EVs@ZIF-8载药体系在成骨细胞自噬抑制环境下促进MC3T3-E1早期成骨分化OA

BACKGROUND:Autophagy is an essential step in the mineralization of osteoblasts in vitro,and dysregulation of autophagy activity can affect the mineralization ability of osteoblasts.Exosomes can promote osteogenesis by regulating autophagy to eliminate harmful substances.However,how to stably transport and release exosomes remains challenging. OBJECTIVE:To investigate whether the successfully synthesized extracellular vesicles(EVs)@zeolitic imidazolate framework-8(ZIF-8)drug-loaded system has an osteogenic effect and whether autophagy may be involved in this process. METHODS:(1)Material synthesis:The plasma exosomes were extracted by an extraction and purification kit,and the successful extraction of exosomes was verified by western blot.ZIF-8 was synthesized via a hydration method,followed by the one-pot synthesis of the EVs@ZIF-8 drug-loaded system.The nano-drug-loaded system was characterized and tested using scanning electron microscopy and X-ray diffraction.(2)Materials promote osteogenesis:MC3T3-E1 cells were co-cultured with different concentrations of EVs@ZIF-8 and exosome groups,and cell proliferation and viability assays were conducted to verify the activity of exosomes and screen for the optimal concentration of EVs@ZIF-8.Subsequently,cells were co-cultured with blank,ZIF-8,and EVs@ZIF-8 groups.After osteogenic induction,alkaline phosphatase detection was used to verify the early osteogenic differentiation ability of MC3T3-E1 promoted by the drug-loaded system.The expression levels of osteoprotegerin,Runx2,and type Ⅰ collagen genes in the samples were measured.(3)Materials promote osteogenesis via autophagy:MC3T3-E1 cells were co-cultured with different concentrations of autophagy inhibitor 3-methyladenine,and the optimal concentration of 3-methyladenine was determined through cell proliferation and viability assays.Subsequently,cells were co-cultured with 3-methyladenine,3-methyladenine+ZIF-8,and 3-methyladenine+EVs@ZIF-8 groups.After osteogenic induction,alkaline phosphatase detection was performed to verify the early osteogenic differentiation ability of MC3T3-E1 promoted by the drug-loaded system. RESULTS AND DISCUSSION:(1)Under scanning electron microscopy,EVs@ZIF-8 exhibited a uniform spherical encapsulation structure with a particle size of(232.5±22.0)nm.(2)The results of cell proliferation and viability experiments indicated that 50 μg/mL EVs@ZIF-8 significantly promoted the proliferation of MC3T3-E1 cells,while 5.0 mmol/L 3-methyladenine exerted its function without excessively inhibiting cell proliferation.Therefore,the aforementioned concentrations were selected for subsequent cell experiments.(3)After osteogenic induction,the alkaline phosphatase activity and osteogenic gene expression in the EVs@ZIF-8 group were higher than those in the other two groups(P＜0.05).After the addition of 3-methyladenine,an autophagy inhibitor,the proliferation,alkaline phosphatase activity,and osteogenic gene expression in the EVs@ZIF-8 group were all still higher than those in the other two groups.These results indicated that cell proliferation was inhibited in the presence of autophagy inhibitors and EVs@ZIF-8 partially antagonized this inhibitory effect,suggesting that autophagy may be involved in the process by which the EVs@ZIF-8 drug delivery system promotes osteogenic differentiation of cells.