DEVELOPMENT OF LIPOSOMES FOR ULTRASOUND-MEDIATED DRUG DELIVERY

In the last decades, ultrasound (US) has been widely investigated for diagnostic and therapeutic applications in the medical field. In particular, focused ultrasound (FUS) is considered to be a promising non-invasive therapeutic technique that exploits the focalization of acoustic waves to induce a thermal or mechanical effect at the focal point by energy transfer after penetration into target tissues of the body. In this study, US was used to enhance the delivery of an active molecule entrapped in liposomes to cardiac myofibroblast (H9C2) cells, allowing a fast and controlled drug release upon US exposure. This thesis project fits in a broader project aiming at exploring US-mediated drug delivery for the treatment of ventricular arrhythmias by fibrosis reduction due to cardiac miofibroblasts (CMF) death. In the first part of this experimental work, liposomes incorporating 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), identified as the key player to achieve US responsivity, were successfully formulated by thin-film hydration followed by extrusion, a conventional technique for the preparation of uniform liposomes. Furthermore, a technology transfer to microfluidic was developed in sight of an optimization of the accuracy and efficiency of the formulation process. Physico-chemical characterization in terms of size, size distribution, surface potential, encapsulation efficiency, and morphology of the prepared liposomes was performed, validating the reproducibility of the developed methods. Conventional liposomes were loaded with a model drug, doxorubicin (DXR), and characterized in terms of in vitro US-triggered drug release, and of in vitro drug retention upon exposure to diluted serum (10 % FBS). Liposomes US-mediated release profile showed that after 8 minutes of continued US exposure more than 90 % of DXR was released, while a slower release profile was obtained in the buffer supplemented with FBS, in which 60 % of the drug could escape from the inner core of the liposomes only after 24 hours. These studies proved the retention ability of the nanosystems upon injection, allowing for a selective release upon US exposure in a temporally- and spatially-controlled manner. The second part of this thesis project focused on in vitro studies on the H9C2 cell line to investigate potential differences on the cytotoxic effect of the drug and its ability to be internalized when administered in its free form or when encapsulated in the liposome inner core. Cell viability evaluated the cytotoxic effect of DXR on H9C2 cells, with no significant differences between the cytotoxic effect of the drug after 4 and 24 hours of incubation of DXR in its free form or when encapsulated. Finally, confocal microscopy and flow cytometry analyses showed the effective and time-dependent cell uptake of DXR-loaded liposomes.