白红艳 刘妍 刘宜鑫 刘书伟 朱智

摘要：目的 观察不同实验条件对红细胞膜纳米载体制备效率和功能的影响，并探讨制备红细胞膜效率最佳时所需的低渗胀破时间。方法 取小鼠血液分离得到红细胞（RBC），分别低渗胀破20 min、1 h、2 h，依次经离心、超声和脂质体挤出等操作，制成红细胞膜纳米载体。通过透射电子显微镜（TEM）、ZS90纳米粒径电位分析仪完成形态表征，采用Western blotting法分析CD47膜蛋白，以及定量分析膜蛋白提取效率。结果 20 min、1 h、2 h三组不同时间下制备出的红细胞膜纳米载体形态规则，并成功保留发挥免疫逃逸作用的CD47蛋白，三组试验蛋白定量分析结果分别为0.95、1.25、1.328 mg/ml。与20min组比较，1h、2h组蛋白定量水平均增高（P均<0.05），1h组蛋白定量指标增长最明显（P<0.05）。结论 不同的实验条件对红细胞膜纳米载体的形态结构及功能均无影响，可以影响膜蛋白提取效率。其中延长低渗胀破时间，蛋白定量结果增长缓慢，以1 h组制备效率最高。

关键词：纳米载体;红细胞膜;仿生;制备

中图分类号：R314 文献标志码：A

Abstract： Objective To elevate the effect of different experimental conditions on the efficiency and function of the preparation of erythrocyte membrane nanocapsules， and to investigate the time of hypotonic rupture when the efficiency of erythrocyte membrane is the best. Methods Red blood cells （RBC's） were isolated from the blood of mice， which were hypoosmotic and burst for 20 min， 1 h and 2 h， respectively. After centrifugation， ultrasound and liposome extrusion， the RBC membranes were made into nanocrystals. Morphology was characterized by transmission electron microscopy （TEM） and ZS90 nanoparticle potential analyzer， and CD47 membrane protein was analyzed by Western blotting method and the extraction efficiency of membrane protein was quantitatively analyzed. Results The morphology of the erythrocyte membrane nanocarriers prepared at different time of 20 min， 1 h and 2 h were regular， and the CD47 proteins that played the role of immune escape were successfully retained. The quantitative results of the three groups of proteins were 0.95， 1.25 and 1.328 mg/ mL， respectively. Compared with group 20 min， the quantitative levels of proteins were increased at 1h and 2h （both P<0.05）， and the most significant increase was observed at group 1h （P<0.05）. Conclusion Different experimental conditions have no effect on the morphology， structure and function of erythrocyte membrane nanocapsules and can affect the extraction efficiency of membrane protein. With the prolongation of the hypotonic burst time， the quantitative results of protein increase slowly. And group 1h has the highest preparation efficiency.

Key words： nano-carrier; erythrocyte membrane; bionic; preparation

針对微纳米载体在体内运输过程中所面临的免疫清除难题，受自然细胞系统的启发，研究人员引入红细胞、巨噬细胞、细菌、血小板和干细胞[1-3]等天然细胞，分离出具有独特生物学性能的细胞膜，以构建具有长循环特性、良好生物相容性的药物传递系统。作为血液中数量最多且寿命最长的血细胞，红细胞具有可塑变形性、渗透脆性、悬浮稳定性等特点，易于分离，是制备纳米载体的良好材料。更值得一提的是，CD47作为红细胞膜表面蛋白，可以发出“Don't eat me”信号，逃避体内巨噬细胞的监视和清除，这为红细胞膜（RBCM）应用于纳米载体提供了坚实的理论依据。……