Ultrasound mediates iRGD targeted liposome-microbubble complex for thrombolytic therapy in vitro

Abstract

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 (3 878.5±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. Key words: Ultrasound targeted microbubble destruction; Urinary plasminogen activator; Thrombolytic therapy; iRGD-LMC