目的 构建iRGD靶向载尿激酶脂质体-微泡复合物(iRGD-LMC),探讨其联合超声靶向微泡破坏技术(UTMD)的体外溶栓作用.方法 薄膜-水化法制备靶向微泡,冻融法制备载尿激酶(uPA)脂质体,两者通过生物素-亲和素偶联获得iRGD-LMC.共聚焦显微镜观察其形态特征,颗粒计数仪测定其粒径和浓度,BCA试剂盒测定载药量,Vevo 2100超声成像仪对其进行体外超声成像.设置不同的超声强度及时间,比较药物释放率.制作体外血栓模型,观察iRGD-LMC联合超声的溶栓效果.结果 制备的iRGD-LMC表现为普通微泡形态,浓度为(0.51±0.03)×109/ml,粒径为(2.62±0.12)μm,载药量为每108个复合物载uPA(3878.5±97.8)μg.超声下iRGD-LMC可显影并释放药物.在体外溶栓实验中,iRGD-LMC联合超声组的溶栓效率为(87.66±1.69)％,溶栓效果最好,与阴性对照组 、 单纯超声组 、 单纯尿激酶组 、 单纯iRGD-LMC组比较差异均有统计学意义(P<0.05).结论 成功构建了iRGD-LMC,并结合了脂质体高载药量 、 超声溶栓及微泡助溶的优点.体外实验证实,联合UTMD可促进药物释放,取得了显著的溶栓效果.%Objective To fabricate iRGD targeted liposome-microbubble complex containing uPA ( iRGD-LMC) ,and to improve the thrombolytic efficiency and reduce the risk of thrombolysis by iRGD-LMC combining with ultrasound targeted microbubbles destruction ( UTMD ) to release drug into the thrombus site with the help of microbubble cavitation effect . Methods Biotinylated iRGD-MBs were fabricated by thin-film rehydration method .Biotinylated liposomes containing uPA were fabricated by freeze-thaw method and were conjugated to the biotinylated iRGD-MBs surface through biotin-avidin linkage . The iRGD-LMC was subjected to confocal microscopy to determine the particle morphology . The concentration , average diameter and size distribution were determined by particle sizing instrument . The uPA loading efficiency was measured by BCA Protein Assay Kit . Ultrasound imaging was performed using a Vevo 2100 ultrasound imaging system . The iRGD-LMC was irradiated by different ultrasound time and intensity to release drug . Thrombolytic effect in vitro of iRGD-LMC combined with UTMD was observed on the thrombosis model which was extracted from mouse blood . Results iRGD-LMC was successfully prepared . iRGD-LMC was exhibited a well-defined spherical morphology and homogeneous distribution ,like ordinary microbubbles . The concentration of iRGD-LMC was ( 0 .51 ± 0 .03 ) × 109 / ml and average diameter was ( 2 .62 ± 0 .12) μm . Drugs loading efficiency was ( 3878 .5 ± 97 .8) μg uPA per 108 microbubbles . iRGD-LMC could achieve contrast-enhanced ultrasound imaging in vitro . The thrombolytic effect of iRGD-LMC +US group ( 87 .66 ± 1 .69) ％ was the best in vitro ,and had significant difference with others groups ( P <0 .05) ,followed by iRGD-LMC group ( 53 .32 ± 4 .86) ％ and uPA group ( 51 .09 ± 9 .01) ％ ,Compared with PBS group ,US group ( 23 .56 ± 9 .46) ％ had thrombolytic effect . Conclusions iRGD-LMC is successfully prepared ,which has the advantages of high drug loading of liposomes and good acoustic properties of microbubbles . iRGD-LMC combined with UTMD achieves a significant thrombolytic effect in vitro .
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