Microfluidic manufacture of rt-PA -loaded echogenic liposomes

Echogenic liposomes (ELIP), loaded with recombinant tissue-type plasminogen activator (rt-PA) and microbubbles that act as cavitation nuclei, are under development for ultrasound-mediated thrombolysis. Conventional manufacturing techniques produce a polydisperse rt-PA-loaded ELIP population with only a small percentage of particles containing microbubbles. Further, a polydisperse population of rt-PA-loaded ELIP has a broadband frequency response with complex bubble dynamics when exposed to pulsed ultrasound. In this work, a microfluidic flow-focusing device was used to generate monodisperse rt-PA-loaded ELIP (mu tELIP) loaded with a perfluorocarbon gas. The rt-PA associated with the mu tELIP was encapsulated within the lipid shell as well as intercalated within the lipid shell. The mu tELIP had a mean diameter of 5 mu m, a resonance frequency of 2.2 MHz, and were found to be stable for at least 30 min in 0.5% bovine serum albumin. Additionally, 35 % of mu tELIP particles were estimated to contain microbubbles, an order of magnitude higher than that reported previously for batch-produced rt-PA- loaded ELIP. These findings emphasize the advantages offered by microfluidic techniques for improving the encapsulation efficiency of both rt-PA and perflurocarbon microbubbles within echogenic liposomes.