KGDS血栓靶向超声造影剂的制备及其初步评价  

Preparation and preliminary evaluation of KGDS-targeted ultrasound contrast agent

作　　者：高峰[1,2] 丁燕飞[3] 盛小茜[3] 王维[1] 梁琪[1] 罗卓琼[4] 周平[4] 李辉[5] 

机构地区：[1]中南大学湘雅三医院细胞移植与基因治疗中心,长沙410013 [2]中南大学.湘雅三医院超声科,长沙410013 [3]中南大学药学院药剂学系,长沙410013 [4]中南大学湘雅三医院超声科,长沙410013 [5]中南大学湘雅二医院麻醉科,长沙410011

出　　处：《中南大学学报（医学版）》2009年第12期1255-1260,共6页Journal of Central South University ：Medical Science

基　　金：湖南省自然科学基金(09JJ3062)~~

摘　　要：目的:探讨制备靶向结合活化血小板的脂质超声造影剂的新方法,评价制备的靶向超声造影剂体外与血栓靶向结合的能力。方法:首先合成荧光标记的,能与血小板膜糖蛋白GPⅡb/Ⅲa受体特异性结合的赖氨酸-甘氨酸-天冬氨酸-丝氨酸多肽-棕榈酸化合物(KGDS-Palm)。采用"超声-高速剪切"法,以带荧光FITC的KGDS-Palm为主要原料之一,制备靶向脂质超声造影剂。马尔文公司粒径测定仪检测微泡大小及分布;Coulter计数仪分析浓度;荧光显微镜下观察KGDS多肽与微泡的结合情况;流式细胞仪评价多肽与微泡的结合效率;观察靶向微泡在体外的稳定性;采用体外血栓模型,检测靶向微泡与血栓靶向结合的特异性。结果:KGDS靶向超声造影剂呈淡黄色混悬液,微泡浓度约为1.5×109/mL,平均粒径为1.5μm,98%的微泡小于5μm。荧光显微镜显示靶向超声造影剂表面呈明亮的绿色荧光;流式细胞仪检测KGDS结合效率为90.04%;体外4℃保存48h后微泡浓度及粒径无显著改变;体外靶向及其拮抗实验证实,靶向超声造影剂与血栓特异性结合。结论:采用"超声-高速剪切法"制备靶向超声造影剂,方法简单易行,有利于靶向造影剂的制备及纯化;KGDS靶向超声造影剂稳定性好、靶向结合特异性强。Objective To prepare a thrombus-targeted ultrasonic contrast agent and to investigate its targeted ability to fresh blood clots.Methods We first synthesized FITC-KGDS-Palm compound,and then prepared thrombus-targeted microbubbles using ＂ultrasound ＆ high speed shearing method＂.Fluorescence labeling thrombus-specific peptides and KGDS,directed at the activated glycoprotein（GP）Ⅱb/Ⅲa receptor of platelets were attached to the surface of lipid microbubbles.The concentration and size of TUCA were measured by Malvern Zeta Sizer Nano-ZS590 and Coulter counter.Immunofluorescence was applied to confirm the conjugation.The conjunct ratio was assessed by flow cytometer（FCM）.Results The KGDS-TUCA was straw yellow turbid liquor,and the concentration was 1.5×109/mL,and the average size was 1.5 μm.The targeted microbubbles conjugated with the thrombus-specific peptides showed bright green rings by fluorescence microscope.FCM demonstrated that the wavelength of shell of KGDS-TUCA changed greatly,and the conjunct ratio was 90.04%.In vitro study showed KGDS-TUCA remained stable for 48 h at 4 ℃ and target-attached to blood clots and showed good stability.Conclusion The ultrasound ＆ high speed shearing method to prepare TUCA is easy and in favor of purification.KGDS-TUCA has high specific biological activity.The conjunct ratio and stability of KGDS-TUCA are excellent.

关 键 词：靶向超声造影剂 血栓 分子影像学 

分 类 号：R943[医药卫生—药剂学]