Fluorescent dye incorporation causes weakened gene association and intracellular aggregate formation in nonviral carriers.

The successful application of nonviral gene transfer technologies requires both improved understanding and control with respect to intracellular trafficking and release. However, the intracellular space is highly complex and hence well-defined, stable structures are necessary to probe the stages of the delivery pathway. Fluorescent labeling is a regularly used approach to monitor nonviral delivery and release, yet few studies investigate the effects of label incorporation on the structure and activity of gene-containing vehicles. In the present study, the impacts of label incorporation on the assembly and gene transfer capacity of DNA polyplexes were determined through the utilization of a model DNA-polyethylenimine (PEI) delivery system. PEI was fluorescently labeled with the Oregon Green® dye prior to polyplex formation and delivery to CHO-K1 cells. The present study provides evidence showing that routine labeling strategies for polyplexes weakened DNA binding affinity, produced large quantities of extracellular structures and significantly increased intracellular polyplex aggregation. Additionally, cellular internalization studies showed that increased labeling fractions led to reductions in polyplex uptake as a result of weakened complexation. These results not only provide insight into the assembly of these structures, but also help to identify labeling strategies sufficient to preserve activity at the same time as enabling detailed studies of trafficking and disassembly.