纳米级脂质微泡超声造影剂肿瘤被动靶向研究

Passive targeting study of nanoscale lipid ultrasound contrast agents on tumors

目的观察纳米微泡通过肿瘤血管内皮间隙实现被动靶向的可行性。方法20只SKOV3细胞皮下种植荷瘤裸鼠分为A组（超声显像组10只）及B组（冰冻切片组10只，再分为B1组及B2组）。制备DiI标记的纳米微泡及微米微泡并调整至相同浓度。A组：各裸鼠先后经尾静脉注射相同浓度微米微泡及纳米微泡（35μl／只，间隔1．5h），分别进行超声显像观察。B组：B1组及B2组各裸鼠分别注射相同浓度微米微泡及纳米微泡（约10μl／只），1．5h后对裸鼠进行心脏灌注后切取肿瘤组织及肌肉组织进行冰冻切片、Hoechst核染，激光共聚焦显微镜下观察不同微泡在两组织中的滞留情况。结果超声显示：纳米微泡组达峰时间及消退时间均长于微米微泡组，而峰值强度低于微米微泡组。冰冻切片观察：纳米微泡组肿瘤细胞周围见明显红色荧光聚集，微米微泡组肿瘤、肌肉组织中及纳米微泡组肌肉组织中均未见明显红色荧光。结论纳米级脂质微泡可通过肿瘤血管内皮间隙，即实现了肿瘤被动靶向。

Objective To tested the passive targeting of nanobubbles penetrating tumor vascular endothelial cells gap. Methods Twenty female BALB/c nude mice subcutaneously bearing human ovary cancer SKOV3 were devided into two groups：group A （ultrasound imaging） and group B （frozen sections： B1 and B2）. DiI labled nanobubbles and microbubbles were prepared and adjusted into the same concentrations. Group A： Microbubbles and nanobubbles of 35 μl were injected into the tail vein of every mouse respectively （1.5 h interval）. Ultrasound imaging were acquired. Group B： Nanobubbles and microbubbles of 10 μl were injected into the tail vein of mice in Group B1 and Group 132 respectively. Heart perfusion by PBS or 0.9% normal saline was carried out 1.5 h after bubbles injection to clear the free bubbles in blood circulation. And the tumor and muscle of right lower limb were immediately cut off for frozen slices （3μm） ,which were stained by Hoechst 33342 to mark the nucleus. Images were obtained with a confocal microscope. Results In vivo ultrasound imaging, the time to peak and clearance time of nanobubbles were longer than those of microbubbles,whereas the intensity of enhancement was lower than microbubbles. Frozen sections showed：with the confocal laser scanning microscopy imaging,quite a number of Diblabeled nanobubbles existed in the intercellular space of SKOV3 tumor, whereas there were few nanobubbles in skeletal muscle sections. In the control, rare DiI-labeled microbubbles were observed in tumors and skeletal muscle. Conclusions Self-made lipid nanobubbles were small enough to pass through the tumor vascular endothelial gap, namely achieve the tumor passive targeting.