Macrophage membrane-encapsulated methacrylated hyaluronic acid hydrogel microspheres and uses thereof

Abstract

The application discloses macrophage membrane-coated methacrylated hyaluronic acid hydrogel microspheres and application, relates to the technical field of drug synthesis, and technical solution points are that a kind of macrophage membrane-coated methacrylated hyaluronic acid hydrogel microspheres EMM@HMs can efficiently neutralize key proinflammatory factors, the proinflammatory factor includes TNF-alpha and IL-1 beta, to relieve joint inflammation.It is good in the curative effect and biocompatibility in OA model, especially its effect in the regulation of macrophage polarization, inhibition of chondrocyte hypertrophy and neutralization of proinflammatory factors.EMM@HMs can not only significantly reduce inflammation by targeting the key link in the pathogenesis of OA, but also has the potential to delay disease progression, providing a new strategy with a breakthrough significance for the treatment of OA.

Description

Technical Field

[0001] This invention relates to the field of drug synthesis technology, and more specifically, to methacrylamide hyaluronic acid hydrogel microspheres encapsulated in macrophage membranes and their applications. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative joint disease characterized by cartilage degeneration, synovitis, bone remodeling, ligament dysfunction, and osteophyte formation. Clinical manifestations primarily include joint pain, functional impairment, and deformity. The etiology of OA is complex, involving multiple factors such as genetic susceptibility, sports injuries, aging, and obesity; despite extensive research, its exact pathogenesis remains incompletely understood. OA can affect various joints, most commonly the knee, hip, hand, and spine. As one of the most prevalent orthopedic diseases, OA currently affects approximately 7.6% of the global population (about 595 million cases) and is on a continuous upward trend, leading to severe lower limb disability and decreased quality of life. With the accelerating aging of the population, the disease burden caused by OA is constantly increasing, becoming a significant global public health challenge.

[0003] The interaction between macrophages and chondrocytes plays a crucial role in the development and progression of osteoarthritis (OA). Infiltrating monocytes can differentiate into M1 or M2 macrophages, mediating pro-inflammatory responses and tissue repair, respectively. Activated M1 macrophages secrete large amounts of pro-inflammatory factors (such as IL-1β and TNF-α), which are highly expressed in OA joints and can accelerate synovial inflammation, promote chondrocyte senescence, and degrade the extracellular matrix (ECM) by inducing matrix metalloproteinases (MMPs). Furthermore, enhanced glycolytic activity in M1 macrophages leads to excessive accumulation of reactive oxygen species (ROS), further promoting the synthesis of inflammatory factors and exacerbating cartilage damage. In contrast, M2 macrophages possess anti-inflammatory and reparative functions, promoting inflammation resolution. Macrophage membranes naturally express multiple receptors capable of adsorbing cytokines in the inflammatory microenvironment, but their expression levels are limited, reducing the efficiency of neutralizing pro-inflammatory factors. To enhance the therapeutic potential of macrophage membranes, we constructed engineered macrophage membranes (EMMs) that overexpress TNF-α and IL-1β receptors to more efficiently bind and neutralize pro-inflammatory factors, thereby alleviating joint inflammation and protecting cartilage.

[0004] Organoid technology has attracted much attention in recent years. Organoids are formed through the directed differentiation of stem cells or progenitor cells, possessing self-renewal and self-organization capabilities. Their three-dimensional structures can mimic key structural and functional characteristics of natural tissues, demonstrating great potential in disease modeling, drug screening, and regenerative medicine. Cartilage organoids can effectively reproduce the extracellular matrix composition and mechanical properties of natural cartilage, and possess corresponding functional advantages, thus providing biologically relevant microenvironmental support for cartilage repair and regeneration. Summary of the Invention

[0005] The purpose of this invention is to provide macrophage membrane-encapsulated methacrylamide hyaluronic acid hydrogel microspheres and their applications. A macrophage membrane-encapsulated methacrylamide hyaluronic acid hydrogel microsphere (EMM@HMs) can efficiently neutralize key pro-inflammatory factors (including TNF-α and IL-1β), thereby relieving joint inflammation.

[0006] The above-mentioned technical objective of this invention is achieved through the following technical solution: methacrylamide hyaluronic acid hydrogel microspheres encapsulated by macrophage membranes, wherein the methacrylamide hyaluronic acid hydrogel microspheres encapsulated by macrophage membranes are EMM@HMs, and their preparation method is as follows:

[0007] S1. Preparation of engineered macrophage cell membranes: IL1R2 and TNFR2 overexpression shuttle plasmid vectors were constructed; the prepared plasmids were transfected into HEK293T cells using Lipofectamine 3000. After culturing for 48 hours, the cell supernatant was filtered through a 0.45 µm filter, followed by centrifugation at 20,000 rpm for 120 minutes at 4°C; the pellet was collected and resuspended, and viral titer was determined by qPCR; macrophages were transfected with lentivirus, and the cells were collected and washed three times with PBS, then resuspended in homogenization buffer containing 75 mM sucrose, 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl, and protease and phosphatase inhibitors. Cells were lysed by mechanical homogenization 20 times; the lysate was centrifuged at 3200 × g for 5 minutes to remove cell debris, the supernatant was collected, and then centrifuged at 20,000 × g for 25 minutes to remove the pellet. Finally, the supernatant was centrifuged at 100,000 × g. Centrifuge at 35 min for 35 min and collect the cell membrane pellet; the concentration of membrane proteins is quantified using a BCA kit.

[0008] S2. Preparation of EMM@HMs: The microfluidic device was set up with an inner needle of 30 G and an outer needle of 27 G. The inner phase was a 150 kDa PBS solution containing 2% HAMA, 0.25% LAP photoinitiator and EMM, and the outer phase was mineral oil containing 3% Span 80 emulsifier. Microsphere droplets were generated under the conditions of an inner phase flow rate of 5 µL / min and an outer phase flow rate of 100 µL / min. The microsphere droplets were cross-linked and cured by irradiation with 405 nm blue light for 15 seconds. The cured microspheres were washed to remove the oil phase and surfactant, and finally stored in PBS at 4°C. The micromorphology of the synthesized microspheres was observed using a bright field microscope, a confocal microscope and a scanning electron microscope (SEM). The particle size distribution was analyzed by a Malvern Mastersizer 2000 and the zeta potential was measured by a Zetasizer Nano ZS90.

[0009] The present invention further describes the application of methacrylamide hyaluronic acid hydrogel microspheres encapsulated in macrophage membranes in the preparation of drugs for treating OA.

[0010] The present invention is further configured such that the effective component of the drug is methacrylamide hyaluronic acid hydrogel microspheres encapsulated in macrophage membranes.

[0011] The present invention is further configured such that the drug is used to regulate macrophage polarization.

[0012] The present invention is further configured such that the drug is used to inhibit chondrocyte hypertrophy.

[0013] The present invention is further configured such that the drug is used to neutralize pro-inflammatory factors.

[0014] In summary, the present invention has the following beneficial effects:

[0015] This invention presents a macrophage membrane-encapsulated methacrylamide hyaluronic acid hydrogel microsphere (EMM@HMs) that can efficiently neutralize key pro-inflammatory factors (including TNF-α and IL-1β), thereby alleviating joint inflammation. Its efficacy and biocompatibility in OA models are excellent, particularly its role in regulating macrophage polarization, inhibiting chondrocyte hypertrophy, and neutralizing pro-inflammatory factors. By targeting key aspects of OA pathogenesis, EMM@HMs not only significantly reduce inflammation but also have the potential to slow disease progression, providing a groundbreaking new strategy for OA treatment. Attached Figure Description

[0016] Figure 1This is a correlation analysis of histological assessment and synovial fluid cytokine levels in an embodiment of the invention. (A) X-ray imaging showed significant narrowing of the medial joint space in OA patients. (B) H&E staining and (C) S&F staining revealed cartilage degeneration characteristics in OA. (D) Synovial fluid ELISA results showed that TNF-α and IL-1β levels gradually increased with the progression of OA. p<0.05, ** p<0.01.

[0017] Figure 2 This document describes the preparation and characterization of EMM@HMs in this embodiment of the invention. (A) Schematic diagram of the construction of IL1R2 and TNFR2 lentiviral vectors. (B) Immunofluorescence showing the expression of IL1R2 and TNFR2 in transfected RAW264.7 macrophages. (C) Western blot confirming increased protein levels of IL1R2 and TNFR2. (D) Bright-field and scanning electron microscopy images of HMs and EMM@HMs. (E) Confocal microscopy showing Did-labeled EMM@HMs. (F) SDS-PAGE analysis verifying successful cell membrane encapsulation. (G) Zeta potential detection indicating changes in the surface charge of EMM@HMs. (H) Analysis of microsphere swelling properties and (I) degradation characteristics. ** p < 0.01.

[0018] Figure 3 This embodiment of the invention demonstrates how EMM@HMs inhibits the hypertrophy of cartilage organoids. (A) Schematic diagram of the cartilage organoid construction and co-culture system. (B) Bright-field microscopy shows the uniform morphology of the cartilage organoids. (C) Alixin blue staining and COL2 immunofluorescence show the synthesis of cartilage matrix in the organoids. (D) qPCR detection of genes related to cartilage matrix synthesis and degradation. (E) Western blot analysis of protein level changes. (F) Immunofluorescence further verifies the expression of cartilage matrix markers. * p<0.05, ** p<0.01, NS indicates no statistical difference.

[0019] Figure 4 In this embodiment of the invention, EMM@HMs regulate macrophage polarization. (A) Immunofluorescence showed that EMM@HMs reduced the expression of the M1 marker CD86 and increased the expression of the M2 marker CD206. (B) Quantitative analysis showed that EMM@HMs promoted the transformation of macrophages to the M2 phenotype. (C) Flow cytometry further confirmed the changes in polarization profile. (D) Statistical results showed that the proportion of M1 decreased and the proportion of M2 increased. (E) ELISA detection showed that EMM@HMs reduced the levels of IL-1β and TNF-α. * p<0.05, ** p<0.01, NS indicates no statistical difference.

[0020] Figure 5This invention demonstrates how EMM@HMs protect joint structures and inhibit inflammation. (A) Flowchart of rat OA modeling, intra-articular drug administration, and subsequent analysis. (B) EMM@HMs reduce IL-1β and TNF-α levels in synovial fluid. (C) X-rays show joint space recovery and reduced osteophyte formation in the treatment group. (D) Quantitative analysis shows a significant improvement in joint space width. (E) Micro-CT results show improvement in osteophyte formation, cartilage damage, and bone degeneration. (F) Micro-CT quantitative analysis. * p < 0.05, ** p < 0.01, NS indicates no statistical difference.

[0021] Figure 6 This is a histological and immunohistochemical assessment of the cartilage-protective effect of EMM@HMs in this embodiment of the invention. (A) S&F staining and (B) H&E staining showed that the OA group had severe cartilage degeneration, while the EMM@HMs group maintained better integrity. (C) OARSI score showed that EMM@HMs significantly reduced cartilage damage. (D) COL2 immunohistochemical results showed preservation of cartilage matrix. (E) COL10 and (F) MMP13 immunohistochemicals showed that EMM@HMs inhibited cartilage hypertrophy and matrix degradation. (G) TRAP staining indicated reduced bone resorption activity. (H) Quantitative results confirmed that EMM@HMs effectively protected cartilage and delayed degeneration. * p<0.05, ** p<0.01, NS indicates no statistical difference.

[0022] Figure 7 In this embodiment of the invention, EMM@HMs alleviated synovial inflammation and regulated macrophage polarization in osteoarthritis (OA). (A) H&E staining showed significant synovial thickening and inflammatory cell infiltration in the OA group, while EMM@HMs significantly improved the pathological manifestations. (B) Synovitis score results. (C) CD31 immunohistochemistry showed increased angiogenesis in the OA group, and EMM@HMs significantly inhibited its expression. (D) CD86 and (E) CD206 immunohistochemistry showed that EMM@HMs reduced the proportion of M1 macrophages and increased the proportion of M2 macrophages. (F) Quantitative analysis results of CD31, CD86, and CD206. * p<0.05, ** p<0.01, NS indicates no statistical difference.

[0023] Figure 8This is a transcriptomic analysis of EMM@HMs treatment for OA in this embodiment of the invention. (A) DEGs heatmap shows that the EMM@HMs treatment group showed significant transcriptional changes related to cartilage repair and ECM remodeling compared to the OA group. (B) GO enrichment analysis suggests enhanced ECM tissue and chondrocyte differentiation pathways. (C) Reactome pathway analysis further confirms key signaling pathways. (D) GSEA shows significant enrichment of cartilage repair-related pathways. (E) Heatmap shows upregulation of key cartilage differentiation genes such as COL2A1 and SOX9. (F) SOX9 immunohistochemistry further verifies enhanced cartilage repair. Detailed Implementation

[0024] The following is in conjunction with the appendix Figure 1-8 The present invention will be described in further detail below.

[0025] Example:

[0026] The methacrylamide hyaluronic acid hydrogel microspheres encapsulated in the macrophage membrane are EMM@HMs, and their preparation method is as follows:

[0027] S1. Preparation of engineered macrophage cell membranes: IL1R2 and TNFR2 overexpression shuttle plasmid vectors were constructed; the prepared plasmids were transfected into HEK293T cells using Lipofectamine 3000. After culturing for 48 hours, the cell supernatant was filtered through a 0.45 µm filter, followed by centrifugation at 20,000 rpm for 120 minutes at 4°C; the pellet was collected and resuspended, and viral titer was determined by qPCR; macrophages were transfected with lentivirus, and the cells were collected and washed three times with PBS, then resuspended in homogenization buffer containing 75 mM sucrose, 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl, and protease and phosphatase inhibitors. Cells were lysed by mechanical homogenization 20 times; the lysate was centrifuged at 3200 × g for 5 minutes to remove cell debris, the supernatant was collected, and then centrifuged at 20,000 × g for 25 minutes to remove the pellet. Finally, the supernatant was centrifuged at 100,000 × g. Centrifuge at 35 min for 35 min and collect the cell membrane pellet; the concentration of membrane proteins is quantified using a BCA kit.

[0028] S2. Preparation of EMM@HMs: The microfluidic device was set up with an inner needle of 30 G and an outer needle of 27 G. The inner phase was a 150 kDa PBS solution containing 2% HAMA, 0.25% LAP photoinitiator and EMM, and the outer phase was mineral oil containing 3% Span 80 emulsifier. Microsphere droplets were generated under the conditions of an inner phase flow rate of 5 µL / min and an outer phase flow rate of 100 µL / min. The microsphere droplets were cross-linked and cured by irradiation with 405 nm blue light for 15 seconds. The cured microspheres were washed to remove the oil phase and surfactant, and finally stored in PBS at 4°C. The micromorphology of the synthesized microspheres was observed using a bright field microscope, a confocal microscope and a scanning electron microscope (SEM). The particle size distribution was analyzed by a Malvern Mastersizer 2000 and the zeta potential was measured by a Zetasizer Nano ZS90.

[0029] The methacrylamide-encapsulated hyaluronic acid hydrogel microspheres encapsulated in macrophage membranes showed certain effects in regulating macrophage polarization, inhibiting chondrocyte hypertrophy, and neutralizing pro-inflammatory factors. The following are the results of their efficacy and biocompatibility assessment in an OA model:

[0030] The correlation between synovial inflammatory factors and cartilage degeneration in OA patients:

[0031] Anteroposterior and lateral X-rays showed that the patient had typical medial compartment osteoarthritis, characterized by significant narrowing of the medial joint space compared to the lateral side. Figure 1 (A). Hematoxylin-eosin (H&E) and safranin O-fast green (S&F) staining of decalcified cartilage sections revealed varying degrees of cartilage structural damage. Figure 1 (B, C). The degree of cartilage damage was assessed based on the Osteoarthritis Research Institute (OARSI) scoring system. Enzyme-linked immunosorbent assay (ELISA) revealed that the levels of TNF-α and IL-1β in synovial fluid gradually increased with the progression of osteoarthritis (OA). Figure 1 These results indicate that elevated levels of pro-inflammatory cytokines (IL-1β, TNF-α) in synovial fluid are closely associated with OA cartilage degeneration.

[0032] Construction and characterization of EMM@HMs:

[0033] To enhance the inflammatory factor neutralization capacity of macrophage membranes, we first constructed a lentiviral vector with high expression of TNF-α and IL-1β receptors. Figure 2 The receptor (A) was transfected into RAW264.7 macrophages. Immunofluorescence and Western blot confirmed a significant upregulation of receptor expression. Figure 2 (B, C)

[0034] Morphological observation showed that HAMA microspheres (HMs) had smooth and transparent surfaces under scanning electron microscopy (SEM), while EMM@HMs encapsulated with macrophage membranes had irregular, rough, and opaque surfaces. Figure 2 (D). Cell membrane labeling was performed using the lipophilic fluorescent dye Did, and laser confocal microscopy clearly showed the localization of Did-labeled EMM@HMs. Figure 2 (E). Electrophoresis and Coomassie brilliant blue staining further confirmed the presence of membrane proteins in the microspheres. Figure 2 (F). Zeta potential testing showed that the surface potential of HMs was approximately -8 mV, while that of EMM@HMs dropped to approximately -17 mV, indicating that the surface charge properties of the microspheres changed after film encapsulation. Figure 2 (G). Functional testing showed that both HMs and EMM@HMs possessed excellent water absorption capacity, and the swelling rate tended to stabilize after 3 hours. Figure 2 In the degradation experiment, both showed similar mass loss trends (H); Figure 2 (I). These results demonstrate that EMM@HMs have a stable structure and successfully encapsulate membrane proteins.

[0035] EMM@HMs inhibited M1-conditioned medium (M1-CM)-induced chondroid organoid hypertrophy:

[0036] To evaluate the role of EMM@HMs in an OA environment, we established a cartilage organoid model ( Figure 3 (A). The obtained organoids appear as uniform spheres under a bright-field microscope. Figure 3 (B), Alixin blue staining and immunofluorescence analysis confirmed the expression of the cartilage-specific marker COL2 ( Figure 3 Functional assays showed that, compared with the M0 conditioned medium (M0-CM) group, the M1-CM group significantly inhibited the expression of cartilage marker genes (COL2A1, ACAN, SOX9) and upregulated the expression of cartilage degradation-related genes (COL1A1, COL10A1, RUNX2). EMM@HMs treatment partially restored the expression of cartilage marker genes and inhibited the upregulation of degradation genes. Figure 3 (D). Western blotting and immunofluorescence analysis further validated the above results. Figure 3 (E, F). In summary, EMM@HMs can promote cartilage matrix synthesis and inhibit cartilage degradation in an inflammatory microenvironment, thereby exerting a protective effect on cartilage organoids.

[0037] EMM@HMs regulate macrophage polarization:

[0038] We further evaluated the effects of EMM@HMs on macrophage polarization. Immunofluorescence staining results showed that M1 conditioned medium (M1-CM) treatment enhanced CD86 expression and decreased CD206 expression, indicating enhanced M1 polarization; while EMM@HMs treatment decreased CD86 expression and increased CD206 expression, thereby promoting M2 polarization. Figure 4 (A, B). Flow cytometry analysis further confirmed that M1-CM treatment led to an increase in the proportion of M1 macrophages and a decrease in the proportion of M2 macrophages, while EMM@HMs reversed this trend. Figure 4 (C, D). Furthermore, ELISA analysis showed that M1-CM treatment significantly increased IL-1β and TNF-α levels, while EMM@HMs effectively reduced these inflammatory factors (C, D). Figure 4 These results indicate that M1-CM promotes macrophage polarization toward the M1 type, while EMM@HMs can inhibit this effect and promote M2 polarization, thereby improving the inflammatory microenvironment.

[0039] EMM@HMs delay cartilage degeneration and alleviate synovitis:

[0040] A rat OA animal model was established by anterior cruciate ligament transection (ACLT) combined with medial meniscus transection (MMT), and rats were treated with HMs, EMMs, and EMM@HMs, respectively. Figure 5 (A). ELISA results showed that, compared with the normal control (NC), the levels of IL-1β and TNF-α in the synovial fluid of the OA model group were significantly increased; HMs, EMMs, and EMM@HMs could all reduce their levels, among which EMM@HMs had the most significant anti-inflammatory effect. Figure 5 (B) Imaging evaluation showed significant narrowing of the joint space in the OA group; in contrast, EMM and EMM@HMs treatments restored joint space width ( Figure 5 (C, D). Micro-CT three-dimensional reconstruction results showed osteophyte formation and trabecular bone structure destruction in the OA group; after EMM@HMs treatment, bone volume fraction (BV / TV) and trabecular thickness (Tb.Th) increased, while osteophyte volume and trabecular separation (Tb.Sp) decreased. Figure 5 E, F). Histological and immunohistochemical results further confirmed the protective effect of EMM@HMs. S&F and H&E staining showed severe cartilage loss and matrix degradation in the OA group, while EMM@HMs maintained cartilage integrity and significantly reduced OARSI scores ( ). Figure 6 (A–C). Immunohistochemical results showed that EMM@HMs could enhance the expression of the cartilage matrix marker COL2 and inhibit the expression of COL10 and MMP13. Figure 6D–F). TRAP staining results indicated that bone resorption activity was enhanced in the OA group, while EMM@HMs treatment effectively reduced bone resorption levels (D–F). Figure 6 (G, H).

[0041] In the synovial tissue, the OA group showed synovial thickening and significant inflammatory cell infiltration, while EMM@HMs treatment significantly alleviated the above pathological changes. Figure 7 (A, B). Immunohistochemical analysis showed an increase in the number of CD31 and CD86 positive cells in the OA group (suggesting enhanced angiogenesis and M1 polarization), while the number of CD206 positive cells (M2 macrophages) decreased. EMM@HMs treatment reduced the proportion of CD31 and CD86 positive cells while increasing the number of CD206 positive cells, indicating that it can inhibit angiogenesis, improve inflammatory response, and restore immune balance. Figure 7 (C–F). In summary, EMM@HMs can effectively alleviate joint inflammation, delay cartilage degeneration, inhibit bone resorption, and regulate synovial immune balance, highlighting their potential as a novel treatment strategy for OA.

[0042] Key molecular pathways by which EMM@HMs regulate cartilage repair and chondrocyte differentiation:

[0043] To further explore the molecular mechanisms of EMM@HMs in OA treatment, we performed transcriptomic and bioinformatics analyses. The experiment was divided into three groups: the NC group (normal control), the OA group, and the EMM@HMs treatment group. The differentially expressed gene (DEG) heatmap showed significant transcriptional differences between the OA group and the EMM@HMs treatment group. Figure 8 (A). GO analysis and Reactome pathway enrichment results ( Figure 8 (B, C) showed that EMM@HMs significantly upregulated functional pathways related to extracellular matrix (ECM) structure organization, collagen binding, and chondrocyte differentiation, suggesting their key role in promoting cartilage repair. Further gene set enrichment analysis (GSEA) revealed that pathways closely related to cartilage repair (such as collagen synthesis, cartilage regeneration, and ECM remodeling) were significantly enriched in the EMM@HMs treatment group. Figure 8 (D). Gene expression analysis showed that cartilage-specific genes such as COL2A1 and SOX9 were significantly upregulated in the EMM@HMs treatment group. Figure 8 Immunohistochemical analysis further validated this finding, indicating that EMM@HMs treatment enhanced the protein levels of COL2 and SOX9, with SOX9 expression being particularly significant in the treatment group. Figure 6 D, Figure 8(F). In summary, EMM@HMs can promote cartilage repair and functional recovery by activating molecular pathways related to ECM remodeling and chondrocyte differentiation, thereby revealing their key mechanism of action in OA treatment.

[0044] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. Methacrylamide hyaluronic acid hydrogel microspheres encapsulated in macrophage membranes, characterized by: The methacrylamide hyaluronic acid hydrogel microspheres encapsulated in the macrophage membrane are EMM@HMs, and their preparation method is as follows: S1. Preparation of engineered macrophage membrane EMM: IL1R2 and TNFR2 overexpression shuttle plasmid vectors were constructed; the prepared plasmids were transfected into HEK293T cells using Lipofectamine 3000. After culturing for 48 hours, the cell supernatant was filtered through a 0.45 µm filter, followed by centrifugation at 20,000 rpm for 120 minutes at 4°C; the pellet was collected and resuspended, and viral titer was determined by qPCR; macrophages were transfected with lentivirus, and the cells were collected and washed three times with PBS, then resuspended in homogenization buffer containing 75 mM sucrose, 20 mM Tris-HCl, 2 mM MgCl2, 10 mM KCl, and protease and phosphatase inhibitors. Cells were lysed by mechanical homogenization 20 times; the lysate was centrifuged at 3200 × g for 5 minutes to remove cell debris, the supernatant was collected, and then centrifuged at 20,000 × g for 25 minutes to remove the pellet. Finally, the supernatant was centrifuged at 100,000 × g. Centrifuge at 35 min for 35 min and collect the cell membrane pellet; the concentration of membrane proteins is quantified using a BCA kit. S2. Preparation of EMM@HMs: The microfluidic device size was set with an inner needle of 30 G and an outer needle of 27 G. The inner phase was a PBS solution containing 2% 150 kDa methacryloyl hyaluronic acid, 0.25% LAP photoinitiator, and EMM. The outer phase was mineral oil containing 3% Span 80 emulsifier. Microsphere droplets were generated under the conditions of an inner phase flow rate of 5 µL / min and an outer phase flow rate of 100 µL / min. The microsphere droplets were crosslinked and cured by irradiation with 405 nm blue light for 15 seconds. The cured microspheres were washed to remove the oil phase and surfactant, and finally stored in PBS at 4°C.

2. The use of the macrophage membrane-encapsulated methacrylamide hyaluronic acid hydrogel microspheres according to claim 1 in the preparation of a drug for treating osteoarthritis.

3. The application according to claim 2, characterized in that: The active ingredient of the drug is methacrylamide hyaluronic acid hydrogel microspheres encapsulated in macrophage membranes.

4. The application according to claim 3, characterized in that: The drug is used to regulate macrophage polarization.

5. The application according to claim 3, characterized in that: The drug is used to inhibit chondrocyte hypertrophy.

6. The application according to claim 3, characterized in that: The drug is used to neutralize pro-inflammatory factors.

Images