Quantitative colocalization analysis of DNA delivery by PEI-mediated cationic polymers in mammalian cells

Abstract

Although cationic polymers are widely used for DNA delivery, the relationship between the properties of the formed complexes and their biological activity is not fully understood. Here, we propose a novel procedure consisting of superresolved images coupled with quantitative colocalization to analyse DNA release in living cells. This work compares the different workflows available in a quantitative colocalization study of DNA delivery using polyethylenimine as transfection reagent. A nimble workflow with deconvolution in three-dimensional images was developed. Among the different colocalization coefficients, Manders' colocalization coefficient was the best to track the complexes. Results showed that DNA/polyethylenimine complexes were tightly interacting at the time of transfection and their disassembly was observed between 2 and 10 h after their uptake. Heterogenicity was found in the intracellular fate of each complex. At 24 h, some complexes were still present underneath the nuclear envelope. Overall, this study opens the door for particle tracking assessment with three-dimensional imaging at intracellular level. LAY DESCRIPTION: DNA delivery technologies in living cells are of high relevance in the biotechnology field. The transient expression of a gene of interest enables the production of a wide range of new therapeutic candidates for clinical purposes. However, the introduction of an exogenous DNA construct into a cell culture requires the use of certain vehicles that protect the DNA from host cell DNases and deliver it into the cell nucleus. From the different systems available, polyethylenimine (PEI) has been extensively used in transient gene expression strategies for the last three decades. However, the intracellular fate of the formed DNA/PEI complexes and the DNA release from the complexes is still poorly understood. In this work, we propose the application of combined superresolved images through mathematical deconvolution to colocalization studies of DNA/PEI complexes evolution in living mammalian cell cultures. Both specimens were covalently labelled with Cy3 and Cy5 dye, respectively, and the kinetics of its disassembly process within the cells was tracked over the time. Because of the specific features of the formed-complexes, a comparative study of the different colocalization coefficients was performed towards optimizing the analysis of these particles with confocal microscopy. Besides, the 3D imaging of the process allowed the direct visualization of a partial DNA/PEI complexes disassembly and the location of those complexes underneath the nuclear envelope during the cell production phase (24 h after the uptake).

Keywords: 3D imaging; Characterization; DNA/PEI complexes; confocal microscopy; deconvolution; transient transfection.