巨噬细胞膜仿生纳米给药系统制备及性能

Preparation and properties of macrophage membrane biomimetic nano drug delivery system

摘要:

心血管疾病严重威胁人类生命健康，动脉粥样硬化是引起心血管疾病的主要病理学基础，然而现有治疗动脉粥样硬化的药物易引发诸多不良反应，探索新的治疗动脉粥样硬化的策略十分必要。该文首先以聚酰胺-胺（PAMAM）树枝状聚合物，乙二胺核，5.0代溶液（PAMAM G5.0）作为基体，将β-环糊精（β-CD）修饰在PAMAM G5.0表面，构成CD-G5载药腔。然后，将疏水性抗炎药姜黄素（CUR）作为模型药物负载于CD-G5内，得到负载CUR的纳米粒子（CURNPs）。最后，在CURNPs外包裹具有免疫逃逸反应和药物缓释作用的巨噬细胞膜（MM），制备了仿生纳米给药系统（MM@CURNPs）。采用FTIR、1HNMR对CD-G5进行结构表征；通过TEM和纳米粒度分布仪对MM@CURNPs的微观形貌和粒径分布进行测定，基于药物释放实验和体外细胞毒性、巨噬细胞摄取实验，进行MM@CURNPs的缓释性能和体外活性评价。结果表明，CURNPs经MM包覆后，MM@CURNPs平均粒径（162.2 nm）增大13.9 nm；MM@CURNPs放置1 d时平均粒径为162.2 nm，10 d时平均粒径为238.6 nm；CURNPs载药率9.60%，MM@CURNPs体外释药速率较CURNPs慢，72 h累积释放量达到约50%；MM@CURNPs比PAMAM G5.0和CURNPs的毒性小，即使在浓度1 mmol/L下，小鼠单核巨噬细胞白血病细胞（Raw 264.7细胞）仍具有90%的活性；MM上保留有巨噬细胞固有膜蛋白和功能，可以帮助MM@CURNPs具备巨噬细胞免疫逃逸功能，4 h时，Raw 264.7细胞对荧光标记的MM@CURNPs粒子摄取的平均荧光强度为72，Raw 264.7细胞对荧光标记的CURNPs粒子摄取的平均荧光强度为80。

Abstract:

Cardiovascular disease is a serious threat to human life and health, atherosclerosis is the main pathological basis of cardiovascular disease, however, the existing drugs for the treatment of atherosclerosis are prone to cause many adverse reactions, and it is necessary to explore new strategies for the treatment of atherosclerosis. In this paper, firstly, a polyamide-amine (PAMAM) dendritic polymer with ethylenediamine nucleus, 5.0 generation solution (PAMAM G5.0) was used as a substrate, and β-cyclodextrin (β-CD) was modified on the surface of PAMAM G5.0 to constitute a CD-G5 drug-carrying cavity. Then, the hydrophobic anti-inflammatory drug curcumin (CUR) was loaded as a model drug within CD-G5 to obtain CUR-loaded nanoparticles (CURNPs). Finally, a bionic nanodrug delivery system (MM@CURNPs) was prepared by wrapping macrophage membranes (MM) with immune escape response and drug slow release outside the CURNPs. FTIR and 1HNMR were used to characterize the structure of CD-G5; the microscopic morphology and particle size distribution of MM@CURNPs were determined by TEM and nano-particle size distribution meter, and the evaluation of the slow-release performance and in vitro activity of MM@CURNPs was carried out based on the drug release assay and in vitro cytotoxicity and macrophage uptake assay. The results showed that the average particle size (162.2 nm) of MM@CURNPs increased by 13.9 nm after CURNPs were coated by MM; the average particle size of MM@CURNPs was 162.2 nm when placed for 1 d, and the average particle size of MM@CURNPs was 238.6 nm when placed for 10 d; the drug loading rate of CURNPs was 9.60%, and the in vitro drug release rate of MM@CURNPs was slower than that of CURNPs, and the cumulative release reached about 50% at 72 h; MM@CURNPs were less toxic than PAMAM G5.0 and CURNPs, with 90% activity in mouse monocyte macrophage leukemia cells (Raw 264.7 cells) even at a concentration of 1 mmol/L; MM retained macrophage-intrinsic membrane proteins and functions on the MM, which could help MM@CURNPs to with macrophage immune escape function, and at 4 h, the average fluorescence intensity of Raw 264.7 cells to fluorescently labeled MM@CURNPs particles uptake was 72, and the average fluorescence intensity of Raw 264.7 cells to fluorescently labeled CURNPs particles uptake was 80.