目的 利用大肠杆菌表达P-选择素靶向肽 (P-selectin banding peptide,PBP) 与载脂蛋白A1 (apolipoprotein A1,ApoA1) 的融合蛋白PBP-ApoA1,将其制备获得包载姜黄素 (curcumin,Cur) 的重组高密度脂蛋白 (high-density lipoprotein,HDL) PA-rHDL-Cur,用于动脉粥样硬化部位的靶向递药。方法 采用谷胱甘肽巯基转移酶 (glutathione S-transferase,GST) 标签共表达的策略实现PBP-ApoA1的可溶性表达,将PBP-ApoA1纯化后与磷脂、胆固醇采用薄膜水化法包载姜黄素制备得到PA-rHDL-Cur载药纳米粒。利用粒度仪、紫外分光光度计表征其理化性质,透析法分析其释药稳定性。体外细胞实验考察PA-rHDL-Cur的生物相容性和细胞摄取效率,通过血小板黏附实验验证PA-rHDL对活化血小板的靶向性。探究PA-rHDL-Cur对RAW264.7巨噬细胞的抗氧化功能、胆固醇泵出效果和减少氧化型高密度脂蛋白的摄取能力。结果 通过摇瓶发酵和纯化PBP-ApoA1产量达1.33 g·L-1,PA-rHDL-Cur大小均一,粒径为 (165.3±29.6) nm,Zeta电位 (-2.19±1.28) mV,生物相容性较好。体外实验证实,PA-rHDL-Cur可靶向到动脉粥样硬化病灶部位,释放Cur降低病灶部位泡沫细胞内的氧化应激,显著提高Cur的生物利用度。PA-rHDL本身含有的ApoA1可通过胆固醇逆向转运促进胆固醇外排,进而延缓动脉粥样硬化的发展进程。结论 本研究设计的仿生重组高密度脂蛋白纳米递药系统为开发抗动脉粥样硬化的新型纳米递送体系提供了设计思路与理论依据。
Abstract
OBJECTIVE To express recombinant protein PBP-ApoA1 by fusion of P-selectin banding peptide (PBP) and apolipoprotein (ApoA1) by Escherichia coli, and PBP-ApoA1 was applied to further prepare a recombinant high-density lipoprotein (HDL) loading with curcumin (Cur), named PA-rHDL-Cur, for the effective treatment of atherosclerosis (AS) by targeting to activated platelets. METHODS The soluble expression of PBP-ApoA1 was achieved using a co-expression strategy with glutathione S-transferase (GST) tag. The purified PBP-ApoA1, phospholipid and cholesterol were encapsulated with Cur to prepare PA-rHDL-Cur by thin-film hydration method. The physicochemical properties of PA-rHDL-Cur were characterized by particle size analyzer and UV spectrophotometer, while the release stability was evaluated using dialysis method. Cell viability and cellular uptake efficiency of PA-rHDL-Cur were assessed in vitro. Platelet adhesion experiments were conducted to confirm the targeting ability of PA-rHDL towards activated platelets. Furthermore, the antioxidant activity, cholesterol efflux effect, and reduction in oxidized high-density lipoprotein uptake capacity of RAW264.7 macrophages treated with PA-rHDL-Cur were investigated. RESULTS The yield of PBP-ApoA1 obtained by shake flask fermentation and purification was 1.3 g·L-1. The resulting PA-rHDL-Cur exhibited uniform particle size with an average diameter of (165.3±29.6) nm and the Zeta potential of (-2.19±1.28) mV. The biocompatibility of this drug delivey system was satisfactory. In vitro cell experiments demonstrated that PA-rHDL-Cur effectively targeted atherosclerotic lesions, releasing curcumin to reduce oxidative stress within foam cells at the lesion site, significantly enhancing the bioavailability of Cur. Additionally, the presence of ApoA1 in PA-rHDL facilitated cholesterol efflux, thereby delaying the progression of atherosclerosis. CONCLUSION This design of biomimetic recombinant high-density lipoprotein nano-drug delivery system provides a new approach and theoretical basis for the development of novel nanocarriers against atherosclerosis.
关键词
载脂蛋白A1 /
重组高密度脂蛋白 /
融合蛋白 /
动脉粥样硬化 /
靶向递药
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Key words
apolipoprotein A1 /
recombinant high-density lipoprotein /
fusion protein /
atherosclerosis /
targeted drug delivery
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脚注
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基金
国家自然科学基金项目资助(32101072); 江苏省自然科学基金项目资助(BK20210473)
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