炎症微环境响应性仿高密度脂蛋白纳米药物的构建及其体外抗动脉粥样硬化作用评价

Construction of an inflammatory microenvironment-responsive HDL-mimetic nanoparticle and evaluation of its in vitro anti-atherosclerotic activity

目的基于活性响应性材料,采用仿生策略构建具有炎症响应性功能、抗动脉粥样硬化作用的仿高密度脂蛋白(high-density lipoprotein, HDL)纳米药物。方法通过化学键合的方式将对羟甲基苯硼酸频那醇酯(phenylboronic acid pinacol ester, PBAP)与β-环糊精(β-cyclodextrin,β-CD)反应,合成具有氧化敏感性的材料OXbCD。采用自组装纳米沉淀法和共挤出法制备仿HDL纳米粒(HDL-mimetic nanoparticles, mHDL NP)并通过红外光谱、核磁、电镜等方法对材料及纳米粒进行表征。选用RAW264.7细胞系进行细胞实验,利用流式细胞仪和激光共聚焦考察细胞对纳米粒吞噬作用,并通过ELISA测定细胞培养上清炎症因子TNF-α和MCP-1,评价纳米粒对细胞产生炎症因子的影响;选用DCFH-DA活性氧探针检测纳米粒对细胞活性氧产生水平的改变;并通过多种方法评价纳米粒对细胞胆固醇外排和泡沫细胞形成的影响。结果光谱学分析表明约5个PBAP键合于每个β-CD分子;电镜和粒径分布结果表明所制备的纳米粒为规则类球形,粒径分布均匀,平均粒径约200 nm。细胞实验证明RAW264.7细胞对纳米粒的吞噬具有时间和剂量依赖性,且能显著抑制炎症因素刺激下细胞炎症因子的分泌和活性氧的产生(P<0.05),具有明显的抗炎和抗氧化作用;mHDL NP能明显促进细胞胆固醇外排(P<0.05),且细胞外排率与细胞和mHDL NP孵育的时间以及mHDL NP的浓度具有相关性;mHDL NP亦能显著抑制巨噬细胞泡沫化的进程。结论基于活性氧响应性材料OXbCD成功合成mHDL NP,该NP同时具有抗氧化和胆固醇逆转运的作用,并在细胞水平证明该纳米粒具有抗动脉粥样硬化的能力。

Objective To construct an inflammation-responsive high-density lipoprotein(HDL)-mimetic nanoparticle with anti-atherosclerotic activity using a bioinspired strategy based on a oxidation-responsive material. Methods 4-(hydroxymethyl) phenylboronic acid pinacol ester(PBAP) was chemically conjugated to β-cyclodextrin(β-CD) to synthesize a reactive oxygen species(ROS)-sensitive material. Nanoprecipitation and coextrusion methods were used to prepare HDL-mimetic nanoparticles(mHDL NP). Spectroscopy was employed to verify the structure of the synthesized material, and microscopy was utilized to observe the morphology of the nanoparticles. We observed phagocytosis of the nanoparticles by murine RAW264.7 macrophages using flow cytometry and confocal microscopy, and examined the release of the inflammatory factors tumor necrosis factor-α(TNF-α) and monocyte chemoattractant protein-1(MCP-1) in the supernatant by the macrophages cultured in the presence of the nanoparticles using ELISA. We also used a fluorescent ROS probe DCFH-DA to detect the changes of ROS generated in RAW264.7 macrophages in response to the nanoparticles. Different methods were used to assess the effects of the nanoparticles on cholesterol efflux and foam cell formation in RAW264.7 cells. Results Spectroscopy analysis showed that about 5 PBAP units were conjugated to each β-CD molecule. The prepared nanoparticles had a well-defined spherical shape with a narrow size distribution and a mean diameter of about 200 nm. In vitro experiments showed that RAW264.7 cells phagocytosed the nanoparticles in a time-and dose-dependent manner. mHDL NP significantly inhibited the secretion of the inflammatory factors and ROS production by RAW264.7 cells in response to inflammatory stimulation(P<0.05), which demonstrated potent anti-inflammatory and antioxidant activities of the nanoparticles. mHDL NP time-and concentration-dependently promoted cholesterol efflux(P<0.05) and significantly inhibited foam cell formation in RAW264.7 macrophages. Conclusion We successfully synthesize HDL-mimetic nanoparticles with antioxidant and reverse cholesterol transport capacities. Our preliminary in vitro cellular evaluations demonstrate obvious anti-atherosclerotic activity of the synthesized nanoparticles.