Intracellular trafficking pathways involved in the gene transfer of nano-structured calcium phosphate-DNA particles.

Abstract

Nano-structured calcium phosphate (NanoCaP) particles have been proven to be a powerful means of non-viral gene delivery. In order to better understand the mechanisms through which NanoCaPs-mediated mammalian cell transfection is achieved, we have sought to define the intracellular trafficking pathways involved in the cellular uptake and intracellular processing of these particles. Previous work has indicated that NanoCaP-DNA complexes are most likely internalized via endocytosis, however the subsequent pathways involved have not been determined. Through the use of specific inhibitors, we show that endocytosis of NanoCaP particles is both clathrin- and caveolae-dependent, and suggest that the caveolaer mechanism is the major contributor. We demonstrate colocalization of NanoCaP-pDNA complexes with known markers of both clathrin-coated and caveolar vesicles. Furthermore, through the use of quantitative flow cytometry, we present the first work in which the percent internalization of CaP-DNA complexes into cells is quantified. The overall goal of this research is to foster the continued improvement of NanoCaP-based gene delivery strategies.