eliminating possible interference.

[0067] The freeze-dried hydrogel microsphere powder was directly subjected to scanning electron microscopy to evaluate the microstructure of the microspheres. Figure 2 As shown, the porosity of HAMA microspheres is significantly reduced compared to other examples, and the pore diameter is larger, indicating that its drug loading performance and potential cell adhesion effect are weaker.

[0068] Example 4: In vitro and in vivo responsiveness detection of diselenide-crosslinked hyaluronic acid hydrogel microspheres:

[0069] According to Example 1, 50 mg of hydrogel microspheres were weighed and encapsulated and then placed in a 6-well plate. These microspheres were incubated with 3 mL of a composite solution, which was either MMP13 protein (5 units / mL) or 0.1% H2O2 or a combination of MMP13 protein and 0.1% H2O2. At predetermined time intervals (6, 12, 18, 24 h, and 1, 3, 5, 7, 14, 21 d), 1.5 mL of sample solution was extracted, then supplemented with an equal volume of buffer and added to the plate to evaluate the concentration of organic valent selenium. The supernatant was analyzed spectrophotometrically at a wavelength of 490 nm using a spectrophotometer (Bio-Tek, Winooski, VT, USA). Under the same conditions, a standard curve of selenium free radical concentration was generated, and the drug concentration was deduced based on the absorbance values ​​obtained to evaluate the in vitro responsive release of the hydrogel. Figure 3 As shown, HSPHR exhibited good responsive release in H2O2, MMP13 protein and the mixed solution of the two.

[0070] Adult SD rats were selected to undergo medial meniscus ligament destabilization surgery (DMM), and the sham operation group (Sham) was used as the control. Two weeks after the surgery, the hydrogel microspheres in Example 1 were taken, and after being fully infiltrated with PBS, the concentration of the hydrogel microspheres was adjusted to 2 mg / mL. The microspheres were probe-labeled with CY5.5 as a marker dye, and the above two groups of SD rats were injected into the articular cavity. After the injection, the SD rat knee joints were imaged by infrared imaging using a small animal infrared in vivo imager (once a day for 14 days) to evaluate the in vivo response release of the hydrogel microspheres. Figure 4 As shown, HSPHR exhibited more obvious and persistent infrared fluorescence in the joint cavity of DMM mice, indicating that it can achieve effective response in the arthritis environment.

[0071] Example 5 In vitro anti-osteoarthritis effects of hydrogel microspheres of different systems

[0072] Chondrocytes were cultured in F12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C. When the confluence reached 100%, the hydrogel microspheres described in Example 1 and Comparative Example 1 were prepared. HSPHR and HAMA groups were set up, and high-concentration cell suspensions were inoculated onto the two hydrogel microspheres. When the cells were fully symbiotic with the microspheres, F12 complete medium containing 10 ng / mL LiL-1β was added and induction was continued for 72 hours. Figure 5As shown, the cells and microspheres were fixed in a co-mixed system. Immunofluorescence staining of the cells on the microspheres using MMP13 and COL2A proteins was then performed to evaluate the protective effects of the different microsphere systems on inflammatory cells. This experiment was repeated three times. The results demonstrated that HSPHR microspheres can effectively regulate the matrix synthesis and degradation homeostasis of chondrocytes in vitro, achieving a significant anti-osteoarthritis effect.

[0073] Example 6 Safranin Fast Green Staining and COL2A and MMP13 Immunohistochemical Staining of Rat Knee Joints

[0074] Adult rats were selected and housed in an SPF environment. The experimental groups were sham, diabetic mucin (DMM), HAMA, and high-spine spondylolisthesis (HSPHR) groups. A rat OA model was established by anesthetizing the rats and immobilizing them in a flexed knee position. The skin, subcutaneous muscle, and patellar ligament of the knee joint were sequentially incised to expose the joint cavity. The meniscus connecting ligament was severed using a sharp blade or severing the ligament. After severance, the joint cavity and skin were closed sequentially with absorbable sutures. Analgesics and antibiotics were administered postoperatively to relieve pain and prevent infection.

[0075] Rat knee joint injection: Two weeks after the rat OA model was established, the two microspheres were taken and fully infiltrated with PBS to a final concentration of 2 mg / mL. The total amount of solution injected into each joint was 100 μL. The injections were continued once a week until the rats were euthanized at the 8th week. The knee joints were then removed for subsequent evaluation.

[0076] Pathological evaluation of rat knee arthritis: Specimens were fixed for 48 hours and then decalcified with 10% EDTA for approximately 2 months. After decalcification, the samples were dehydrated and embedded in paraffin. Tissue blocks were sectioned at a thickness of 6 μm. These sections were dewaxed in xylene, dehydrated through a graded ethanol series, and stained with Safranin O / Fast Green. Subsequently, the sections were immunohistochemically stained for COL2A and MMP13. Tissue samples were observed using a light microscope, as shown in Figure 2. Figure 6 As shown, the HSPHR group showed better cartilage protection compared with the HAMA group, indicating that it can correct the inflammatory environment in the early stage of arthritis.

[0077] Example 7 Gross Assessment of Cartilage Defects in Rats and Safranin Fast Green Staining

[0078] Adult rats were selected and housed in an SPF environment. The experimental groups were sham, dilated hamster ovary syndrome (DMM), hamster necrosis factor (HAMA), and high-spine spondylosis (HSPHR) groups. A rat cartilage defect model was prepared: the rats were anesthetized and fixed in a flexed knee position. The skin, subcutaneous muscle, and patellar ligament of the knee joint were sequentially incised to expose the lower end of the femur. An electric drill was used to create a 1.5 mm diameter and 1.5 mm deep defect in the cartilage of the femoral trochanteric groove. The residual material was flushed with saline, the patella was repositioned, the joint cavity was closed, and the tissue layers were sutured sequentially. In the sham group, only the joint cavity was opened and sutured. Analgesics and antibiotics were injected postoperatively to relieve pain and prevent infection.

[0079] Rat knee joint injection: Two weeks after the rat OA model was established, the two microspheres were taken and fully infiltrated with PBS to a final concentration of 2 mg / mL. The total amount of solution injected into each joint was 100 μL. The injections were continued once a week until the rats were euthanized at the 8th week. The knee joints were then removed for subsequent evaluation.

[0080] Pathological evaluation of rat knee arthritis: The specimens were fixed for 48 h, and the femoral defect of the specimens was macroscopically evaluated using a micro single-lens camera, such as Figure 7 As shown. Decalcification was then performed with 10% EDTA for approximately 2 months. After decalcification, the samples were dehydrated and embedded in paraffin. Tissue blocks were sectioned at a thickness of 6 μm. These sections were dewaxed in xylene, dehydrated through a graded ethanol series, and stained with Safranin O / Fast Green. Tissue samples were observed using a light microscope, as shown. Figure 7 As shown in the results, the HSPHR group had a better repair effect on cartilage defects compared with the HAMA group, indicating that it can provide targeted treatment for cartilage destruction in the late stage of arthritis.

[0081] Biomaterials such as HA and its derivatives HAMA and HSPHR have excellent biocompatibility and biodegradability, which introduce an additional network structure layer into the hydrogel, enhance its mechanical strength, and provide conditions for the material to fully respond in the local microenvironment.

[0082] The occurrence and progression of osteoarthritis are complexly associated with the dynamic changes of pathological factors, including ROS, MMPs, and other components of the joint microenvironment. Although appropriate selenium supplementation can effectively restore intracellular protein metabolism and intra-articular redox homeostasis, it is worth noting that the therapeutic window of selenium is quite narrow. Selenium-selenium bonds have been widely reported to be effective responses to ROS and have unique functions. Based on this, the present invention has developed a diselenyl hyaluronic acid structure grafted with an MMP13-responsive peptide, which can achieve a focal dual response to ROS and MMP13, thereby intelligently promoting the release of selenium. Through the "environmental signal-structural dissociation" feedback loop, intelligent release of selenium is achieved, simulating the adaptive regulation of biological systems. Compared with traditional selenium supplementation technology, HSPHR has better biosafety and in vitro antioxidant activity.

[0083] Injectable functionalized microspheres have significant repair potential by reducing articular cartilage friction and regulating cartilage matrix metabolic homeostasis, thereby promoting the healing of soft defects. Our injectable hydrogel microspheres provide a minimally invasive option and offer greater operability compared to traditional surgical implantation methods.

[0084] The injectable hydrogel microspheres of the present invention have excellent injectability and stability, and can respond to the pathological microenvironment of osteoarthritis (OA), provide sustained release of the trace element organic selenium, restore the redox balance of cartilage, subchondral bone, and synovial cells, and achieve repair of the cartilage matrix. They can be preferably used in the damage and repair process of osteoarthritis.

[0085] It is easy to understand that those skilled in the art can combine, split, reorganize, etc. the embodiments of the present application based on one or several embodiments provided in the present application to obtain other embodiments, and these embodiments do not exceed the scope of protection of the present application.

[0086] In short, the above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the patent of the present invention.

Claims

1. A method for preparing injectable hydrogel microspheres containing organic diselenide bonds, characterized in that: The following steps are involved: Hyaluronic acid HA is reacted with an alkyne-containing reagent to generate alkyne-containing HA; After methylation and reduction activation, selenocysteine ​​Sec was grafted onto alkyne-containing HA via a thiol-alkyne click reaction, and then oxidized to form diselenide-crosslinked HS. The carboxyl groups of HS were activated with EDC / NHS, and the MMP13-responsive peptide KCGPQG was added for amidation reaction to obtain MMP13-responsive hyaluronic acid HSR; Methacryl-modified hyaluronic acid HAMA is prepared by reacting hyaluronic acid HA with methacrylic anhydride; HSR, HAMA and methacryloyl RGD peptide are mixed as an aqueous phase, and hydrogel microspheres are prepared using a microfluidic device. The hydrogel microspheres are then cured by ultraviolet light, washed and freeze-dried to obtain the injectable hydrogel microspheres HSPHR.

2. The method for preparing injectable hydrogel microspheres containing organic diselenide bonds according to claim 1, characterized in that: The alkyne-containing reagent is an alkyne isocyanate Alkyne-NCO, the molar ratio of HA to Alkyne-NCO is 1:1.5-3, and the preparation method of the alkyne-containing HA comprises: HA was dissolved in PBS at pH 7.0-8.0, and after deoxygenation by nitrogen, the pH was adjusted to 8.0-9.

0. Alkyne-NCO was added and reacted at 0-4°C for 2-6 h. The pH was adjusted to 6.5-7.5, and the HA containing alkyne groups was obtained by dialyzing and freeze-drying.

3. The method for preparing injectable hydrogel microspheres containing organic diselenide bonds according to claim 1, characterized in that: The method for methylating and reducing selenocysteine ​​Sec comprises: Dissolve Sec in PBS at pH 7.0-8.0, add triethylamine (TEA) first, then slowly add CH3I, and react in the dark for 4-6 hours to generate thiol methylation product. Then add TCEP and adjust the pH to 9.0-9.5, and stir at room temperature for 1-2 hours to generate thiolate Sec-S. - The molar ratio of Sec to TEA is 1:1~3, the molar ratio of Sec to CH3I is 1:1~3, and the final concentration of TCEP is 0.1~1.0 M.

4. The method for preparing injectable hydrogel microspheres containing organic diselenide bonds according to claim 3, characterized in that: The preparation method of the diselenide bond cross-linked HS comprises: The HA containing alkyne groups was combined with Sec-S - Dissolve in PBS at pH 7.0-8.0, deoxygenate with nitrogen, add CuSO4 and sodium ascorbate, and react in the dark for 16-24 hours. After dialysis to remove free Sec, lyophilize to obtain the monoselenium-modified product HA-Sec. HA-Sec was dissolved in PBS at pH 7.0-8.0, H2O2 was added and reacted at room temperature for 2-4 h, the reaction was terminated with Na2S2O3, the unreacted H2O2 was removed by dialysis, and then freeze-dried to obtain HS cross-linked with diselenide bonds; The molar ratio of the alkyne-containing HA to Sec-S- is 1:1-5, the concentrations of CuSO4 and sodium ascorbate are 50-100 μM and 5-10 mM, respectively, and the concentration of H2O2 is 1-3% w / v.

5. The method for preparing injectable hydrogel microspheres containing organic diselenide bonds according to claim 1, characterized in that: During the preparation of the MMP13-responsive hyaluronic acid HSR, the molar ratio of HS, EDC, and NHS is 1-2:5:5, the concentration of the MMP13-responsive peptide KCGPQG is 0.5-1.0 mg / mL, the amidation reaction temperature is 60° C., and the reaction time is 6-8 h.

6. The method for preparing injectable hydrogel microspheres containing organic diselenide bonds according to claim 1, characterized in that: During the preparation of hydrogel microspheres using a microfluidic device, HSR, HAMA, and methacryloyl-RGD peptide were mixed in a mass ratio of 1-6:2-5:1 to form the aqueous phase, and Span 80 and sorbitol isopropyl ester were mixed in a mass ratio of 1:9 to form the oil phase. The flow rate ratio of the aqueous phase to the oil phase was controlled at 500:

15.

7. An injectable hydrogel microsphere containing an organic diselenide bond, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the injectable hydrogel microspheres containing organic diselenide bonds according to claim 7 in the preparation of a drug for treating early osteoarthritis and late cartilage defects.

9. The use according to claim 8, characterized in that The hydrogel microspheres are used to treat early osteoarthritis and late cartilage defects by removing active oxygen in and outside the body and correcting selenoprotein metabolic disorders.

10. A pharmaceutical composition, characterized in that The injectable hydrogel microspheres comprising the organic diselenide bonds according to claim 7.

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