CAJ | 학술논문

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-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-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(0.5 mg/ml)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.
목적 제비파향인혈관내피세포생장인자수체2(VEGFR2)적납미액태불탄구낭,진행체외파향결합여초성현영적연구.방법 이용유화/증발법제비생물소수식적납미액태불탄구낭(LNCs),채용생물소-련매친화소련접법진일보우련생물소수식적anti-VEGFR2항체,제비VEGFR2파향납미불탄구낭(V2-LNCs).관찰기형태、대소급결구,측량기립경화표면전위.용면역형광법검측V2-LNCs여인혈관내피세포(HUEVC)적파향결합정황.관찰납미액태불탄구낭체외초성현영효과,병진행통계학분석.결과 제비료평균립경위(98.45±0.07)nm적V2-LNCs;V2-LNCs여배체결합후적형광면역염색시험위양성;체외심파시험현시V2-LNCs능교호지점부재HUEVC세포상,반복세조불도,용anti-VEGFR2항체예봉폐HUEVC세포가성공조단기파향결합;이작위대조적LNCs경반복세조후납미구낭여세포완전분리,무결합;체외초성현영효과명현증강,재교저농도(0.5 mg/ml)즉가현저증강초성현영효과.결론 성공제비출VEGFR2파향납미액태불탄구낭초성조영제,해납미구낭여체외HUEVC유교강결합력차능현저증강초성현영성능,위진일보적혈관외초성파향현영연구획득료실험실기출수거.
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 perfluorocarbons-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 antiVEGFR2 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 perfluorocarbons-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.