Thrombolysis efficiency of urokinase plasminogen activator-loaded anionic lipid microbubbles combined with low-frequency ultrasound in vitro / 中华核医学与分子影像杂志

ABSTRACT

Objective To prepare urokinase plasminogen activator (uPA)-loaded anionic lipid microbubbles (uPA-MBs) for thrombolysis with low-frequency ultrasound in vitro.Methods Anionic microbubbles 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 (C3F8) were prepared by the mechanical vibration method.Then,the resulting anionic microbubbles were incubated with uPA.uPA-MBs were obtained via electrostatic 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-polyacrylamide gel electrophoresis (SDS-PAGE) was used for confirming the binding of uPA protein and anionic microbubbles.The encapsulation efficiency of uPA-MBs was determined by bicinchoninic acid (BCA) protein assay 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 analysis 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 significantly higher than that of P-MBs(-36.64±0.21) mV vs (-66.33±2.38) mV (t =21.538,P＜0.05).Fluorescence microscope showed a green shell of FITC-labeled uPA-MBs.The encapsulation efficiency of uPAMBs with the added dosage of 10 000 U was (42.01±2.02) ％,which was significantly higher than those of 50000 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 significantly better thrombolysis effect when combined with low-frequency ultrasound.