Microscopy Meets Nanomedicine: The Challenge of Liposomes. Selecting, Understanding, and Adapting Imaging Techniques to Localize and Characterize Nanocarriers

Nanocarriers have brought several medical advances through better diagnostics and improved drug therapy. Yet, many promising preclinical findings were never translated into clinical success, consequently slowing drug development, and increasing its financial burden. By reliably predicting the nanoparticle fate at in vitro stages, the disappointment of suboptimal in vivo outcome could be avoided. To tackle the challenges of in vitro settings, this project aimed at gaining deeper insight on the interaction between nanocarriers and biological environment. Specifically, advance microscopy tools were used to visualize, characterize, and follow the biological fate of nanocarriers. As a back-to-basics approach, liposomes were chosen as model nanocarriers for their versatility, biocompatibility, and clinical relevance. When including a fluorescent molecule in the liposomal formulation, fluorescence dye and nanocarrier were found to affect each other’s properties in a manner dependent on environmental conditions (e.g., temperature, time, medium, and dye-specific chemistry). The fluctuations of fluorescence in the sample were further analyzed through image processing algorithms to obtain super resolution information from a diffraction-limited multi-frame acquisition. In parallel, to overcome some of the disadvantages often linked to the use of fluorescence, quantitative phase microscopy was optimized as a complementary label-free technique for the localization and characterization of liposomes in their hydrate state. Finally, fluorescence and label-free imaging were combined to determine the integrity of liposomes in nanofibers for topical administration. To understand the behavior of liposomes in cell culture, their internalization was followed using high throughput screenings, based on flow cytometry. These batch-mode results were validated in flow imaging and confocal microscopy. The behavior of liposomes, with and without PEG-coating, was compared in terms of intracellular localization and overall cellular response, resorting to the combination of fluorescence and label-free imaging (here, confocal and electron microscopy). Volumetric image correlation was then attempted, discussing benefits and limitations of the methods involved