Dual frequency ultrasound-enhanced tissue plasminogen activator thrombolysis in an in vitro human clot model

Introduction: Stroke causes death and disability in patients throughout the world. At present, the only FDA- approved drug for ischemic stroke is recombinant tissue plasminogen activator (rt- PA). Unfortunately, rtPA can cause intracerebral hemorrhage and must use within limited time window (within 3-4.5 hour after onset of stroke). Ultrasound with rtPA loaded liposomes (rtPA_L) was suggested as adjuvant therapy that reduce the dose of rt-PA and increase its efficiency. Different frequency protocols such as kHz and MHz were used for sonothrombolysis. In this study, we suggested a protocol including both kHz and MHz with rtPA_L The kHz wave could fragment rtPA_Ls and release rtPA, the MHz enhances lytic efficiency of released rt-PA by stable cavitation and its microstreaming. This protocol may reduce bioeffect of ultrasound wave and simultaneously at least maintain rt-PA lytic effect.   Materials and Methods: The rt-PA loaded liposomes were prepared by hydrating lipid film with rt-PA and freezing liposomes under air pressure (4 atm). Cylindrical human whole blood clots (1 mm diameter) formed in and around micropipette at 37 ˚C. In combination with rtPA_L ([rtPA]=50 μg/ml) and human plasma, three protocol were used: 1 MHz (1.5 W/cm2_ 30 minutes), 130 kHz ( 0.01 W/cm2_10 s) + 1 MHz (0.5 W/cm2_ 30 minutes) and control. Clots were imaged under microscope and clot lysis were quantified by measuring clot diameters before and after sonication. Results: Statistical analysis of clot diameter varation between two sonication groups (1 MHz (1.5 W/cm2_ 30 minutes), 130 kHz ( 0.01 W/cm2_10 s) + 1 MHz (0.5 W/cm2_ 30 minutes)) do not show significant difference. Conclusion: Not significant difference between two sonication groups indicates that the dual frequency protocol by using less energy and consequently lower bioeffects may have similar effect as single frequency protocol.