【摘要】 目的 制备巨噬细胞膜包覆的载姜黄素仿生微泡（C@MMBs），探讨其体外靶向能力及对巨噬细胞内活性氧（ROS）的清除效果。方法 采用差速离心法提取RAW264.7巨噬细胞膜，运用旋转蒸发结合机械振荡法制备载姜黄素脂质微泡（C@MBs），并通过超声辅助膜融合技术制备C@MMBs。通过光学显微镜观察其形态，马尔文粒径分析仪测定粒径及Zeta电位；采用SDS-PAGE考马斯亮蓝染色检测C@MMBs膜蛋白保留情况，紫外分光光度法计算姜黄素包封率；采用CCK-8法评价生物相容性。将RAW264.7巨噬细胞分别与C@MMBs和C@MBs孵育4 h（C@MMBs组和C@MBs组），流式细胞术检测细胞的平均荧光强度，评估细胞在体外对C@MMBs和C@MBs的摄取能力；另将RAW264.7巨噬细胞分别与C@MMBs、C@MBs、无血清培养基共孵育4 h，随后加入H₂O₂孵育2 h以建立氧化应激模型，分别对应C@MMBs+H₂O₂组、C@MBs+H₂O₂组及H₂O₂组，采用DCFH-DA荧光探针检测各组细胞内ROS水平，并对平均荧光强度进行定量分析。结果 成功制备结构稳定的C@MMBs，光学显微镜下其呈均匀分布的规则球形结构。C@MMBs粒径及Zeta电位分别为（946.30±38.50）nm、（-19.860±0.122）mV，较C@MBs[（882.50±14.44）nm、（-7.196±2.819）mV]略有变化；SDS-PAGE考马斯亮蓝染色证实其保留了巨噬细胞膜的主要蛋白成分，提示细胞膜成功包覆。紫外分光光度法显示，姜黄素包封率最高可达（81.07±0.66）%。CCK-8法结果显示，C@MMBs在140μg/ml磷脂浓度以下对RAW264.7巨噬细胞增殖无抑制作用。流式细胞术检测显示，C@MMBs组巨噬细胞平均荧光强度显著高于C@MBs组（P<0.01），提示仿生修饰增强了微泡对巨噬细胞的同源靶向能力。在H₂O₂诱导的氧化应激模型中，C@MMBs+H₂O₂组平均荧光强度显著低于C@MBs+H₂O₂组（P<0.05），表明C@MMBs显著降低了细胞内ROS水平，抗氧化效果优于C@MBs。结论 本实验成功制备了C@MMBs，其在体外模型中表现出良好的稳定性和生物相容性，可增强巨噬细胞摄取能力和ROS清除效果。更多还原

【Abstract】 Objective To prepare macrophage membrane curcumin-loaded biomimetic microbubbles（C@MMBs）and to investigate their targeting ability and scavenging effect of reactive oxygen species（ROS） in macrophages in vitro.Methods The RAW264.7 macrophage membranes were extracted using differential centrifugation.Curcumin-loaded lipid microbubbles（C@MBs） were prepared via rotary evaporation combined with mechanical oscillation,and C@MMBs were subsequently constructed using ultrasound-assisted membrane fusion technique.The morphology of the C@MMBs was observed by optical microscope,and the particle size and Zeta potential were measured using a Malvern particle size analyzer. The retention of C@MMBs membrane proteins was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDSPAGE） with Coomassie brilliant blue staining. The encapsulation efficiency of curcumin was calculated using ultraviolet spectrophotometry.The biocompatibility was evaluated using a CCK-8 assay.The RAW264.7 macrophages were co-incubated with the C@MMBs and C@MBs for 4 h,respectively（C@MMBs group and C@MBs group）.The mean fluorescence intensity was then measured by flow cytometry to assess the cellular uptake capability of C@MMBs and C@MBs in vitro. To establish an oxidative stress model,the RAW264.7 macrophages were co-incubated with C@MMBs,C@MBs,and serum-free medium for4 h,respectively,then followed by H₂O₂ incubation for 2 h.The groups were designated as C@MMBs+H₂O₂ group,C@MBs+H₂O₂ group,and H₂O₂ group,respectively.Intracellular ROS levels were detected using the DCFH-DA fluorescent probes,and the mean fluorescence intensity was quantitatively analyzed.Results C@MMBs with stable structure were successfully prepared,appearing as regular spherical structures and uniform distribution under light microscope.The particle size and Zeta potential of C@MMBs were（946.30±38.50）nm,（-19.860±0.122）mV,respectively,showing slight variations compared to C@MBs[（882.50±14.44）nm,（-7.196±2.819）mV].SDS-PAGE with Coomassie brilliant blue staining confirmed the retention of major macrophage membrane protein components,indicating successful membrane coating.Ultraviolet spectrophotometry revealed that the encapsulation efficiency of curcumin reached a maximum of（81.07±0.66）%.CCK-8 assays demonstrated that C@MMBs had no significant inhibitory effect on the proliferation of RAW264.7 macrophages at phospholipid concentrations below 140 μg/ml.Flow cytometry detected that the mean fluorescence intensity of the C@MMBs group was significantly higher than that of the C@MBs group（P<0.01）,suggesting that biomimetic modification enhanced the homologous targeting ability of the microbubbles.In the H₂O₂-induced oxidative stress model,the mean fluorescence intensity of the C@MMBs+H₂O₂ group was significantly lower than that of the C@MBs+H₂O₂ group（P<0.05）,indicating a significant reduction in intracellular ROS levels and superior antioxidant efficacy compared to C@MBs.Conclusion This study successfully prepare C@MMBs characterized by good stability and biocompatibility in the in vitro models.This could enhance macrophages’ uptake capability and the scavenging effect of ROS levels.更多还原