Live Cell Imaging of GFP-Rab Gtpases allows for Intracellular, Spatiotemporal Tracking of Surface Functionalized Lipid-DNA Complexes

Abstract

Cationic lipid-nucleic acid complexes are used in vitro [1] and also in clinical trials for both gene delivery and gene silencing [2]. Steric stabilization of cationic lipid-DNA (CL-DNA) complexes is required for their use in vivo, but PEGylation (PEG; polyethylene glycol) of CL-DNA complexes reduces their efficacy as gene delivery vectors in vitro [3]. To improve the uptake as well as allow for active targeting of PEGylated CL-DNA complexes, we developed CL-DNA complexes with an RGD motif present at the distal end of PEG for use in vivo and studied their efficacy and mechanism of entry in vitro. RGD-tagged CL-DNA complexes showed superior uptake but low gene expression, suggesting that endosomal escape was a major barrier to transfection.In order to understand the fate of intra-endosomal CL-DNA complexes, we used GFP constructs of a class of membrane bound GTPases called Rabs which control budding, trafficking and fusion of vesicles along the endocytic pathway [4]. GFP labeling of Rabs allows for detection of the spatiotemporal state of endocytosed CL-DNA complexes by directly labeling endosomes. We will present quantitative analysis using GFP-Rab5 (an early endosome marker) which shows CL-DNA complexes colocalizing with early endosomes suggesting that macropinocytosis or clathrin mediated endocytosis as the route of entry. Cells expressing GFP-Rab5-Q79L (a mutant which slows early endosome maturation) showed enlarged endosomes containing multiple CL-DNA complexes. A thorough understanding of the intracellular fate of CL-DNA complexes will allow for optimization of gene delivery vectors.Supported by NIH-GM59288 and and NSF DMR-1101900.[1] Ewert, K.K. et al.; Topics Current Chemistry, 2010, 296, 191-226[2] www.wiley.com/legacy/wileychi/genmed/clinical[3] Chan, C.L.; Majzoub, R. N. et al.; Biomaterials 2012, 33, 4928-4935[4] Zerial, M., McBride, H.; Nat. Rev. Mol. Cell Biol., 2001, 2(2), 107-117