摘要
目的制备靶向人血管内皮细胞生长因子受体2（VEGFR2）的纳米液态氟碳球囊，进行体外靶向结合与超声显影的研究。方法利用乳化／蒸发法制备生物素修饰的纳米液态氟碳球囊（LNCs），采用生物素一链霉亲和素链接法进一步偶联生物素修饰的anti—VEGFR2抗体，制备VEGFR2靶向纳米氟碳球囊（V2一LNCs）。观察其形态、大小及结构，测量其粒径和表面电位。用免疫荧光法检测V2-LNCs与人血管内皮细胞（HUEVC）的靶向结合情况。观察纳米液态氟碳球囊体外超声显影效果，并进行统计学分析。结果制备了平均粒径为（98．45±0．07）nm的V2-LNCs；V2-LNCs与配体结合后的荧光免疫染色试验为阳性；体外寻靶试验显示V2-LNCs能较好地黏附在HUEVC细胞上，反复洗涤不掉，用anti-VEGFR2抗体预封闭HUEVC细胞可成功阻断其靶向结合；而作为对照的LNCs经反复洗涤后纳米球囊与细胞完全分离，无结合；体外超声显影效果明显增强，在较低浓度（0．5mg／m1）即可显著增强超声显影效果。结论成功制备出VEGFR2靶向纳米液态氟碳球囊超声造影剂，该纳米球囊与体外HUEVC有较强结合力且能显著增强超声显影性能，为进一步的血管外超声靶向显影研究获得了实验室基础数据。
Objective To prepare human vascular endothelial growth factor receptor 2 （VEGFR2）- targeted liquid perfluorocarbons-filled nanocapsules ultrasound contrast agent,and to investigate its ability of targeting human umbilical endothelial vein cells（HUEVC） and the peculiarity of the enhancing ultrasound imaging in vitro. Methods Rotary evaporation/emulsion technique was used to prepare biotinylated liquid perfluorocarhons-filled nanocapsules （LNCs）. VEGFR2-tageted LNCs （V2-LNCs） was further made through conjugating the ligand （biotinylated anti-VEGFR2 antibody） to the surface of LNCs by biotin-avidin system and its appearance,distribution,size and zeta-potential properties were assessed. Immunofluorescent staining assay was used to identify the combination of ligand with the nanocapsules. The ability of V2-LNCs targeting with HUEVC in vitro was evaluated under the fluorescence microscope. Quantitative and statistical analysis were performed to evaluate the ultrasound contrast enhancement of the nanocapsules in vitro. Results The V2-LNCs are uniform and stabilized with size about （98.45 -＋ 0.07）nm. The ligation of anti- VEGFR2 antibody and nanocapsules was positive in immunofluorescent straining assay. In vitro, targeting ability research showed the V2-LNCs could actively adhere to HUEVC, while the control was negative. At the same time, using anti-VEGFR2 antibody to pre-incubation with HUVEC could effectively block the interaction between V2-LNCs and HUEVC. The ultrasound images proved both V2-LNCs and LNCs can significantly enhanced ultrasound imaging contrast agent effect at a concentration of 5 mg/ml in vitro. Conclusions A stable VEGFR2-targeted liquid perfluoroearbons-filled nanocapsules was prepared successfully with biotin-avidin method and effectively bound to HUVEC specially in vitro. Also the liquid perfluorocarbons-filled nanocapsules can strongly enhance the ultrasound contrast in vitro, which might be taken as a kind of vascular ultrasound contrast agent and to get the basic experimental data for the later research.
出处
《中华超声影像学杂志》
CSCD
北大核心
2014年第1期71-75,共5页
Chinese Journal of Ultrasonography
关键词
微气泡
纳米囊
人脐静脉内皮细胞
血管内皮生长因子受体2
Microbubbles
Nanocapsules
Human umbilical vein endothelial cells
Vascularendothelial growth factor receptor-2
