DOI: 10.25549/usctheses-c3-201066
title: Analysis of endocytic and trafficking pathways of potential candidates for drug delivery in HeLa and lacrimal gland acinar cells

description: When identifying potential candidates for drug delivery or biomedical diagnostic tools, it is of utmost importance to delineate, as best as possible, their uptake and intracellular trafficking pathways. Here we investigated these pathways, in a simple cell model first, followed by a more complex and physiologically-relevant cell model, for three potential theranostic and drug delivery candidates. The first is a nanoparticle based on difluoroboron dibenzoylmethane-poly(lactic acid), that exhibits unique molecular-weight dependent emission properties. We showed that these nanoparticles are highly photostable, in vitro and in situ, resisting laser-induced photobleaching under conditions that destroy the fluorescence associated with a common photostable probe, LysoTracker Blue. Also, we showed that the internalized nanoparticles do not accumulate in acidic compartments such as late endosomes and lysosomes, but rather in a non-lysosomal perinuclear compartment. Additionally, we demonstrated that their uptake utilizes actin filaments and microtubules. These findings demonstrate the feasibility of using these nanoparticles with unique emission properties for in situ, live cell imaging. ❧ The second candidate examined is a cyclotide, MCoTI-I. Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose cyclic structure makes them remarkably stable and resistant to denaturation and degradation. Using real-time confocal microscopy imaging, we showed that MCoTI-I is readily internalized in HeLa and lacrimal gland acinar cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I is achieved primarily through fluid-phase endocytosis in HeLa as evidenced by its significant colocalization with 10,000 MW dextran, but also through other pathways as well as evidenced by its colocalization with cholera toxin-B and epidermal growth factor. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway did not affect MCoTI-I uptake. In lacrimal gland acinar cells, endocytosis is also achieved primarily through fluid-phase endocytosis, albeit to a lesser extent. In HeLa, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments where MCoTI-I-containing vesicles continue to exhibit microtubule-associated movements. In contrast, almost no MCoTI-I reaches acidic compartments in lacrimal gland acinar cells in the same time frame. These findings demonstrate internalization of MCoTI-I through multiple pathways that may be dominant in the cell type investigated, and suggest that this cyclotide has ready access to general endosomal pathways. ❧ The third and final candidate investigated is Adenovirus 5. The established method of Adenovirus 5 infection in most cells is by a penton-dependent mechanism. Here, we demonstrated that while the penton base remains on the surface of lacrimal gland acinar cells, fiber is readily internalized, suggestive of a fiber-dependent entry mechanism. We also determined that macropinocytosis is not the prevalent route of uptake. Data from studies involving various endocytosis inhibitors suggest that uptake of Adenovirus 5 in lacrimal gland acinar cells may rely on multiple pathways other than primarily clathrin-mediated endocytosis. Additionally, binding of Adenovirus 5 to its surface receptor appears to be cholesterol-dependent. Also, the inward movement of internalized Adenovirus 5 seems to rely on the microtubule network. Taken together, these studies indicate that the internalization and intracellular trafficking of Adenovirus 5 in lacrimal gland acinar cells follow a different pathway than what is reported in the literature, and may involve very complex and compensatory mechanisms. ❧ Together, these findings demonstrate the unique challenges encountered on the path to assessing the utility and feasibility of potential theranostics and drug delivery candidates. They also highlight the importance of using physiologically- and disease-relevant cell models, especially since information obtained from one cell type is likely not applicable in another.