An injectable hydrogel microsphere for local treatment of osteoarthritis and a preparation method thereof

Abstract

The application discloses an injection hydrogel microsphere for local treatment of osteoarthritis and a preparation method thereof. The microsphere takes aldehyde-modified methylacrylated hyaluronic acid and polyethylene glycol diacrylate as a water-phase matrix and is prepared through microfluidic technology; magnesium-aluminum layered double hydroxide nanoparticles loaded with diclofenac are embedded in the inner layer, and paeonol is loaded on the outer layer. The microsphere realizes specific covalent adhesion to damaged cartilage through Schiff base reaction between aldehyde groups and amino groups on the surface of cartilage; in the acidic microenvironment of osteoarthritis, diclofenac is responsively and rapidly released to realize early precise anti-inflammation, and paeonol is continuously and slowly released to realize long-term anti-oxidation and cartilage repair, and the two realize therapeutic effects in a time sequence. The microsphere has uniform particle size, good injectability and excellent biocompatibility and can be used for local precise treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and tissue engineering, and relates to an injectable hydrogel microsphere for local treatment of osteoarthritis and its preparation method. Background Technology

[0002] Osteoarthritis (OA) is a chronic joint disease characterized by degenerative changes in articular cartilage, synovial inflammation, and subchondral bone remodeling, clinically manifesting as pain, functional impairment, and even disability. Current clinical treatments include oral nonsteroidal anti-inflammatory drugs (NSAIDs), intra-articular injection of hyaluronic acid (HA), and late-stage surgical replacement. However, oral NSAIDs require long-term, high-dose administration, which can easily cause gastrointestinal damage, liver and kidney dysfunction, and cardiovascular risks. Directly injected drugs or HA preparations are easily diluted and cleared by synovial fluid, with a half-life of only a few hours to a few days, making it difficult to maintain effective therapeutic concentrations. The pathogenesis of OA is complex, involving multiple pathological processes such as overexpression of inflammatory factors, oxidative stress, chondrocyte ferroptosis, and extracellular matrix degradation; single anti-inflammatory or lubricating therapies are insufficient to achieve fundamental cartilage repair.

[0003] In recent years, hydrogel microspheres based on microfluidic technology have attracted widespread attention due to their controllable particle size, good injectability, and good biocompatibility. Among them, methacryloyl hyaluronic acid (HAMA), as a derivative of HA, retains the bioactivity of natural HA and introduces photocrosslinking ability, making it an ideal raw material for constructing cartilage repair carriers. However, conventional HAMA microspheres lack active targeting capabilities, and single drug loading is insufficient to meet the time-series requirements of multi-stage OA treatment.

[0004] Therefore, developing an injectable hydrogel microsphere system that combines active lesion targeting, inflammatory response drug release, and multi-mechanism synergistic therapy is of great clinical significance for the local precision treatment of OA. Summary of the Invention

[0005] The purpose of this invention is to construct an injectable hydrogel microsphere with active cartilage adhesion and inflammatory microenvironment-responsive sequential drug release function for the local treatment of osteoarthritis (OA).

[0006] To achieve the above objectives, this invention employs a two-step chemical modification method, sequentially introducing aldehyde groups (–CHO) and methacryloyl groups (C=C) onto the hyaluronic acid (HA) molecular chain to prepare aldehyde-modified methacryloyl hyaluronic acid (HA-ALD-MA). Simultaneously, the anti-inflammatory drug diclofenac (DCF) is intercalated and loaded into magnesium-aluminum layered double hydroxide (LDH) nanoparticles via a co-precipitation method to obtain LDH-DCF. Using HA-ALD-MA and polyethylene glycol diacrylate (PEGDA) as an aqueous matrix, LDH-DCF is dispersed and paeoniflorin (PAE) is dissolved. Water-in-oil emulsion droplets are prepared using a flow-focusing microfluidic chip, and then cross-linked and cured under ultraviolet light to obtain dual-drug-loaded composite microspheres. The morphology, particle size, swelling ratio, degradation behavior, drug loading, encapsulation efficiency, and in vitro drug release characteristics of the microspheres are characterized, and their biocompatibility and proliferative effects are evaluated through rat chondrocyte co-culture experiments.

[0007] Results: The prepared microspheres exhibited uniform particle size and good injectability. At a HA-ALD-MA to PEGDA mass ratio of 3:1, the paeoniflorin loading was 20.78% and the encapsulation efficiency was 75.21%; the diclofenac loading was 25.94% and the encapsulation efficiency was 61.65%. In vitro release experiments showed that diclofenac achieved a cumulative release rate of 87.3±4.2% at pH 6.8 (simulating the OA inflammatory microenvironment) after 48 h, exhibiting a significant pH-responsive rapid release; paeoniflorin maintained continuous release for over 25 days at pH 7.4 (cumulative release >80%), exhibiting typical sustained-release characteristics. Cell experiments demonstrated that the microspheres showed no significant cytotoxicity (cell viability ≥80%) and significantly promoted chondrocyte proliferation, with effects superior to conventional hydrogels.

[0008] Conclusion: This study successfully constructed a hydrogel microsphere drug delivery system that combines active cartilage adhesion, pH-responsive rapid release of diclofenac, and long-term sustained release of paeoniflorin. The microspheres exhibit controllable preparation process, uniform performance, and excellent biocompatibility, enabling synergistic treatment of "early anti-inflammatory and long-term cartilage repair," providing a new strategy and experimental basis for the precise local treatment of osteoarthritis.

[0009] The more specific technical solutions are as follows: A method for preparing hydrogel microspheres for local treatment of osteoarthritis includes the following steps: (1) Preparation of aldehyde-modified methacrylamide hyaluronic acid; (2) The aldehyde-modified methacrylamide hyaluronic acid obtained in step (1) is mixed with polyethylene glycol diacrylate at a mass ratio of 1~5:1, and the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite is added as the aqueous phase; (3) Diclofenac was intercalated and loaded into magnesium-aluminum layered double hydroxide nanoparticles by coprecipitation to obtain drug-loaded nanoparticles; (4) Disperse the drug-loaded nanoparticles obtained in step (3) in the aqueous phase of step (2), add paeoniflorin, and form an oil-in-water emulsion droplet through a microfluidic device; (5) The droplets obtained in step (4) are subjected to ultraviolet light crosslinking and curing, the microspheres are collected, washed and dried to obtain the hydrogel microspheres.

[0010] Preferably, the preparation method of the aldehyde-modified methacrylamide hyaluronic acid includes: first, oxidizing hyaluronic acid with sodium periodate to obtain aldehyde-modified hyaluronic acid, and then esterifying the aldehyde-modified hyaluronic acid with methacrylic anhydride to obtain aldehyde-modified methacrylamide hyaluronic acid.

[0011] Preferably, the mass ratio of the aldehyde-modified methacrylamide hyaluronic acid to polyethylene glycol diacrylate is 3:1.

[0012] Preferably, the photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite accounts for 0.3-1% of the aqueous phase by mass.

[0013] Preferably, the preparation method of the drug-loaded nanoparticles includes: dissolving magnesium nitrate and aluminum nitrate in water to obtain a mixed salt solution; dissolving diclofenac in NaOH solution to obtain a drug alkali solution; mixing the mixed salt solution and the drug alkali solution under nitrogen protection and vigorous stirring at 55-65°C, and reacting the mixture while maintaining the pH of the system at 10-12 with NaOH; finally, subjecting the reaction system to hydrothermal crystallization treatment, collecting the precipitate by centrifugation, washing, and drying to obtain magnesium-aluminum layered double hydroxide nanoparticles loaded with diclofenac.

[0014] Preferably, the molar ratio of magnesium nitrate to aluminum nitrate is 1-3:1, and the diclofenac accounts for 1-5% of the mass of the mixed salt.

[0015] Preferably, the drug-loaded nanoparticles account for 1-5% of the mass of the aqueous phase, and the concentration of paeoniflorin in the aqueous phase is 1-5 mg / mL.

[0016] Preferably, the aqueous phase flow rate of the microfluidic device is 0.3~0.8 mL / min, and the oil phase flow rate is 0.5~1.2 mL / min.

[0017] Preferably, the conditions for ultraviolet crosslinking curing are: wavelength 365 nm, light intensity 15 mW / cm², and irradiation time 1.5 min.

[0018] The present invention also provides the application of the hydrogel microspheres in the preparation of topical therapeutic agents for osteoarthritis.

[0019] Compared with the prior art, the present invention has the following significant advantages: (1) Active targeted adhesion to prolong joint cavity retention: By introducing aldehyde groups onto the HA molecular chain, the prepared HA-ALD-MA / PEGDA composite microspheres can form Schiff base covalent bonds with the amino groups exposed on the surface of damaged cartilage, achieving specific adhesion. This "chemical anchoring" mechanism significantly prolongs the residence time of the microspheres in the joint cavity and improves the local bioavailability of the drug.

[0020] (2) Time-sequential dual-drug controlled release, matching the disease progression: An innovative two-stage delivery system of "microsphere-encapsulated nanoparticles" was constructed. The inner layer, LDH-DCF, responsively degrades in the weakly acidic microenvironment of OA (pH ~6.5), enabling preferential and rapid release of diclofenac (release rate >78% at 48 h) for early and precise anti-inflammatory effects. The outer hydrogel matrix, containing paeoniflorin, achieves sustained release through a diffusion barrier (cumulative release >80% at 25 days), providing long-term antioxidant, anti-ferroptosis, and cartilage repair effects. This temporal cascade strategy achieves synergistic treatment of "early anti-inflammatory - long-term repair".

[0021] (3) Precise microfluidic preparation with uniform and controllable performance: Utilizing a flow-focusing microfluidic chip, the microsphere particle size (100–300 μm), monodispersity (PDI < 0.3), and structural integrity can be precisely controlled. The prepared microspheres exhibit good injectability (passable through a 26G needle), suitable swelling rate (700%–1000%), and controllable degradation behavior (8–10 weeks), while simultaneously supporting chondrocyte adhesion and proliferation.

[0022] (4) Excellent biocompatibility and synergistic treatment through multiple mechanisms: Hyaluronic acid and PEGDA are both FDA-approved materials. The combination of LDH with diclofenac and paeoniflorin showed no significant cytotoxicity in cell experiments (cell viability ≥80%). Diclofenac inhibits the COX-2 pathway to reduce inflammation, while paeoniflorin inhibits ferroptosis and oxidative stress through the circ-PREX1 / miR-140-3p axis. Mg²⁺ + Ions promote cartilage differentiation, and the three work synergistically to promote cartilage matrix regeneration. Attached Figure Description

[0023] Figure 1 Infrared spectrum of aldehyde-modified methacrylamide hyaluronic acid.

[0024] Figure 2 Particle size distribution of diclofenac magnesium aluminum layered double hydroxide nanoparticles.

[0025] Figure 3UV spectra of diclofenac, layered double hydroxides, and drug-loaded nanoparticles.

[0026] Figure 4 : Physical image of the dual-drug-loaded hydrogel microspheres and an illustration of their injectability.

[0027] Figure 5 Morphological images of dual-drug-loaded hydrogel microspheres under optical microscopes at different magnifications.

[0028] Figure 6 Comparison of swelling rates of different hydrogel microspheres.

[0029] Figure 7 : In vitro degradation curve of hydrogel microspheres.

[0030] Figure 8 Cumulative drug release curves of diclofenac and paeoniflorin under different pH conditions. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. However, the following embodiments are only for illustrating the present invention and should not be considered as limiting the present invention. The concept of the present invention is as follows: First, introducing aldehyde groups into methacryloyl hyaluronic acid hydrogels can increase cartilage adhesion, thereby promoting the attachment of intra-articular mesenchymal stem cells, but it degrades rapidly and has low mechanical strength. Therefore, polyethylene glycol diacrylate is introduced in combination with it. Their high water content and structural similarity to the extracellular matrix support a hydrated, cell-friendly microenvironment. Moreover, they can form hydrogels with tunable mechanical strength through chain growth polymerization in the presence of photoinitiators, enhancing mechanical strength and injectability. Secondly, most nonsteroidal anti-inflammatory drugs (NSAIDs), such as diclofenac, have short intra-articular half-lives, are easily and rapidly eliminated, and have significant side effects, which is detrimental to cartilage repair. Utilizing the layered structure and anion exchange properties of magnesium-aluminum layered double hydroxide nanoparticles, diclofenac is intercalated and loaded between the layers of the layered double hydroxide via co-precipitation. The layered double hydroxide is structurally stable in normal synovial fluid, resulting in slow drug release; however, in the acidic inflammatory microenvironment (lowered pH) of the osteoarthritis lesion site, the layered double hydroxide gradually degrades, thereby triggering a responsive release of diclofenac.

[0032] Third, using microfluidic technology, a bilayer-loaded, dual-drug-loaded injectable hydrogel microsphere was constructed. Diclofenac was loaded into layered double hydroxide nanoparticles and dispersed in the hydrogel core, allowing for preferential and rapid release in response to the acidic inflammatory environment, achieving early "precise and potent anti-inflammatory effects." Simultaneously, paeoniflorin, which has antioxidant and ferroptosis-inhibiting effects, was loaded into the outer layer of the hydrogel matrix, enabling long-term sustained release through the barrier effect of the hydrogel matrix, achieving long-term "cartilage protection and repair." This sequential cascade release strategy combines early anti-inflammatory effects with long-term cartilage protection, achieving synergistic treatment through multiple mechanisms and multiple targets.

[0033] Example 1: Preparation of dual-drug-loaded injectable hydrogel microspheres Step 1: Preparation of Aldehyde-Modified Methacrylamide Hyaluronic Acid (HA-ALD-MA) Dissolve 1 g of hyaluronic acid (molecular weight 200 kDa) in 100 mL of deionized water and stir at 350 rpm until completely dissolved. Slowly add 5 mL of 0.5 M sodium periodate solution, and react in the dark with stirring at 250 rpm for 2 h. Add 1 mL of ethylene glycol and continue stirring for 1 h to quench the reaction. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 8-14 kDa, dialyze at room temperature for 72 h, and lyophilize to obtain aldehyde-modified hyaluronic acid (HA-ALD).

[0034] Dissolve 1 g of HA-ALD in 50 mL of deionized water, and slowly add 20 mL of methacrylic anhydride under ice bath conditions, stirring at 250 rpm. Using a micro-injection pump, add 20 mL of 5 M NaOH solution at a uniform rate over 2 h, maintaining the pH of the reaction solution at 8-9. After reacting overnight at 4°C, centrifuge at 7000 rpm for 15 min, collect the supernatant, dialyze for 72 h, and lyophilize to obtain a white spongy HA-ALD-MA.

[0035] Infrared spectroscopy ( Figure 1 The results showed that HA-ALD was at 1730 cm. - The characteristic peak of the aldehyde group appears at ¹; HA-ALD-MA shows a peak at 1700 cm⁻¹. - The enhanced absorption peak near ¹ is attributed to the superposition vibration of the aldehyde group and the carbon-carbon double bond, indicating the successful introduction of the aldehyde group and the methacryloyl group.

[0036] Step 2: Preparation of diclofenac magnesium aluminum layered double hydroxide nanoparticles (LDH-DCF) 5.13 g (0.02 mol) magnesium nitrate hexahydrate and 3.75 g (0.01 mol) aluminum nitrate nonahydrate were dissolved in 40 mL of deionized water to obtain a mixed salt solution. 0.3 g diclofenac was dissolved in 10 mL of 0.15 M NaOH solution to obtain a drug alkali solution. Under nitrogen protection and vigorous stirring in a 60℃ water bath, the mixed salt solution and drug alkali solution were simultaneously added dropwise to a reaction flask containing 10 mL of deionized water, maintaining the pH at 11.0 ± 0.5 with 5 M NaOH. After the addition was complete, stirring was continued for 30 min. The slurry was then transferred to a 100 mL high-pressure reactor and hydrothermally treated at 80℃ for 6 h. After natural cooling, the precipitate was collected by centrifugation at 10000 rpm for 10 min, washed three times each with deionized water and anhydrous ethanol, and lyophilized to obtain a reddish-brown LDH-DCF powder.

[0037] Figure 2 The results showed that LDH-DCF particles had a diameter of approximately 258 nm, a PDI of 0.248, and good dispersibility; UV spectroscopy confirmed successful intercalation and loading of diclofenac. Figure 3 ).

[0038] Step 3: Preparation of dual-drug-loaded hydrogel microspheres using microfluidic methods Aqueous phase preparation: HA-ALD-MA and polyethylene glycol diacrylate (PEGDA) were mixed at a mass ratio of 3:1 to prepare an aqueous solution with a total concentration of 5% (w / w). 0.5% (w / w) of phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite (LAP) photoinitiator was added, followed by 2% (w / w) of LDH-DCF and 3 mg / mL of paeoniflorin. The mixture was then ultrasonically dispersed until homogeneous.

[0039] Oil phase preparation: Dissolve 6% (v / v) Span 80 in paraffin oil.

[0040] Microfluidic parameters: A polydimethylsiloxane (PDMS) flow-focusing microfluidic chip with a channel diameter of 200 μm was used. The aqueous phase flow rate was 0.5 mL / min, the oil phase flow rate was 0.8 mL / min, and the formed oil-in-water droplets were collected.

[0041] Photocrosslinking curing: The receiving tube was placed under a 365 nm UV lamp (15 mW / cm²) for 1.5 min to solidify the droplets into microspheres. The microspheres were collected by centrifugation at 6000 rpm for 5 min, washed three times with isopropanol, washed with PBS, and stored at 4℃.

[0042] Figure 4 The results showed that the obtained microspheres were white, uniform in size (approximately 182.1 μm), and could be injected through a 26G needle, demonstrating good injectability. Figure 5 The microspheres appear as regular spheres under an optical microscope and exhibit good dispersibility.

[0043] Step 4: Characterization of Microsphere Performance 1. Swelling rate determination: Weigh W0 of the lyophilized microspheres, immerse them in pH 7.4 PBS at 37℃ for 24 h to swell, remove them, absorb the surface moisture, and weigh W. s Swelling rate = (W) s -W0) / W0× 100%. Figure 6 The results showed that the swelling rate of the HA-ALD-MA / PEGDA composite microspheres was 800.4%, which is close to the 851.1% of the single HA-ALD-MA microspheres. The high swelling rate helps to absorb inflammatory exudates and maintain a moist environment.

[0044] 2. Degradation experiment: The microspheres were placed in pH 7.4 PBS and degraded at 37℃. Samples were taken every 2 weeks, lyophilized and weighed. Figure 7 The results showed that the composite microspheres gradually degraded within 8 to 10 weeks.

[0045] 3. Drug loading and encapsulation efficiency: Lyophilized microspheres were accurately weighed, ground, and extracted with PBS by ultrasonication for 30 min. The contents of paeoniflorin and diclofenac were determined by HPLC. Table 1 shows that when the HA-ALD-MA / PEGDA ratio was 3:1, the paeoniflorin drug loading was 20.78% and the encapsulation efficiency was 75.21%; the diclofenac drug loading was 25.94% and the encapsulation efficiency was 61.65%.

[0046] Table 1: Effect of different proportions of PEGDA on the drug loading performance of hydrogel microspheres

[0047] 4. In vitro release experiment: The drug-loaded microspheres were placed in dialysis bags and immersed in PBS release media at pH 7.4 and pH 6.8 respectively (37℃, 100 rpm shaker), and samples were taken at regular intervals for HPLC analysis. Figure 8 The results showed that diclofenac was released rapidly at pH 6.8, with a cumulative release rate of 87.3±4.2% after 48 hours, while the release rate decreased significantly at pH 7.4, with only 38.5±3.1% released after 48 hours, exhibiting significant pH responsiveness and sustained-release characteristics. Meanwhile, paeoniflorin release showed less pH dependence and exhibited sustained-release characteristics. These characteristics not only facilitate long-term cartilage protection and repair but also enable rapid release of diclofenac under acidic inflammatory conditions, which is crucial for controlling inflammation and promoting cartilage regeneration.

[0048] 5. Chondrocyte proliferation experiment: Rat primary chondrocytes were co-cultured with different groups of hydrogels, and the OD450 value was detected by CCK-8 assay. Table 2 shows that the OD value of the HA-ALD-MA / PEGDA group on day 5 was significantly higher than that of the HA / sodium alginate group, gelatin / sodium alginate group, and gelatin / HA group, indicating that the composite gel material of the present invention has a better ability to promote chondrocyte proliferation and is an ideal drug carrier for the treatment of osteoarthritis.

[0049] Table 2: Effects of different types of hydrogel microspheres on chondrocyte proliferation

[0050] In addition, the effect of PEGDA dosage on the proliferation of primary rat chondrocytes was investigated by setting different mass ratios of HA-ALD-MA to PEGDA. Table 3 shows that with the increase of PEGDA dosage, chondrocyte proliferation showed a trend of first increasing and then decreasing. When the mass ratio of HA-ALD-MA to PEGDA was 3:1, the chondrocyte proliferation capacity was stronger.

[0051] Table 3: Effects of different HA-ALD-MA:PEGDA ratios on chondrocyte proliferation

[0052] Example 2: Step 1: Preparation of aldehyde-modified methacrylamide hyaluronic acid Dissolve 0.8g of hyaluronic acid in 100mL of deionized water and stir until completely dissolved. Slowly add 3mL of 0.5M sodium periodate solution, protect from light, and stir for 2h. Add 1mL of ethylene glycol and continue stirring for 1h to quench the reaction. Transfer the reaction solution to a dialysis bag and dialyze at room temperature for 72h. Freeze dry to obtain aldehyde-modified hyaluronic acid (HA-ALD).

[0053] Dissolve 0.5 g HA-ALD in 50 mL deionized water, and slowly add 15 mL methacrylic anhydride under ice bath conditions, stirring at 250 rpm. Add 5 M NaOH solution using a micro-syringe pump to maintain the pH of the reaction solution at 8-9, react at 4 °C, centrifuge at 7000 rpm for 15 min, collect the supernatant, dialyze for 72 h, and lyophilize to obtain aldehyde-modified methacryloyl hyaluronic acid (HA-ALD-MA).

[0054] Step 2: Preparation of diclofenac magnesium aluminum layered double hydroxide nanoparticles (LDH-DCF) 0.03 mol magnesium nitrate and 0.01 mol aluminum nitrate were dissolved in 40 mL of deionized water to obtain a mixed salt solution. 0.3 g diclofenac was dissolved in 10 mL of 0.15 M NaOH solution to obtain a drug alkali solution. Under nitrogen protection and vigorous stirring in a 65 °C water bath, the mixed salt solution and drug alkali solution were simultaneously added dropwise to a reaction flask containing 10 mL of deionized water, while maintaining the pH at 11.0 ± 0.5 with 5 M NaOH. After the addition was complete, stirring was continued for 30 min. The slurry was then transferred to a 100 mL high-pressure reactor and hydrothermally treated at 90 °C for 5 h. After natural cooling, the precipitate was collected by centrifugation at 10,000 rpm for 10 min, washed three times each with deionized water and anhydrous ethanol, and then lyophilized to obtain LDH-DCF powder.

[0055] Step 3: Preparation of dual-drug-loaded hydrogel microspheres using microfluidic methods Aqueous phase preparation: HA-ALD-MA and polyethylene glycol diacrylate (PEGDA) were mixed at a mass ratio of 2:1 to prepare an aqueous solution with a total concentration of 5% (w / w). 0.8% (w / w) LAP photoinitiator was added, followed by 5% (w / w) LDH-DCF and 2 mg / mL paeoniflorin. The mixture was then ultrasonically dispersed until homogeneous.

[0056] Oil phase preparation: Dissolve 6% (v / v) Span 80 in paraffin oil.

[0057] Microfluidic parameters: A polydimethylsiloxane flow-focusing microfluidic chip with a channel diameter of 200 μm was used. The aqueous phase flow rate was 0.6 mL / min, the oil phase flow rate was 1 mL / min, and the formed oil-in-water droplets were collected.

[0058] Photocrosslinking curing: The receiving tube was placed under a 365nm UV lamp (15mW / cm²) for 1.5 min to solidify the droplets into microspheres. The microspheres were collected by centrifugation at 6000rpm for 5 min, washed three times with isopropanol, washed with PBS, and stored at 4℃.

Claims

1. A method for preparing hydrogel microspheres for local treatment of osteoarthritis, characterized in that, Includes the following steps: (1) Preparation of aldehyde-modified methacrylamide hyaluronic acid; (2) The aldehyde-modified methacrylamide hyaluronic acid obtained in step (1) is mixed with polyethylene glycol diacrylate at a mass ratio of 1-5:1, and the photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl hypophosphite is added as the aqueous phase; (3) Diclofenac was intercalated and loaded into magnesium-aluminum layered double hydroxide nanoparticles by coprecipitation to obtain drug-loaded nanoparticles; (4) Disperse the drug-loaded nanoparticles obtained in step (3) in the aqueous phase of step (2), add paeoniflorin, and form an oil-in-water emulsion droplet through a microfluidic device; (5) The droplets obtained in step (4) are subjected to ultraviolet light crosslinking and curing, the microspheres are collected, washed and dried to obtain the hydrogel microspheres.

2. The preparation method according to claim 1, characterized in that, The preparation method of the aldehyde-modified methacrylamide hyaluronic acid includes: first, oxidizing hyaluronic acid with sodium periodate to obtain aldehyde-modified hyaluronic acid, and then esterifying the aldehyde-modified hyaluronic acid with methacrylic anhydride to obtain aldehyde-modified methacrylamide hyaluronic acid.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the aldehyde-modified methacrylamide hyaluronic acid to polyethylene glycol diacrylate is 3:

1.

4. The preparation method according to claim 1, characterized in that, The photoinitiator, phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, accounts for 0.3-1% of the aqueous phase by mass.

5. The preparation method according to claim 1, characterized in that, The preparation method of the drug-loaded nanoparticles includes: dissolving magnesium nitrate and aluminum nitrate in water to obtain a mixed salt solution; dissolving diclofenac in NaOH solution to obtain a drug alkali solution; mixing the mixed salt solution and the drug alkali solution under nitrogen protection and vigorous stirring at 55-65°C, and reacting the mixture while maintaining the pH of the system at 10-12 with NaOH; finally, subjecting the reaction system to hydrothermal crystallization treatment, collecting the precipitate by centrifugation, washing, and drying to obtain magnesium-aluminum layered double hydroxide nanoparticles loaded with diclofenac.

6. The preparation method according to claim 5, characterized in that, The molar ratio of magnesium nitrate to aluminum nitrate is 1-3:1, and the diclofenac accounts for 1-5% of the mass of the mixed salt.

7. The preparation method according to claim 1, characterized in that, The drug-loaded nanoparticles account for 1-5% of the mass of the aqueous phase, and the concentration of paeoniflorin in the aqueous phase is 1-5 mg / mL.

8. The preparation method according to claim 1, characterized in that, The microfluidic device has an aqueous phase flow rate of 0.3~0.8 mL / min and an oil phase flow rate of 0.5~1.2 mL / min.

9. The preparation method according to claim 1, characterized in that: The conditions for UV crosslinking curing are: wavelength 365 nm, light intensity 15 mW / cm², and irradiation time 1.5 min.

10. The use of a hydrogel microsphere in the preparation of a topical treatment formulation for osteoarthritis, wherein the hydrogel microsphere is prepared according to any one of claims 1-9.

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