树枝状聚合物纳米载体靶向结合损伤心肌细胞的体外实验研究

Targeted adhesion of dendrimer nanocarriers to hypoxia-injured primary cardiomyocyte cultures

目的 制备一种可靶向于缺氧损伤心肌细胞的纳米载体,检测其基本理化性质和细胞毒性作用,并实现其与损伤原代心肌细胞的靶向结合. 方法 化学合成树枝状聚合物纳米载体（非靶向纳米载体）和血管紧张素Ⅱ1型（angiotensinⅡType 1,AT1）多肽修饰的树枝状聚合物纳米载体（靶向纳米载体）,测定其结构、基因负载能力和负载基因后的粒径、电位,观察其形貌,检测其对心肌细胞的毒性.将SD乳鼠原代心肌细胞采用随机数字表法分为3组：缺氧损伤＋非靶向组（A组,原代心肌细胞加入非靶向纳米载体后在缺氧条件下培养）,正常＋靶向组（B组,加入靶向纳米载体,在常氧条件下培养）,缺氧损伤＋靶向组（C组,加入靶向纳米载体,在缺氧条件下培养）,观察并定量两种纳米载体与心肌细胞的结合情况. 结果 合成的靶向纳米载体与基因质量比为6∶1时可完全负载基因,其粒径为（180±55） nm,zeta电位为＋5.4 mV,透射电子显微镜下近球形.当靶向纳米载体的浓度小于200 mg/L时,不同时间点靶向纳米载体作用的细胞活性均大于85％.分别与缺氧损伤＋非靶向组（荧光强度为7 102±134）和正常＋靶向组（8 120±235）相比,缺氧损伤＋靶向组（12 350±495）心肌细胞膜周围的红色荧光均显著增强（P＜0.05）. 结论 AT1多肽修饰的树枝状聚合物纳米载体毒性小,基因负载能力强,并可与体外高表达AT1受体的心肌细胞靶向结合.该靶向纳米载体有望为心肌缺血的基因治疗提供新的递送策略.

Objective To prepare nanocarriers capable of targeting hypoxia-injured cardiomyocytes and to investigate its physicochemical characteristics,cytotoxicity and efficiency of targeted binding to injured primary cardiomyocytes.Methods The dendrimer nanocarriers （non-targeting nanocarriers） and angiotensin Ⅱ Type 1 （AT1）-modified dendrimer uanocarriers （targeting nanocarriers） were synthesized.The chemical structure,gene compact capacity,particle sizes and zeta potentials of the targeting nanocarriers were determined and the morphology was observed.The primary cardiomyocytes of neonatal SD rats were cultured in 3 different conditions：with non-targeting nanocarriers under hypoxia,with targeting nanocarriers under hypoxia,and with targeting nanocarriers under normal culture condition.The targeted adhesion of the nanocarriers to injured cardiomyocytes and normal cardiomyocytes were observed under a laser scanning confocal microscope and quantified with flow cytometry.The toxicity of nanocarriers to cardiomyocytes was also assessed.Results Non-targeting nanocarriers and targeting nanocarriers were successfully synthesized.When the weight ratio of targeting nanocarriers to gene was 6∶1,the gene was completely incorporated,and formed spherical particles with a diameter of （180±55） nm and a zeta potential of ＋5.4 mV.When the concentrations of nanocarriers were less than 200 mg/L,the cell viability was more than 85% at a series of time points.Compared with hypoxia injury＋non-targeted group and normal＋targeted group,the targeted adhesion efficiency of myocardial cell membrane was significantly enhanced in hypoxia injury＋ targeted group.Conclusions AT1-modified dendrimer nanocarrier was successfully prepared,which can effectively bind to the injured cardiomyocytes overexpressing angiotensin Ⅱ type 1 receptors （AT1R） with low cytotoxicity.It may provide a new targeted delivery strategy for gene therapy of perioperative myocardial ischemia.