Fluorescence cross‐correlation spectroscopy as a valuable tool to characterize cationic liposome‐DNA nanoparticle assembly

Ana I. Gómez‐Varela, Ricardo Gaspar, Adelaide Miranda, Juliane L. Assis, Rafael H.F. Valverde, Marcelo Einicker‐Lamas, Bruno F. B. Silva and Pieter A.A. De Beule

The development of nonviral gene delivery vehicles for therapeutic applications requires methods capable of quantifying the association between the genes and their carrier counterparts. Here we investigate the potential of fluorescence cross‐correlation spectroscopy (FCCS) to characterize and optimize the assembly of nonviral cationic liposome (CL)‐DNA complexes based on a CL formulation consisting of the cationic lipid DOTAP and zwitterionic lipid DOPC. We use a DNA plasmid for lipoplex loading encoding the Oct4 gene, critically involved in reprogramming somatic cells into induced pluripotent stem cells. We demonstrate that FCCS is able to quantitatively determine the extent of the association between DNA and the liposomes and assess its loading capacity. We also establish that the cationic lipid fraction, being proportional to the liposome membrane charge density, as well as charge ratio between the CLs and anionic DNA play an important role in the degree of interaction between the liposomes and DNA.