载尿激酶阴离子脂质微泡联合低频超声体外溶栓的效率研究

目的制备载尿激酶（uPA）阴离子脂质微泡，探讨其联合低频超声的体外溶栓效果。方法利用二硬脂酸磷酯酰胆碱（DSPC）、二棕榈酰磷酯酰甘油（DPPG）、PEG2000修饰二硬脂酸磷酯酰乙醇胺（DSPE-PEG2000）3种磷脂，通过机械振荡法制备阴离子微泡，再将微泡与uPA混合孵育，两者结合即得载药微泡，用粒径分析仪测定其粒径及分布。用FITC标记uPA，制得荧光标记的载药微泡，在荧光显微镜下观察微泡的形态及uPA与微泡的黏附情况。用Zeta电位仪分别测定载药微泡与未载药微泡的表面电荷；用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）对载药微泡进行验证；测量加入10000、50000和100000UuPA时微泡的包封率。抽取大鼠血液制作体外血栓模型，测定载药微泡联合低频超声的体外溶栓效果。采用两样本t检验、单因素方差分析及Bonferroni法进行数据比较。结果载药微泡的平均粒径为（1．76±0．29）μm，在荧光显微镜下可以观察到FITC标记的uPA成功结合到阴离子微泡表面。载药微泡的表面电荷明显低于未载药微泡的表面电荷：（-36．64±0．21）mV与（-66．33±2．38）mV，t=21．538，P〈0．05；SDS—PAGE显示载药微泡的泳道有明显的蛋白质条带，而未载药微泡没有；当uPA加入量分别为10000、50000和100000U时，药物包封率分别为（42．01±2．02）％、（33．24±1．95）％和（33．10±1．65）％（F=22．340，P〈0．05）。生理盐水组、生理盐水±超声组、未载药微泡±超声组、载药微泡±超声组、单纯uPA组的体外溶栓效率分别为（4．09±0．80）％、（8．50±1．48）％、（14．27±1．59）％、（35．72±6．31）％、（16．87±0．46）％，载药微泡超声组最高（F=48．783，t=-8．613、-7．273、-5．942及-6．908，均P〈0．05）。结论阴离子微泡能成功搭载uPA，该微泡联合低频超声的溶栓效果良好。

Thrombolysis efficiency of urokinase plasminogen activator-loaded anionic lipid microbubbles com bined with low-frequency ultrasound in vitro

Objective To prepare urokinase plasminogen activator （uPA）-loaded anionic lipid mi- crobubbles （uPA-MBs） for thrombolysis with low-frequency ultrasound in vitro. Methods Anionic micro bubbles composing of 1,2-distearoyl-sn-glycero-3-phosphocholine （ DSPC ）, 1,2-dipalmitoyl-sn-glycero-3- phospho-（ l＇-rac-glycerol ） （ DPPG ）, 1, 2-distearoyl-sn-glycero-3-phosphoethanol amine-N （ succinyl PEG2000） （DSPE-PEG2000） and perfluoropropane （ C3Fs ） were prepared by the mechanical vibration method. Then, the resulting anionic microbubbles were incubated with uPA. uPA-MBs were obtained via e lectrostatic adsorption. Bubble size and distribution were measured by particle size analyzer. FITC-labeled uPA-MBs were obtained and observed under fluorescence microscope. The surface potential of uPA-MBs and plain microbubbles （P-MBs） were detected by Zeta potential analyzer. Sodium dodecyl sulfonate-polyacryl amide gel electrophoresis （SDS-PAGE） was used for confirming the binding of uPA protein and anionic mi crobubbles. The encapsulation efficiency of uPA-MBs was determined by bicinchoninic acid （BCA） proteinassay kit under three different dosages of uPA （ 10 000, 50 000 and 100 000 U）. The thrombolysis efficiency of uPA-MBs combined with low-frequency ultrasound was examined in vitro. Two-sample t test, one-way anal ysis of variance and Bonferroni test were performed to analyze the data. Results UPA-MBs were successfully obtained with the mean particle size of （1.76±0.29） μm. The surface potential of these bubbles was signifi cantly higher than that of P-MBs ： （-36.64±0.21） mV vs （-66.33±2.38） mV （t = 21.538, P〈0.05）. FIu orescence microscope showed a green shell of FITC-labeled uPA-MBs. The encapsulation efficiency of uPA MBs with the added dosage of 10 000 U was （42.01_±2.02）% ,which was significantly higher than those of 50 000 and 100 000 U （（33.24±1.95）% and （33.10±1.65）% respectively, F=22.340,P〈0.05）. The thrombolysis efficiency by saline was （4.09±0.80）%, saline ultrasound （8.50±1.48）%, MBs ultrasound （ 14.27± 1.59） %, uPA-MBs ultrasound （35.72±6.31） % and uPA （16.87±0.46） %, respectively （F= 48.783,t=-8.613, -7.273, -5.942, -6.908, all P〈0.05）. Conclusion Anionic microbubbles can successfully load uPA. and achieve sitmificantlv better thrombolvsis effect when combined with low-freunencv ultrasound.