Abstract: BACKGROUND: Previous studies have shown that M2 macrophage membrane-modified nanosystems can achieve efficient aggregation in joint and inflammation-related diseases, thereby significantly alleviating inflammation and tissue damage. 
OBJECTIVE: To investigate the therapeutic effect of M2 macrophage membrane-coated black phosphorus nanosheets (denoted as BPNs) combined with near-infrared photothermal stimulation on osteoarthritis. 
METHODS: (1) BPNs were synthesized using a modified liquid-phase exfoliation method. The BPNs were then modified with interleukin-4-induced M2 macrophage membranes to obtain M2 macrophage membrane-coated black phosphorus nanosheets (denoted as M2M@BPNs). The microstructure, hydration particle size, and photothermal properties of M2M@BPNs were characterized. (2) Mouse bone marrow macrophages were cultured in five groups. The control group received no treatment. The lipopolysaccharide group was induced to polarize to the M1 type and then cultured in PBS for 24 hours. In the BPNs group and the M2M@BPNs group, after inducing cell polarization towards the M1 type, BPNs and M2M@BPNs were added respectively and the cells were cultured for 24 hours. In the M2M@BPNs+near-infrared intervention group, after inducing cell polarization towards the M1 type, M2M@BPNs were added and the cells were cultured for 24 hours, during which near-infrared light was irradiated for 180 seconds. After culture, cell viability was detected by CCK-8 assay. The expression of CD86, inducible nitric oxide synthase, CD206, and arginase 1 mRNA was detected by RT-qPCR. Immunofluorescence staining was used to detect CD86 expression. (3) The supernatant of mouse bone marrow macrophages was collected from the lipopolysaccharide group, BPNs group, M2M@BPNs group, and M2M@BPNs+near-infrared intervention group. After centrifugation, the supernatant was used as conditioned medium to culture mouse knee chondrocytes, with untreated chondrocytes as controls. After 24 hours of culture, Alcian blue and safranin O staining were utilized to observe extracellular matrix deposition. RT-qPCR was used to detect the mRNA expression of aggrecan, type II collagen, matrix metalloproteinase 13, and integrin-metalloproteinase 5. Immunofluorescence staining was applied to measure the expression of matrix metalloproteinase 13. (4) The above four groups of conditioned mediums were employed to culture mouse embryonic fibroblasts, with untreated fibroblasts as controls. After 24 hours of culture, RT-qPCR was used to examine the mRNA expression of type I collagen, type III collagen, and α-smooth muscle actin. Immunofluorescence staining was used to detect type I collagen expression. Scratch assay was used to detect cell migration. (5) Thirty C57BL/6J mice were randomly divided into five intervention groups: The sham-operated group (n=6) received no modeling. The osteoarthritis group (n=6) underwent medial meniscus and tibial ligament transection to establish an osteoarthritis model, followed by intra-articular injection of PBS one week later. The BPNs group (n=6) and the M2M@BPNs group (n=6) received intra-articular injections of BPNs and M2M@BPNs, respectively, one week after modeling. The M2M@BPNs+near-infrared intervention group received intra-articular injection of M2M@BPNs one week after modeling, followed by near-infrared light irradiation (3 times) 24 hours later, with treatment once a week. Eight weeks post-operation, tissue samples were collected for hematoxylin-eosin staining of articular cartilage and synovial tissue, safranin O-fast green and toluidine blue staining of articular cartilage, CD86 and CD206 immunofluorescence staining of synovial tissue, type II collagen and matrix metalloproteinase 13 immunofluorescence staining of cartilage tissue, and hematoxylin-eosin staining of major organs. 
RESULTS AND CONCLUSION: (1) M2M@BPNs exhibited an ultrathin nanosheet morphology with an average particle size of (184.76±0.46) nm, stably increasing temperature by 6°C under near-infrared irradiation, and demonstrated good photothermal stability. (2) CCK-8 assays showed that BPNs, M2M@BPNs, and M2M@BPNs+near-infrared intervention did not affect the survival rate of bone marrow macrophages, indicating good cell compatibility. The experimental results showed that M2M@BPNs combined with near-infrared intervention significantly decreased CD86 and inducible nitric oxide synthase expression and increased CD206 and arginase-1 expression, promoting M2 polarization in macrophages. (3) Comprehensive experimental results showed that M2M@BPNs combined with near-infrared conditioned medium could increase the expression of proteoglycans and type II collagen in chondrocytes, decrease the expression of matrix metalloproteinase 13 and integrin-metalloproteinase 5, and promote cartilage formation. M2M@BPNs combined with near-infrared conditioned medium could decrease the expression of type I collagen, type III collagen, and α-smooth muscle actin in fibroblasts, and inhibit fibroblast migration. (4) Comprehensive experimental results showed that M2M@BPNs combined with near-infrared intervention could improve cartilage structure, retain proteoglycans, decrease the expression of CD86 and matrix metalloproteinase 13, increase the expression of CD206 and type II collagen, alleviate synovial hyperplasia and osteoarthritis degeneration, and had good in vivo biocompatibility. (5) The results indicated that M2M@BPNs combined with near-infrared intervention could regulate macrophage polarization, indirectly maintain the homeostasis of chondrocytes and fibroblasts, and thus exert a cartilage-protective effect in osteoarthritis.

Key words: cell membrane modification, black phosphorus nanosheets, photothermal therapy, macrophage polarization, osteoarthritis, matrix metabolism, near-infrared irradiation