HER2靶向载HSV-TK基因并具有核定位效应的纳米超声造影剂制备

Preparation of HER2-targeted nano-ultrasound contrast agent carrying HSV-TK gene and with nuclear localization effect

目的制备一种以人上皮生长因子受体2(HER2)为靶点同时载有单纯疱疹病毒胸苷激酶(HSV-TK)基因且具有核定位效应的纳米超声造影剂,并探讨其理化特性和体外超声显影效果。方法采用前期课题组构建的胍基化SS-PAAs类阳离子聚合物AGM-CBA,制备AGM-CBA/HSV-TK载体基因复合物并测定其粒径,采用薄膜水化—机械振荡法制备生物素化脂质体,以桥联亲和素—生物素法制备以HER2为靶点的靶向纳米级脂质微泡超声造影剂。对超声造影剂的外观形态、粒径、电位等进行表征;使用流式细胞仪定量检测HER2抗体与造影剂连接情况;使用多普勒彩色超声仪观察靶向纳米造影剂的超声显影效果。结果AGM-CBA/HSV-TK载体基因复合物粒径为(96.73±1.27)nm,所制备的纳米微泡超声造影剂呈相对规则圆整的球形,粒径为(550.93±10.19)nm,Zeta电位为(-9.75±0.74)mV。HER2抗体与造影剂成功连接。体外超声成像显示,纳米超声造影剂呈细腻均匀的点状密集高回声。结论成功制备出HER2靶向载HSV-TK基因并具核定位效应的纳米超声造影剂,且体外超声显影效果理想。

Objective To prepare a human epithelial growth factor receptor 2(HER2)-targeted nano-ultrasound contrast agent carring herpes simplex virus thymidine kinase(HSV-TK)gene and with nuclear localization effect,and to detect its physicochemical properties and ultrasound contrast in vitro effects.Methods The guanidine of SS-PAAs(poly amino amine)containing disulfide bond type of cationic gene carrier guanidine butyl amine monomer(AGM)and N,N'-double acryloyl in AGM-CBA(CBA)polymerization products was used to prepare the AGM-CBA/HSV-TK carrier gene complexes and determine the particle size.The thin-film hydration-mechanical oscillation was used to prepare the of biotinylated liposome,the bridging avidin biotin method was used to prepare nano-scale lipid microbubble ultrasound contrast agents targeting HER2.The appearance morphology,particle size and potential of ultrasonic contrast agent were characterized.Flow cytometry was used to quantitatively detect the connection between HER2 antibody and contrast agent.Doppler color ultrasound instrument was used to observe the ultrasonic development effect of targeted nanometer contrast agent.Re⁃sults The particle size of the vector gene complex AGM-CBA/HSV-TK was(96.73±1.27)nm,and the ultrasound contrast agent prepared was relatively regular and round,with an average particle size of(550.93±10.19)nm and a Zeta potential of(-9.75±0.74)mV.The HER2 antibody was successfully linked to the contrast agent.In vitro ultrasound imaging showed that the nano-ultrasonic contrast agent showed dense and high echo.Conclusion HER2-targeted nano-ultrasonic contrast agent containing HSV-TK gene and with nuclear localization effect has been successfully prepared,and the effect of in vitro ultrasound development is ideal.