Quantitative intracellular localization of cationic lipid–nucleic acid nanoparticles with fluorescence microscopy

Abstract

Current activity in developing synthetic carriers of nucleic acids (NA) and small molecule drugs for therapeutic applications is unprecedented. One promising class of synthetic vectors for the delivery of therapeutic NA is PEGylated cationic liposome (CL)–NA nanoparticles (NPs). Chemically modified PEG-lipids can be used to surface-functionalize lipid–NA nanoparticles, allowing researchers to design active nanoparticles that can overcome the various intracellular and extracellular barriers to efficient delivery. Optimization of these functionalized vectors requires a comprehensive understanding of their intracellular pathways. In this chapter we present two distinct methods for investigating the intracellular activity of PEGylated CL– NA NPs using quantitative analysis with fluorescence microscopy. The first method, spatial localization, describes how to prepare fluorescently labeled CL–NA NPs, perform fluorescence microscopy and properly analyze the data to measure the intracellular distribution of nanoparticles and fluorescent signal. We provide software which allows data from multiple cells to be averaged together and yield statistically significant results. The second method, fluorescence colocalization, describes how to label endocytic organelles via Rab-GFPs and generate micrographs for software-assisted NP–endocytic marker colocalization measurements. These tools will allow researchers to study the endosomal trafficking of CL–NA NPs which can guide their design and improve their efficiency.