自制靶向卵巢癌的纳米超声造影剂特性及裸鼠体内造影增强实验

目的探讨自制靶向卵巢癌的纳米超声造影剂特性和体内造影增强效果。方法应用冷冻干燥-超声法制备普通纳米脂质膜微泡，通过生物素-亲和素桥连法将生物素化的促黄体生成素释放激素（LHRH）与普通纳米脂质膜微泡连接，制备靶向卵巢癌的纳米微泡（LHRH-N-Mb）；观察其镜下形态，计算浓度以及室温下保存时间；以Zeta检测仪检测粒径范围、表面电位；光镜下观察LHRH-N-Mb与LHRH受体过表达的人卵巢癌OVCAR-3细胞靶向结合情况；观察LHRH-N-Mb增强裸鼠卵巢癌移植瘤显像情况。结果LHRH-N-Mb成功制备，光镜下外观圆整，分布均匀，浓度为（2．24±0．42）×10^9／ml，粒径范围为369～618nm，平均（511．76±64．25）nm，表面电位为-14．6mV。室温下保存14天后，LHRH-N-Mb上述理化特性与刚制备时的差异无统计学意义（P〉O．05）。光镜下OVCAR-3细胞周围可见LHRH-N-Mb围绕或黏附，呈花环样结构，这种靶向结合能被生物素化的LHRH抗体预先阻断。LHRH-N-Mb能增强裸鼠卵巢癌移植瘤血管及实质显像效果。结论通过冷冻干燥超声法和生物素-亲和素桥连法制备的LHRH-N-Mb造影剂具有粒径小、稳定性高，体外能靶向高效结合人卵巢癌OVCAR-3细胞，在裸鼠体内有明显增强显像效果。

Characterization and enhanced ultrasound imaging of self-made nanoliposomal microbubbles specifically targeting ovarian cancer

Objective To prepare luteinising-hormone releasing hormone （LHRH） nanoliposomal microbubbles specific- ally targeting ovarian cancer cells, and to characterize their physiochemical properties, in vitro targeting ability and en- hanced ultrasound imaging. Methods Lyophilization/sonication method was used to prepare non-targeting nanoliposomal microbubbles （N-N-Mb）. The biotin-avidin bridge method was used to conjugate biotinylated LHRH antibodies, and N-N- Mb generated biotinylated LHRH nanoliposomal microbubbles （LHRH-N-Mb） targeting specifically ovarian cancer cells. The morphology, concentration and shelf life of microbubbles were detected with optical microscopy, and the particle size range and the surface potential were measured with Zeta detector. The binding of LHRH-N-Mb to human OVCAR-3 ovari- an cancer cells was detected using light mieroseopy. The enhancement duration time, intensity of blood vessel and paren- chyma of ovarian cancer transplanted subcutaneously in nude mice were observed in vivo. Results The rounded and uni- formly distributed LHRH-N-Mb were successfully generated. The particle size ranged from 369--618 nm with a mean of （511.76±64.25） nm for LHRH-N-Mb. The concentration and surface potential of LHRH-N-Mb was （2.24±0.42）× 109/ml and -14.6 mV, respectively. After being kept at room temperature for 14 days, no significant difference of LHRH-N-Mb＇s physiochemical properties was deteeted compared with that of freshly prepared microbubbles （P〈0.05）. Moreover, the rosette formation surrounding OVCAR-3 eells was observed under light microscopy after cells were ineuba- ted with LHRH-N-Mb while pre-incubated biotinylated LHRH antibody blocked the rosette formation. In vivo study showed that the self-made LHRH-N-Mb could enhance ultrasound imaging for vessels and parenchyma of ovarian cancer transplanted subcutaneously in nude mice. Conclusion LHRH nanoliposomal mierobubbles specifically targeting ovarian cancer cells can be successfully prepared through biotin-avidin mediation and lyophilization/sonication method. LHRH nan- oliposomal microbubbles have smaller size, high stability and high efficiency of targeting human OVCAR-3 ovarian cancer cells in vitro and enhanced ultrasound imaging in vivo in nude mice.