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.
Highlights
Gold–polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency
Differential transfection in SK‐BR3 and CT26 To establish the differential transfection efficiency of SK-BR3 and CT26 cells, a green fluorescent protein (GFP) reporter gene was delivered into both cell lines either alone, as PEI/DNA, or as AUPAMAM/DNA
In the “easy to transfect” SK-BR3 cell line, PEI and AuPAMAM both performed significantly better than DNA alone in terms of percent transfection (37 and 60% versus 0.5%, respectively) and mean fluorescence intensity (MFI) (3902 and 8896 versus 32 fluorescence intensity units, respectively)
Summary
Gold–polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. In the former case, genetically modified viral vectors—such as retroviruses or adenoviruses—are used to protect and deliver DNA into cells While these types of vectors are able to achieve high transfection yields, they are often limited in the size of the genetic payload that can be delivered, their cost of production, and their inherent immunogenicity and oncogenicity [1, 2]. To address these limitations, synthetic non-viral vectors are generally used as an alternative transfection method, as they are less expensive to produce, more scalable, and less immunogenic than their viral counterparts [1, 3, 4].
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