A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines

Abstract

BackgroundGold–polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au–PAMAM transfection than others. Here we utilized two representative cell lines—a “difficult to transfect” CT26 cell line and an “easy to transfect” SK-BR3 cell line—and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors.ResultsA comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability.ConclusionsThe cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-017-0271-8) contains supplementary material, which is available to authorized users.

Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression

“…36 Differences in the dynamics of trafficking have been shown in a recent study that also identified CT26.WT mouse line as a low protein expressor compared with breast cancer SK-BR3 cells following transfection with DNA plasmid polyplexes composed of either polyethyleneimine (PEI) or polyamidoamine gold. 37 A high initial polyplex uptake rate was identified as a determining factor for transfection efficiency, similar to what we observe here with an LNP delivering mRNA. The data also highlights, without comment, juxtanuclear polyplexes in SKBR3 cells but scattered localisation in CT26.WT cells.…”

Nanoparticle-Mediated In Situ Molecular Reprogramming of Immune Checkpoint Interactions for Cancer Immunotherapy

“…CT26 is known to be extremely difficult to transfect using ionizable lipids, believed to be due, in part, to a defective endolysosomal system and thus represents a robust challenge to the developed system. , As shown in Supplementary Figure 5, we observe no significant reduction in tumor size or improved mouse survival. This suggests that efficacy may be limited to the highly transfectable cell lines such as B16F10.…”

ROS responsive polyethylenimine-based fluorinated polymers for enhanced transfection efficiency and lower cytotoxicity

“…Efficient cellular uptake is a crucial step for achieving high transfection efficiency [ 32 ]. 293TN cells were transfected by the synthesized gene vectors encapsulating Cy5-labelled pDNA for 6 h. The results in Figure 7B showed that the uptake efficiency for all gene vectors reached almost 95% without significant difference.…”