Overcoming the plasma membrane barrier to improve the efficiency of therapeutic delivery to the cellular cytoplasm

Difficulties in controlling endocytosis limit the success of many nanoparticle-based drug delivery strategies. Therefore, there is a need to both (i) introduce new mechanisms of therapeutic delivery that overcome the limitations of endocytic uptake, and (ii) gain better control of endocytosis by understanding its underlying mechanisms at a molecular level. Towards achieving efficient therapeutic delivery independently of endocytosis, I first report the development of targeted Connectosomes, cell-derived lipid vesicle materials that contain embedded connexons and are capable of forming functional gap junctions with cells. These materials encapsulated diverse molecular cargo, including dyes and drugs. These materials achieved efficient delivery of molecular cargo directly into the cytoplasm of specific populations of target cells, through interactions of embedded multi-functional, multi-domain transmembrane targeting proteins that target cell-specific receptors. By opening direct routes to the cytoplasm, targeted Connectosomes reduced the therapeutically effective dose (LD50) of doxorubicin for target cells by more than an order of magnitude in comparison to the unencapsulated drug, and by several orders of magnitude in comparison to conventional liposomal doxorubicin. These data illustrate the therapeutic importance of direct access to the cell cytoplasm, and highlight the potential of gap junction-mediated cytoplasmic delivery to increase the effectiveness of diverse therapeutics. Towards furthering our basic biophysical understanding of the mechanisms that drive clathrin-mediated endocytosis, I then investigated the curvature sensing abilities of clathrin, a critical question limiting our understanding of how nanoparticles and other molecular cargo are internalized. In particular, my findings demonstrate that clathrin binds preferentially to highly curved membranes, suggesting a possible new explanation for clathrin’s early participation in endocytic vesicle formation. In sum, this work represents key steps towards improving the success of nanoparticle-based drug delivery strategies from both applied and fundamental standpoints.