KGDS血栓靶向超声造影剂的制备及其初步评价

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1.5 μm，98%的微泡小于5 μm。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48 h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。

Preparation and preliminary evaluation of KGDS-targeted ultrasound contrast agent

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 1.5×10~9/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.