载自杀基因靶向超声分子探针制备及其超声显像的实验研究

Preparation and imaging of targeted ultrasound molecular probe carrying suicide gene

目的制备一种包裹液态氟碳及载肝癌治疗基因质粒,并具有叶酸分子靶向性的纳米级超声分子探针,研究其基本特性及超声显像能力。方法通过碳乙亚胺法在聚乳酸-羟基乙酸共聚物(PLGA)表面连接叶酸分子,制成靶向膜材料(FA-PLGA)后,采用双乳化法制备载有全氟戊烷(PFP)的纳米级靶向超声造影剂,将其与多聚赖氨酸孵育后,形成表面带正电荷的阳离子,利用正负电荷吸附作用,使纳米粒表面载上目标质粒S-HSV1-TK,得到S-HSV1-TK/PFP@FA-PLGA。使用低强度聚焦超声(LIFU)辐照的方式处理纳米粒,显微镜下观察其相变情况,于超声造影模式下观察其体外显影效果,流式细胞仪检测其连靶率;用PLGA代替FA-PLGA,使用相同方法制备无叶酸的非靶向造影剂,将靶向造影剂和非靶向造影剂分别与肝癌细胞HepG2孵育,观察其体外寻靶能力。结果成功制备了载有质粒及PFP的纳米级靶向超声造影剂S-HSV1-TK/PFP@FA-PLGA,该纳米粒大小均一,分散性好,平均粒径(200.50±66.34)nm,平均表面电位(37.40±7.08)mV。使用LIFU 5档辐照5 min后,显微镜下可见较多的纳米粒相变,超声造影模式下亦可见显像。流式细胞仪检测纳米粒连靶率约95%;体外细胞实验结果显示该靶向造影剂有明显的靶向作用。结论本实验成功制备了一种载质粒及PFP的纳米级靶向超声造影剂S-HSV1-TK/PFP@FA-PLGA;该纳米粒可用于超声造影显像,并具有肿瘤靶向性,为研究质粒的转染及肿瘤的治疗奠定了良好基础。

Objective To prepare a nanoscale ultrasound molecular probe carrying liver cancer therapeutic gene plasmid and folic acid(FA)targeting,and to study its basic characteristics and ultrasound imaging ability.Methods Pegylated imine was used to connect FA molecules on the surface of PLGA to prepare targeted membrane material(PLGA-FA),then PFPloaded nanoscale targeted ultrasound contrast agent was prepared by double emulsification method,and positively charged cations were formed on the surface after incubation with polylysine,and S-HSV1-TK were loaded on the surface of nanoparticles by positive and negative charge adsorption,then S-HSV1-TK/PFP@FA-PLGA was obtained.The nanoparticles were treated by low intensity focased ultrasound(LIFU)irradiation.The phase change was observed under a microscope,and the image effect in vitro was observed under contrast-enhanced ultrasound mode.The connection rate was detected by flow cytometry.The nontarge ted contrast agent without FA was prepared by PLGA instead of FA-PLGA by the same method.The targeted contrast agent and the non-targeted contrast agent were incubated with HepG2 of liver cancer cells respectively to observe their target-seeking ability in vitro.Results The nanoscale targeted ultrasound contrast agent S-HSV1-TK/PFP@FA-PLGA loaded with plasmid and PFP was successfully prepared.The nanoparticles were uniform in size,good in dispersion,with average particle size of(200.50±66.34)nm and average surface potential of(37.40±7.08)mV.After 5 min of LIFU irradiation,more nanoparticle phase trasitions were observed under the microscope,as well as contrast-enhanced ultrasound.The connection rate of nanoparticle was about 95%by flow cytometry,and the cell experiment results in vitro showed that the targeted contrast agent had an obvious targeting effect.Conclusion A nanoscale targeted ultrasound contrast agent S-HSV1-TK/PFP@FA-PLGA with plasmid and PFP was successfully prepared in this study.The nanoparticles can be used for ultrasound contrast imaging and have tumor targeting,which lays a good foundation for the study of plasmid transfection and tumor treatment.