Effect of covalent fluorescence labeling of plasmid DNA on its intracellular processing and transfection with...

The development of biotechnological pharmaceutics, like macro- and nanocarriers, can benefit greatly from studying their characteristics in situ using advanced fluorescence microscopy methods. While choosing the optimal labeling method for visualizing the carrier or its cargo is crucial, it seldom receives attention. The possibility that high labeling densities alter the intracellular processing of the molecule is considered, but how and at which point this interference happens is not yet studied. The aim of this study was to elucidate the effect of labeling density on the cellular trafficking of labeled pDNA. Due to the drastic effect on expression levels for higher labeling densities, we tried to determine at which steps in the intracellular processing labeled pDNA behaves different than its nonlabeled counterpart. Therefore, different labeling densities, up to the manufacturer's recommended density, were tested. It was found that the cellular uptake remains unaffected, while the affinity for lipids is increased, which affects dissociation from the lipid-based complex and may affect endosomal escape. Also, nuclear injections clearly demonstrated that transcription is affected. The information and methodology, included in this work, could be helpful in determining if the labeling method and density used yields biological relevant results for the intended research question.