仿生微泡超声造影剂制备方法及其生物效能的实验研究

Preparation method and characteristics of biomimetic microbubbles

目的以磷脂杂化法制备系列仿生微泡超声造影剂, 通过检测其理化性质和生物效能, 评估该方法制备仿生微泡的可行性和广谱适用性。方法通过薄膜-水化、磷脂杂化、机械振荡等步骤制备仿白细胞微泡(MBleu)、仿血小板微泡(MBpla)及仿红细胞微泡(MBery), 采用动态光散射检测仿生微泡的粒径、电位, 应用激光共聚焦显微镜观察结构, 流式细胞计数检测MBleu、MBpla、MBery典型细胞膜蛋白, 聚丙烯酰胺凝胶电泳验证仿生微泡所含相应细胞膜蛋白, 并进行体外细胞实验和动物实验检测安全性, 超声成像检测离体和在体成像能力和稳定性, 活体荧光成像检测在体代谢情况, 超声分子成像检测仿生微泡在大鼠缺血-再灌注模型和急性肝炎模型中的应用价值。结果仿生微泡呈圆形、分布均匀、无明显聚集, 细胞膜成功嵌于微泡磷脂膜上。三种仿生微泡的粒径与对照微泡比较差异无统计学意义(均P>0.05), 而电位低于对照微泡(均P<0.05)。离体和在体实验证实仿生微泡生物安全性良好。仿生微泡包含相应细胞膜的主要蛋白。超声造影显示仿生微泡稳定性及在体成像效果良好。活体荧光成像显示仿生微泡膜主要经肝脾代谢。MBleu、MBpla在两种疾病模型中均有良好的靶向成像效果。结论本研究通过磷脂杂化法成功制备三种仿生微泡超声造影剂, 其安全性良好、性能稳定。该方法具有良好的广谱适用性, 是一种便于推广应用的仿生微泡制备技术。

Objective To evaluate the feasibility and applicability of using phospholipid-hybridization method for preparing biomimetic microbubbles(Bio-MBs)ultrasound contrast agents.Methods Leukocyte biomimetic microbubbles(MBleu),platelet biomimetic microbubbles(MBpla)and erythrocyte biomimetic microbubbles(MBery)were prepared by multiple steps:film-hydration,phospholipid-hybridization,mechanical oscillation.The size and zeta potential of Bio-MBs were measured by dynamic light scattering.A laser scanning confocal microscopy experiment was performed to confirm the presence of membrane proteins on the shell of Bio-MBs.The fluorescence of FITC-labeled typical membrane protein was evaluated using a flow cytometer.Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to characterize the membrane protein.Biosafety of Bio-MBs was evaluated by CCK-8 counting kit,blood and major organs.The contrast enhancement effect and stability were observed in vitro and in vivo.An in vivo fluorescence imaging system was performed to evaluate the distribution of Bio-MBs.The application value of biomimetic microbubbles was measured by ultrasound molecular imaging by using ischemia-reperfusion rat models and acute hepatitis rat models.Results Bio-MBs with spherical shape distributed homogenously,without obvious aggregation.The membrane proteins were successfully integrated into the shell of Bio-MBs.The diameter of three Bio-MBs was similar to that of control microbubbles(MBcon)(P>0.05),three Bio-MBs had a lower zeta potential than MBcon(P<0.05).The Bio-MBs had an appreciable performance in vitro and in vivo biosafety.The Bio-MBs retained the main proteins inherited from cell membrane.Contrast enhanced ultrasound imaging in vitro and in vivo showed that the Bio-MBs had a stable imaging ability.MBleu and MBpla have good targeted imaging effect in two disease models.Conclusions A series of Bio-MBs ultrasound contrast agents,which have high stability,biosafety and targeted imaging efficiency,were successfully prepared by using phospholipid-hybridization method.This fabrication method for obtaining Bio-MBs can be applied to different clinical scenarios with different cell types in the future.