An injectable porous hydrogel microsphere containing magnesium targeting cartilage, and a preparation method and application thereof

Abstract

This invention discloses an injectable porous hydrogel microsphere containing magnesium and targeting cartilage, its preparation method, and its application. The preparation method is as follows: Step 1, preparing phosphorylated methacrylamide gelatin; Step 2, thoroughly mixing the product from Step 1 with magnesium oxide nanoparticles and a photoinitiator to form an aqueous phase, and then preparing MgO-GelMA-PS microspheres with an oil phase using a microfluidic device; Step 3, reacting the hydrogel microspheres from Step 2 with a chitosan quaternary ammonium salt solution to obtain M-GP@QCS. The hydrogel microspheres of this invention possess phosphate groups, which can provide binding sites to form stable metal ligand supramolecular bonds with MgO NPs, achieving continuous release of magnesium ions and long-term therapeutic effects on osteoarthritis; a chitosan quaternary ammonium salt coating is constructed on the surface of the microspheres, and the positive charge of the coating binds to the negatively charged cartilage tissue, achieving targeted adhesion of the microspheres to cartilage and realizing precise treatment of osteoarthritis.

Description

An injectable porous hydrogel microsphere containing magnesium and targeting cartilage, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an injectable porous hydrogel microsphere containing magnesium that targets cartilage, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is the most common joint disease, with knee osteoarthritis (KOA) being its main type. KOA is characterized by pain and functional impairment, and its pathology involves damage to multiple structures, including cartilage, synovium, and subchondral bone, potentially leading to disability in advanced stages. Articular cartilage, as an important component of the knee joint, exhibits a characteristic feature of osteoarthritis due to the imbalance between its repair and destruction. Current clinical treatment primarily focuses on relieving symptoms with nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids, but these cannot promote cartilage repair or reverse disease progression.

[0003] Magnesium ions (Mg 2+ Magnesium, an important trace element in the human body, is closely related to knee arthritis (KOA) as research has shown. Magnesium deficiency can exacerbate cartilage damage and pain, while intra-articular injection of magnesium salts can alleviate pain, inhibit apoptosis, and promote matrix synthesis. The mechanism involves multiple signaling pathways, including Wnt / β-catenin and HIF-1α. However, direct injection of magnesium ions has drawbacks such as short intra-articular retention time and rapid clearance, making it difficult to maintain effective therapeutic concentrations.

[0004] Intra-articular injection is the OARSI-recommended treatment strategy for KOA (knockouty arthritis). Intra-articular injection delivers drugs directly to the injury site, offering advantages such as improved bioavailability, reduced systemic exposure, fewer adverse events, and lower overall drug costs. However, conventional formulations like corticosteroids only remain in the joint for a few hours. In recent years, injectable hydrogels have received widespread attention as drug delivery carriers, but they suffer from limitations such as large injection volume, irregular shape, and susceptibility to injection injury. Microfluidic hydrogel microspheres offer advantages such as uniform size, good injectability, and easy dispersion, prolonging drug retention and adapting to joint movement. However, they still face the following challenges: due to gravity and joint movement, microspheres may become trapped in the joint space, hindering their therapeutic effect on damaged cartilage; this is exacerbated by insufficient microsphere injection. Excessive microsphere injection can lead to accumulation in the joint depression, increasing intra-articular friction and causing adverse effects.

[0005] The extracellular matrix (ECM) of chondrocytes is the microenvironment upon which chondrocytes depend for survival. It is composed of a large network of collagen, which is filled with proteoglycans and negatively charged glycosaminoglycans (GAGs), giving cartilage tissue a negative charge. Since GelMA-derived hydrogel microspheres are mostly negatively charged, they may repel the negatively charged cartilage, affecting the drug delivery efficiency of the hydrogel microspheres. Furthermore, because hydrogel microspheres lack cartilage targeting specificity, it may be difficult to efficiently deliver drugs in situ to the cartilage injury site.

[0006] Therefore, there is an urgent need to develop a hydrogel microsphere delivery system with cartilage-targeting function and controlled release of magnesium ions to achieve long-term safe and effective treatment of KOA. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing an injectable porous hydrogel microsphere containing magnesium that targets cartilage, its preparation method, and its application.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The first aspect is to provide a method for preparing magnesium-containing, cartilage-targeting, injectable porous hydrogel microspheres, comprising the following steps:

[0010] Step 1, Preparation of phosphorylated methacrylamide gelatin: Methacrylamide gelatin (60% degree of substitution) is reacted with phosphocreatine in the presence of a condensing agent to form amide bonds between phosphocreatine molecules and methacrylamide gelatin molecules to obtain phosphorylated methacrylamide gelatin. The resulting product is dialyzed and freeze-dried to obtain a white, loose, porous product, denoted as GelMA-PS.

[0011] Step 2, Preparation of magnesium-loaded microspheres:

[0012] The product GelMA-PS obtained in step one was thoroughly mixed with magnesium oxide nanoparticles and a photoinitiator to form an aqueous phase, which was then mixed with an oil phase containing a surfactant to form droplets through a microfluidic device. After crosslinking with ultraviolet light, MgO-GelMA-PS hydrogel microspheres were obtained, denoted as M-GP.

[0013] Step 3, Surface coating treatment:

[0014] The hydrogel microspheres obtained in step two were reacted with a chitosan quaternary ammonium salt solution, and after washing, composite hydrogel microspheres with positively charged QCS coatings on the surface were obtained, denoted as M-GP@QCS.

[0015] Furthermore, in step one, the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and the reaction system is also equipped with the auxiliary activator N-hydroxysuccinimide (NHS).

[0016] More preferably, in step one, the reaction is carried out at room temperature, with EDC added and the reaction lasting 0.25-2 hours, followed by the addition of NHS and a reaction lasting 6-12 hours.

[0017] More preferably, in step one, the mass ratio of the methacrylamide gelatin, creatine phosphate, EDC and NHS is (10-15):(9-11):(6-8):(2-2.5).

[0018] Furthermore, in step one, the reaction is carried out in a MES buffer solution system, wherein the concentration of MES in the MES buffer solution is 0.5M.

[0019] Furthermore, in step one, the dialysis is performed in a dialysis bag (with a molecular weight cutoff of 3.5 kDa or less), and the dialysis operation is performed in deionized water at 4°C for 7-10 days.

[0020] Furthermore, in step two, the concentration of GelMA-PS in the aqueous phase is 50-75 mg / mL, the concentration of magnesium oxide nanoparticles is 15-20 mg / mL, and the photoinitiator is Irgacure 2959 with a concentration of 5-6 mg / mL.

[0021] Furthermore, in step two, the oil phase is a mineral oil containing a surfactant; the surfactant is Span80, and its content is 4-6 v / v.

[0022] Furthermore, in step two, the flow rate ratio of the aqueous phase to the oil phase in the microfluidic device is controlled to be (1.8-2.5):1.

[0023] Furthermore, in step two, the hydrogel microspheres M-GP prepared by the microfluidic device have a particle size range of 70-100 μm.

[0024] Furthermore, in step three, the concentration of the chitosan quaternary ammonium salt solution is 0.5-2 mg / mL; and the contact reaction time is no less than 5 minutes.

[0025] Furthermore, in step three, the particle size range of the composite hydrogel microspheres M-GP@QCS is 70-100 μm.

[0026] The second aspect is to provide an injectable porous hydrogel microsphere containing magnesium and targeting cartilage prepared by the above-mentioned preparation method.

[0027] The third aspect is to provide the application of the above-mentioned magnesium-containing cartilage-targeting injectable porous hydrogel microspheres in the preparation of drugs for treating osteoarthritis.

[0028] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0029] The hydrogel microspheres prepared in this invention have phosphate groups, which can provide binding sites to form stable metal ligand supramolecular bonds with MgO NPs, thereby achieving the effect of continuous release of magnesium ions and realizing the long-term therapeutic effect on osteoarthritis.

[0030] This invention constructs a chitosan quaternary ammonium salt coating on the surface of hydrogel microspheres. The positive charge on the coating can bind to the negatively charged cartilage tissue, enabling the hydrogel microspheres to target and adhere to the cartilage, thus achieving precise treatment of osteoarthritis. Attached Figure Description

[0031] Figure 1 shows the characterization of M-GP@QCS hydrogel microspheres; where (A) is an optical microscope image of M-GP; (B) is the particle size distribution analysis of M-GP; (C) is a SEM image of M-GP; (D) is an optical microscope image of M-GP@QCS; (E) is the particle size distribution analysis of M-GP@QCS; (F) is a SEM image of M-GP@QCS; (G) is the elemental mapping image of M-GP@QCS: nitrogen spectrum, magnesium spectrum, oxygen spectrum, and phosphorus spectrum; (H) is the Fourier transform infrared (FTIR) spectrum of each group of microspheres; (I) is the Mg spectrum of M-GP@QCS. 2+ Cumulative release curves (n = 3); (J) shows the in vitro degradation behavior of M-GP and M-GP@QCS (n = 3); (K) shows the laser scanning confocal microscopy (LSCM) image of M-GP@QCS, with green fluorescence representing FITC-labeled QCS.

[0032] Figure 2 shows the in vitro cartilage targeting effect of M-GP@QCS; (A) and (B) M-GP suspension (yellow) and M-GP@QCS suspension (blue); (C) shows the adhesion of M-GP@QCS to the cartilage surface and the anti-rinsing process; (D) and (E) are images of the cartilage before and after rinsing.

[0033] Figure 3 shows the in vitro cytotoxicity evaluation results of hydrogel microspheres; (A) shows the live / dead cell staining results of the control group, GP group, M-GP group and M-GP@QCS group; (B) shows the quantitative analysis results of the number of live cells after live / dead cell staining in each group (n = 3); (C) shows the CCK-8 detection results on days 1, 3 and 5 (n = 3).

[0034] Figure 4 shows the inhibition of IL-1β-induced chondrocyte degeneration by M-GP@QCS. (A) Alcian blue staining results of ATDC5 cells in the control group, GP group, M-GP group, and M-GP@QCS group under IL-1β treatment; (B) Immunofluorescence staining results of collagen type II (Col II) in ATDC5 cells of each group; (C) Flow cytometry (FCA) analysis results of ATDC5 cells in the control group, GP group, M-GP group, and M-GP@QCS group under IL-1β treatment; (D) Quantitative analysis of Col II expression fluorescence intensity using ImageJ (n = 3); (E) Quantitative analysis of apoptosis rate obtained from FCA detection.

[0035] Figure 5 shows the imaging assessment results of the rat osteoarthritis model; (A) are representative anteroposterior and lateral X-ray images of the knee joint of each group of rats; (B) is the relative quantitative analysis of the joint space width (n = 3); (C) is the Kellgren–Lawrence (KL) grading results of each group based on X-ray imaging assessment (n = 3).

[0036] Figure 6 shows the histological analysis of the rat knee joints in different treatment groups; (A) are representative images of hematoxylin–eosin staining (HE) and safranin-O-green staining of articular cartilage in each group; (B) are the OARSI scores of articular cartilage degeneration (n = 3); and (C) are the relative glycosaminoglycan (GAG) content of each group (n = 3).

[0037] Figure 7 shows HE staining of rat internal organs after treatment in each treatment group, including samples from rat heart, liver, spleen, lung and kidney. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0039] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0040] Example 1

[0041] This embodiment provides an injectable porous hydrogel microsphere containing magnesium and targeting cartilage, and its preparation method, which specifically includes the following steps:

[0042] Step 1, Preparation of phosphorylated methacrylamide gelatin GelMA-PS (GP):

[0043] MES was dissolved in deionized water to prepare a 0.5M MES solution. 1g of methacrylamide gelatin (GelMA) (60% substitution) was dissolved in 50mL of the prepared MES solution. Gentle stirring in a 37°C water bath could accelerate dissolution, ultimately yielding a GelMA MES solution with a concentration of 20mg / mL. 0.9g of creatine phosphate (PS) was dissolved in 50mL of the prepared MES solution, ultimately yielding a PS MES solution with a concentration of 18mg / mL. The prepared PS solution and GelMA solution were mixed and shaken well. Then, 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to the solution, and the reaction was continued to be stirred at room temperature for 0.5 hours. Then, 0.2 g of N-hydroxysuccinimide (NHS) was added, and the reaction was continued to be stirred at room temperature for 6 hours. After the reaction was completed, excess deionized water was added to dilute the reaction mixture and terminate the reaction. Dialysis was performed at 4°C for 7 days using a dialysis bag (molecular weight cutoff of 3.5 kDa) to remove unreacted EDC, NHS, PS and their byproducts. The deionized water in the dialysis system was changed 3 times a day. After dialysis, the solution was freeze-dried to obtain GelMA-PS (GP).

[0044] Step 2, Preparation of Mgo-GelMA-PS microspheres (M-GP)

[0045] We observed that excessive MgO NPs might affect the photocrosslinking effect of the hydrogel microspheres. Therefore, we dissolved 100 mg of the GP prepared in step one in 1 mL of deionized water; then added 40 mg of MgO NPs to 1 mL of deionized water and ultrasonically dispersed it. Immediately afterwards, we poured this into the GP solution and thoroughly mixed and reacted in a 37°C water bath for 2 hours. Then, we added 12 mg of photoinitiator (Irgacure 2959) to prepare the aqueous phase required for microfluidic technology. We also prepared a mineral oil phase containing 5 v / v% Span 80 by adding 5 mL of mineral oil and surfactant Span 80. The aqueous and oil phases were then placed into a microfluidic device, and the flow rate ratio of the aqueous and oil phases was adjusted to 1.8:1 to obtain hydrogel droplets. The droplets were immediately photocrosslinked and cured with a UV lamp (365nm) for 10 minutes. The hydrogel microspheres were then repeatedly washed with isopropanol and deionized water to remove residual mineral oil and other impurities. The microspheres were then freeze-dried to finally obtain MgO-GelMA-PS microspheres (M-GP).

[0046] Step 3, Preparation of M-GP@QCS

[0047] Weigh 50 mg of chitosan quaternary ammonium salt (QCS) and dissolve it in deionized water. Stir thoroughly in a 37°C water bath to dissolve the QCS and prepare a 1 mg / ml QCS solution. Take 10 mg of the hydrogel microspheres M-GP prepared in step two and resuspend the M-GP hydrogel microspheres in 500 μL of deionized water. Slowly add the M-GP hydrogel microsphere suspension to the QCS solution while stirring continuously. After standing for 5 min, aspirate the supernatant and resuspend the microspheres in deionized water. Repeat this process 3 times to finally obtain positively charged QCS-coated hydrogel microspheres M-GP@QCS.

[0048] Example 2

[0049] This embodiment provides an injectable porous hydrogel microsphere containing magnesium and targeting cartilage, and its preparation method, which specifically includes the following steps:

[0050] Step 1, Preparation of phosphorylated methacrylamide gelatin GelMA-PS (GP):

[0051] MES was dissolved in deionized water to prepare a 0.5M MES solution. 1.5g of methacrylamide gelatin (GelMA) (60% substitution) was dissolved in 50mL of the prepared MES solution. Gentle stirring in a 37°C water bath could accelerate dissolution, ultimately yielding a GelMA MES solution with a concentration of 30mg / mL. 1.1g of creatine phosphate (PS) was dissolved in 50mL of the prepared MES solution, ultimately yielding a PS MES solution with a concentration of 22mg / mL. The prepared PS solution and GelMA solution were mixed and shaken well. Then, 0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to the solution, and the reaction was continued to be stirred at room temperature for 1 hour. Then, 0.25 g of N-hydroxysuccinimide (NHS) was added, and the reaction was continued to be stirred at room temperature for 12 hours. After the reaction was completed, excess deionized water was added to dilute the reaction mixture and terminate the reaction. Dialysis was performed at 4°C for 10 days using a dialysis bag (molecular weight cutoff of 3.5 kDa) to remove unreacted EDC, NHS, PS and their byproducts. The deionized water in the dialysis system was changed 3 times a day. After dialysis, the solution was freeze-dried to obtain GelMA-PS (GP).

[0052] Step 2, Preparation of Mgo-GelMA-PS microspheres (M-GP)

[0053] We observed that excessive MgO NPs (magnesium oxide nanoparticles) might affect the photocrosslinking effect of the hydrogel microspheres. Therefore, we dissolved 150 mg of the GP prepared in step one in 1.25 mL of deionized water; then added 30 mg of MgONPs to 0.75 mL of deionized water and ultrasonically dispersed it. This was then immediately poured into the GP solution and thoroughly mixed in a 37°C water bath for 2 hours. Subsequently, 10 mg of photoinitiator (Irgacure 2959) was added to prepare the aqueous phase required for microfluidic technology. 5 mL of mineral oil was added to prepare a mineral oil containing 6 v / v% Span 80 as the oil phase. The aqueous and oil phases were then placed into a microfluidic device, and the flow rate ratio of the aqueous and oil phases was adjusted to 2.5:1 to obtain hydrogel droplets. The droplets were immediately photocrosslinked and cured with a UV lamp (365nm) for 15 minutes. The hydrogel microspheres were then repeatedly washed with isopropanol and deionized water to remove residual mineral oil and other impurities. The microspheres were then freeze-dried to finally obtain MgO-GelMA-PS microspheres (M-GP).

[0054] Step 3, Preparation of M-GP@QCS

[0055] Weigh 100 mg of chitosan quaternary ammonium salt (QCS) and dissolve it in deionized water. Stir thoroughly in a 37°C water bath to dissolve the QCS and prepare a 2 mg / ml QCS solution. Take 10 mg of the hydrogel microspheres M-GP prepared in step two and resuspend the M-GP hydrogel microspheres in 500 μL of deionized water. Slowly add the M-GP hydrogel microsphere suspension to the QCS solution while stirring continuously. After standing for 8 min, aspirate the supernatant and resuspend the microspheres in deionized water. Repeat this process 3 times to finally obtain positively charged QCS-coated hydrogel microspheres M-GP@QCS.

[0056] Example 3 Characterization of hydrogel microspheres and Mg 2+ Release and degradation behavior study

[0057] To systematically evaluate the physicochemical properties of the M-GP@QCS hydrogel microspheres prepared in Example 1, we conducted the following experiments:

[0058] 3.1 Characterization of microsphere morphology and structure

[0059] The macroscopic morphology and particle size distribution of the microspheres were observed using a bright-field microscope (Leica, Germany). After gold sputtering, the freeze-dried microspheres were then examined for their microstructure and elemental distribution using a scanning electron microscope (Hitachi SU 70, Japan) and its associated energy dispersive spectroscopy (EDS). To further trace the distribution of quaternized chitosan (QCS) in the microspheres, FITC-labeled QCS was used to prepare microspheres, which were then observed using a laser confocal microscope (Leica, Germany). The chemical structure of the hydrogel microspheres was analyzed using Fourier transform infrared spectroscopy (Nicolet 6700, USA).

[0060] 3.2 Mg 2+ Release behavior research

[0061] 50 mg of M-GP@QCS microspheres containing MgO NPs were placed in a dialysis bag (MWCO 3500 Da), immersed in 25 mL of deionized water, and released by shaking at 37 °C and 80 rpm. Dialysate was collected at preset time points (1 d, 3 d, 7 d, 14 d, 21 d, and 28 d), and the Mg²⁺ concentration was determined using inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 5110, USA). An equal volume of fresh deionized water was added, and a cumulative release curve was plotted.

[0062] 3.3 Evaluation of in vitro degradation behavior

[0063] Add 5 mg of microspheres to phosphate-buffered saline (PBS) solution (pH 7.4) containing 1 U / mL collagenase II and incubate at 37 °C. Collect the microspheres at 4, 7, 14, 21, 28, and 35 days, freeze-dry them, weigh them, calculate the residual rate based on the initial weight, and plot the degradation curve.

[0064] The experimental results are shown in Figure 1:

[0065] Uniform hydrogel microspheres (M-GP) were successfully prepared using microfluidic technology. Subsequently, the M-GP microspheres were immersed and stirred in a QCS solution to obtain positively charged QCS-coated hydrogel microspheres (M-GP@QCS). Bright-field microscopy results showed that the M-GP@QCS exhibited a well-dispersed spherical morphology (part D of Figure 1), with a uniform particle size distribution and an average diameter of 87.91 ± 2.42 μm (part E of Figure 1). Scanning electron microscopy (SEM) images of the freeze-dried sample (part F of Figure 1) revealed a porous surface structure, which is beneficial for drug loading and release. Energy-dispersive X-ray spectroscopy (EDS) further verified the elemental distribution of M-GP@QCS (part G of Figure 1).

[0066] The chemical structure of the microspheres was analyzed using Fourier transform infrared (FTIR) spectroscopy (part H of Figure 1). GP, M-GP, and M-GP@QCS exhibited similarities to POC (1070 cm⁻¹). -1 The characteristic peaks corresponding to the M-GP bond confirmed the successful grafting of the phosphate group onto GelMA. M-GP and M-GP@QCS showed characteristic peaks at 1570 cm⁻¹. -1 The area also shows characteristic MgO-P peaks, indicating that MgO is effectively coupled to the phosphate group of GP.

[0067] Chitosan quaternary ammonium salt is a natural macromolecule with good biocompatibility and tissue adhesion properties. Rich in positive charges, it can electrostatically interact with the negatively charged GP backbone on the microsphere surface, forming a stable coating layer that facilitates targeting and adhesion to negatively charged cartilage tissue. To verify the uniformity of the coating, hydrogel microspheres M-GP@QCS were constructed using FITC-labeled chitosan quaternary ammonium salt (FITC-QCS) and observed using laser confocal microscopy. The results showed a continuous and uniform green fluorescent coating layer on the microsphere surface (part K in Figure 1), demonstrating the effective construction of the QCS coating.

[0068] Monitoring of Mg in M-GP@QCS microspheres using inductively coupled plasma optical emission spectroscopy (ICP-OES) 2+Release Behavior. As shown in Part I of Figure 1, the microspheres exhibited a relatively rapid release process in the first 21 days, with a cumulative release of 37.397 mg / L. The release rate then gradually slowed down, achieving sustained and stable release by day 28, with a final cumulative release of 38.133 mg / L. This indicates that the M-GP@QCS microspheres possess excellent controlled-release performance, laying the foundation for subsequent in vivo and in vitro studies. To simulate physiological degradation, the microspheres were placed in PBS (pH 7.4) containing 2 μg / mL collagenase II. As shown in Part J of Figure 1, both M-GP and M-GP@QCS microspheres showed gradual degradation within 35 days, with a degradation rate of approximately 70%, indicating good biodegradability.

[0069] Example 4: Validation of Cartilage Targeting Performance

[0070] Fresh cartilage was purchased from the market, and the knee joint surface cartilage was separated using a file. Two food colorings were used to stain the M-GP and M-GP@QCS hydrogel microspheres, with yellow microspheres representing M-GP and blue microspheres representing M-GP@QCS. A suspension of the stained M-GP and M-GP@QCS hydrogel microspheres (concentration 15 mg / mL) was prepared. Using a 1.0 mL syringe, the suspension of M-GP and M-GP@QCS microspheres was drawn and injected onto the cartilage surface. To simulate the fluid flow environment within the joint cavity, the joint surface was rinsed with running water for 6 seconds. Finally, the adhesion effect of the microspheres was observed.

[0071] The experimental results are shown in Figure 2:

[0072] After staining the microspheres (parts A and B of Figure 2) (M-GP in yellow, M-GP@QCS in blue), they were applied to the cartilage surface. The surfaces were then rinsed under running water to simulate the flow environment within the joint cavity (part C of Figure 2). After rinsing, most of the yellow M-GP microspheres were washed away, while the blue M-GP@QCS microspheres remained firmly adhered to the cartilage surface (parts D and E of Figure 2). This result is attributed to the electrostatic interaction between the positive charge of the microspheres and the negatively charged matrix of the cartilage, suggesting that M-GP@QCS has good cartilage-targeting adhesion, can resist the rinsing of synovial fluid, and has a good ability to adapt to the flow environment within the joint cavity.

[0073] Example 5: Cell compatibility evaluation

[0074] To determine the biocompatibility of the hydrogel microspheres (GP, M-GP, M-GP@QCS), we placed the hydrogel microspheres in the upper chamber of a transwell chamber (0.4µm wells, Corning, USA) and seeded 1×10⁶ microspheres in each lower chamber. 4Cell cytotoxicity of the hydrogel was determined by live / dead cell staining assay. On days 1, 3, and 5 of co-culturing cells with hydrogel microspheres, live / dead cell staining reagent was added and incubated at 37°C for 30 min. Cells were observed using a fluorescence microscope (Leica, Germany) and cell density per unit area and at each time point was calculated using ImageJ software.

[0075] The experimental results are shown in Figure 3:

[0076] The cytocompatibility of GP, M-GP, and M-GP@QCS microspheres was evaluated using live / dead staining and CCK-8 assays. As shown in parts A and B of Figure 3, most ADTC5 cells remained viable in all groups, with very few cell deaths, and cell proliferation continued to increase over 1 to 5 days. CCK-8 assays (part C of Figure 3) further showed that cell viability increased in a time-dependent manner in all groups, with no significant differences between groups. In conclusion, GP, M-GP, and M-GP@QCS microspheres all exhibit excellent biocompatibility.

[0077] Example 6: Effects of hydrogel microspheres on chondrocyte formation and apoptosis

[0078] 6.1 In vitro chondrogenic induction

[0079] To investigate the effect of hydrogel microspheres on ATDC5 chondrogenesis, hydrogel microspheres (GP, M-GP, M-GP@QCS) were placed in the upper chamber of a transwell chamber, and 1×10⁶ microspheres were implanted in each lower chamber. 4 ATDC5 cells were co-cultured for a specified number of days to simulate the inflammatory microenvironment of osteoarthritis (OA), and treated with IL-1β (10 ng / ml). On day 7, cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 minutes, followed by incubation with Alcian blue solution (Beyotime, China) for 30 minutes and staining at room temperature for 1 hour. Furthermore, we further assessed Col II expression using immunofluorescence staining. Specifically, cells were fixed with 4% paraformaldehyde (Beyotime, China) for 15 minutes at room temperature, followed by permeabilization with 0.1% Triton X-100 for 20 minutes. Cells were blocked with 5% BSA for 30 minutes, and then incubated overnight at 4°C with rabbit anti-collagen II antibody. Finally, cells were treated with goat anti-rabbit IgG H&L secondary antibody at 25°C for 1 hour. Finally, the ADTC5 was stained with 4,6-diamidinyl-2-phenylindole (DAPI) for 5 minutes, then stained with rhodamine-conjugated phalloidin for 30 minutes, and finally observed under a fluorescence microscope.

[0080] 6.2 Study on anti-apoptotic effects

[0081] Hydrogel microspheres (GP, M-GP, M-GP@QCS) were placed in the upper chamber of the transwell chamber, and 2 × 10⁶ microspheres were seeded in each lower chamber. 5 To simulate the inflammatory environment within the joint cavity, ATDC5 cells were treated with medium containing interleukin-1β (IL-1β) at a concentration of 10 ng / ml. One day later, cells in the lower chamber were treated with EDTA-free trypsin and mixed with the collected cell supernatant. After centrifugation, the cells were washed three times with pre-cooled PBS, and finally resuspended in Binding Buffer. After adding propidium iodide (PI) and annexin V dye, the cells were incubated at room temperature in the dark for 5 minutes, and finally analyzed using flow cytometry (BD).

[0082] The experimental results are shown in Figure 4:

[0083] Chondrocyte differentiation is characterized by increased synthesis of glycosaminoglycans (GAGs) and type II collagen (Col II) in the extracellular matrix (ECM). To mimic the inflammatory microenvironment of osteoarthritis (OA), ATDC5 cells were treated with IL-1β. After 7 days, alcinocyanine staining showed that IL-1β significantly reduced GAG levels compared to the control group. However, magnesium-containing microspheres (M-GP and M-GP@QCS) significantly alleviated this decrease, with GAG staining intensity significantly higher than in the PBS and GP groups (Figure 4, part A). Immunofluorescence further confirmed that IL-1β stimulation led to a decrease in Col II expression, most pronounced in the PBS and GP groups, while the M-GP and M-GP@QCS groups significantly maintained higher Col II levels (Figure 4, parts B and D). These results indicate that magnesium-containing microspheres can alleviate IL-1β-induced ECM degradation and protect chondrocytes from inflammatory damage. Flow cytometry results further showed that IL-1β significantly induced apoptosis in ATDC5 cells, while treatment with M-GP and M-GP@QCS effectively reduced the level of apoptosis (parts C and E of Figure 4).

[0084] Example 7: Therapeutic effect of microspheres in a rat model of osteoarthritis

[0085] 7.1 Animal Model Establishment and Grouping

[0086] Eight-week-old male Sprague-Dawley rats were used. After anesthetizing the rats, the knee joints were prepared and disinfected. The joint capsule was then opened, the medial meniscus was removed, and the anterior cruciate ligament was severed, confirmed by the anterior drawer test. The sham-operated group (n=3) underwent only skin surgery without compromising the integrity of the joint. One week after surgery, the OA rats were randomly divided into four groups: PBS group (treated with PBS solution), GP group (treated with 15 mg / mL GP), M-GP group (treated with 15 mg / mL M-GP), and M-GP@QCS group (treated with 15 mg / mL M-GP@QCS). The corresponding drugs were injected into the joint cavity of the OA model every two weeks.

[0087] 7.2 Radiological and Histological Evaluation

[0088] Eight weeks post-surgery, rats were euthanized under anesthesia, and knee joint specimens were obtained. Anteroposterior (AP) and lateral (LAT) scans of the rat knee joints were acquired using an X-ray imaging system. The severity of osteoarthritis (OA) in the rats was assessed using the joint space width and the Kellgren-Lawrence (KL) scoring system.

[0089] The obtained specimens were fixed with 4% paraformaldehyde for 48 hours, decalcified with 10% EDTA for 2 months, then embedded in paraffin, cut into 5 μm thick slices, and subjected to H&E staining (H&E staining also included samples from rat heart, liver, spleen, lung, and kidney) and safranin-O-fast green staining. The severity of OA in each group of specimens was assessed according to the currently accepted OARSI scale, and the GAG ​​content was assessed using brightfield microscopy and ImageJ software.

[0090] The experimental results are shown in Figure 5-7:

[0091] Eight weeks after modeling, imaging results showed significant narrowing of the medial joint space in the PBS and GP groups, accompanied by obvious osteophyte formation (part A of Figure 5). The relative medial joint space width decreased to 52% and 53.5%, respectively, and the KL scores were 3.67 and 3.33, respectively (parts B and C of Figure 5). In contrast, the M-GP and M-GP@QCS groups significantly alleviated joint space narrowing and osteophyte formation, with relative medial joint space widths of 75.58% and 88.60%, respectively, and KL scores decreased to 1.67 and 0.67, respectively (parts B and C of Figure 5). Among them, the M-GP@QCS group showed the best treatment effect due to the cartilage-targeting properties of M-GP@QCS.

[0092] Histological analysis results were consistent with this. HE and safranin-O-green staining showed that the cartilage in the PBS and GP groups was severely eroded and collapsed, while M-GP and M-GP@QCS treatments could maintain cartilage integrity to some extent. The cartilage surface in the M-GP group was rough and the chondrocytes were arranged in a disordered manner, while the cartilage surface in the M-GP@QCS group was generally smooth and the cells were arranged in a regular manner, similar to the sham-operated group (Figure 6, part A). OARSI scores showed that the M-GP (8.67) and M-GP@QCS (3.33) groups were significantly lower than those in the PBS (21.33) and GP (20.67) groups, with M-GP@QCS showing the most significant improvement (Figure 6, part B). At the same time, safranin-O-green staining results showed that the GAG ​​content in the cartilage of the M-GP@QCS group was significantly increased, approximately 2.67 times that of the PBS group (Figure 6, part C).

[0093] Furthermore, HE staining of major organs such as the heart, liver, spleen, kidneys, and lungs showed no structural abnormalities or inflammatory reactions (Figure 7), suggesting that the microsphere material has good biocompatibility in vivo.

[0094] In summary, the microspheres M-GP@QCS prepared in this invention possess stable magnesium ion sustained-release properties, biodegradability, cartilage targeting, and excellent biocompatibility. They can also inhibit cartilage degeneration and suppress IL-1β-induced apoptosis. In a rat osteoarthritis model, M-GP@QCS effectively alleviated cartilage degeneration, maintained cartilage matrix composition, and significantly reduced OARSI scores. This further demonstrates that M-GP@QCS is a safe and effective injectable biomaterial with good anti-inflammatory and cartilage-protective potential in the treatment of osteoarthritis, providing a new approach to osteoarthritis treatment.

[0095] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing magnesium-containing, cartilage-targeting, injectable porous hydrogel microspheres, characterized in that, The process includes the following steps: Step 1, preparation of phosphorylated methacrylamide gelatin: Methacrylamide gelatin is reacted with creatine phosphate in the presence of a condensing agent to form amide bonds between creatine phosphate molecules and methacrylamide gelatin molecules, resulting in phosphorylated methacrylamide gelatin. The product is dialyzed and freeze-dried to obtain a white, loose, porous product denoted as GelMA-PS. The condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the reaction system also includes the auxiliary activator N-hydroxysuccinimide. The reaction is carried out in a MES buffer solution system. Step 2, preparation of magnesium-loaded microspheres: The product GelMA-PS obtained in Step 1 is thoroughly mixed with magnesium oxide nanoparticles and a photoinitiator to form an aqueous phase. This aqueous phase is then mixed with an oil phase containing a surfactant to form droplets using a microfluidic device. After UV crosslinking, MgO-GelMA-PS hydrogel microspheres are obtained, denoted as M-GP. The photoinitiator is Irgacure 2959. The oil phase contains the surfactant Span. 80% mineral oil; Step 3, surface coating treatment: The hydrogel microspheres obtained in Step 2 are reacted with chitosan quaternary ammonium salt solution, and after washing, composite hydrogel microspheres with positively charged QCS coating are obtained, denoted as M-GP@QCS.

2. The preparation method according to claim 1, characterized in that, In step one, the reaction is carried out at room temperature. After adding EDC, the reaction lasts for 0.25-2 hours, followed by adding NHS and reacting for 6-12 hours.

3. The preparation method according to claim 1, characterized in that, In step one, the mass ratio of the methacrylamide gelatin, creatine phosphate, EDC and NHS is (10-15):(9-11):(6-8):(2-2.5).

4. The preparation method according to claim 1, characterized in that, In step one, the concentration of MES in the MES buffer solution is 0.5M.

5. The preparation method according to claim 1, characterized in that, In step two, the concentration of GelMA-PS in the aqueous phase is 50-75 mg / mL, the concentration of magnesium oxide nanoparticles is 15-20 mg / mL, the concentration of the photoinitiator is 5-6 mg / mL, and the flow rate ratio of the aqueous phase to the oil phase in the microfluidic device is controlled to be (1.8-2.5):

1.

6. The preparation method according to claim 1, characterized in that, In step two, the content of Span 80 in the oil phase is 4-6 v / v.

7. The preparation method according to claim 1, characterized in that, In step three, the concentration of the chitosan quaternary ammonium salt solution is 0.5-2 mg / mL; the contact reaction time is not less than 5 minutes.

8. An injectable porous hydrogel microsphere containing magnesium and targeting cartilage, prepared by the preparation method according to any one of claims 1-7.

9. The use of the magnesium-containing cartilage-targeting injectable porous hydrogel microspheres as described in claim 8 in the preparation of a drug for treating osteoarthritis.

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