Engineering liposomal particles for direct, intracellular delivery of therapeutic molecules

Nanoparticles have been developed in the last few decades in effort to improve delivery of chemotherapeutics and reduce off-target effects. Despite longer circulation times and improved tumor localization, chemotherapeutics still often lack improved efficacy with nanoparticle delivery compared to delivery of free drug. Many nanoparticles enter cells by endocytosis, where degradation in lysosomes or immediate exocytosis often limits cytoplasmic drug release, ultimately reducing bioavailability of drugs within the cell. Towards overcoming these barriers, for my thesis work, I have developed two different approaches for novel liposomal nanoparticles that improve efficiency of intracellular delivery. The first approach relies on the permeability of lipid phase boundaries in ternary lipid compositions to promote effective release. Exploiting this property, I demonstrated delivery of doxorubicin using ternary liposomes that have the potential to undergo membrane phase separation upon contact with the cell surface. My results revealed that ternary, phase separating vesicles improve the performance of doxorubicin by up to 5-fold in comparison to delivery of conventional liposomal doxorubicin. The second approach I examined for improving efficient intracellular delivery of therapeutics harnesses the cellular gap junction network. Gap junctions allow direct, intracellular diffusion of molecules from one cell to another. Our lab has developed connectosomes, cell-derived vesicles that contain gap junction transmembrane proteins enabling the formation of gap junction channels with cells. Doxorubicin delivery via connectosomes dramatically reduced the minimum lethal dose, in vitro, in comparison to conventional liposomal doxorubicin. In my graduate work, I demonstrated that connectosomes also improve doxorubicin delivery to cellular targets in vivo in mouse models by up to 16-fold compared to conventional liposomal doxorubicin. Further, I investigated the potential for connectosomes to deliver not just small molecule therapeutics, but macromolecular therapeutics to cells. I show that polymer-like macromolecules of 10 kDa can diffuse through gap junction channels, and further that they exhibit 95% loading into connectosomes through gap junction hemichannel pores. Overall, my work reveals two novel approaches for improving delivery efficiency to intracellular targets for therapeutic molecules. These two approaches demonstrate mechanisms to overcome the barriers of endocytosis by enabling direct delivery of molecular cargo to the cellular interior.Biomedical Engineerin