红细胞膜定向包裹血红蛋白-白蛋白纳米粒的制备及评价

Preparation and Evaluation of Hemoglobin-Bovine Serum Albumin Nanoparticles with Red Blood Cell Membrane Directional Coating

目的制备红细胞膜定向包裹的血红蛋白-白蛋白纳米粒(RBC-Hb/BSA-NP),并对其进行表征和长循环能力的探究。方法通过溶剂蒸发法制备包含血红蛋白的白蛋白纳米粒(Hb/BSA-NP),然后采用物理挤压的方式制备得到RBC-Hb/BSA-NP,并对其粒径、Zeta电位和外观形态进行表征;通过评价红细胞膜包覆白蛋白纳米粒的完整性筛选出红细胞膜的最佳用量;采用测量制剂表面唾液酸含量确定红细胞膜的正确朝向;使用荧光显微镜与流式细胞仪检测RBCHb/BSA-NP体外抗巨噬细胞吞噬的能力;最后通过体内药代动力学实验来评价纳米制剂在体内的长循环效果。结果制备的RBC-Hb/BSA-NP的平均粒径为(127.7±3.5)nm,平均Zeta电位为(−17.1±0.28)mV,具有清晰的核-壳结构,72 h内稳定性良好。0.8 mL的全血中提取出的红细胞膜能够刚好完整包覆1 mL Hb/BSA-NP(ρBSA=10 mg/mL)。与游离的红细胞相比,RBC-Hb/BSA-NP中的唾液酸含量无显著变化,表明红细胞膜正向包裹在纳米粒的表面。与普通白蛋白纳米粒(BSA-NP)和Hb/BSA-NP相比,RBC-Hb/BSA-NP可显著减少巨噬细胞摄取,且在体内的循环时间显著延长。结论成功制备了红细胞膜定向包裹的血红蛋白-白蛋白纳米粒,并证实该递送系统具有良好的长循环能力。

Objective To prepare hemoglobin-bovine serum albumin nanoparticles with red blood cell membrane directional coating(RBC-Hb/BSA-NP)and to evaluate their physicochemical properties and long circulation capability.Methods The bovine serum albumin nanoparticles modified with human hemoglobin(Hb/BSA-NP)were prepared by the solvent evaporation method.Then,the red blood cell(RBC)membrane was coated on the surface of Hb/BSA-NP by applying physical extrusion.The particle size,zeta potential and morphology of the resulting RBCHb/BSA-NP were characterized.The optimal amount of RBC membrane was determined by evaluating the integrity of the RBC membrane coating on RBC-Hb/BSA-NP,while the orientation of the RBC membrane was determined by measuring the content of sialic acid content on the surface of the RBC-Hb/BSA-NP.The anti-phagocytosis ability against macrophage of the RBC-Hb/BSA-NP was evaluated with fluorescence microscope and flow cytometry in vitro,and the long circulation capability of the nanoparticles was assessed through pharmacokinetic experiment in vivo.Results The RBC-Hb/BSA-NP showed an average size of(127.7±3.5)nm,a zeta potential of(−17.1±0.28)mV,a clear core-shell structure and good stability within 72 h.The RBC membrane extracted from 0.8 mL of whole blood can completely coat 1 mL of Hb/BSA-NP(ρBSA=10 mg/mL).The content of sialic acid on the RBC-Hb/BSA-NP was comparable to that of natural red blood cells without significant change,indicating the correct orientation of RBC membranes on the surface of the nanoparticles.Compared with regular BSA nanoparticles,RBC-Hb/BSA-NP could significantly reduce the uptake in macrophages,and it’s circulation time in vivo was greatly prolonged.Conclusion RBC-Hb/BSA-NP was successfully constructed,and it was shown that the delivery system had prolonged circulation time.