Cellular Uptake Mechanism of Nucleic Acid Nanocapsules and Their DNA-Surfactant Building Blocks

Nucleic acid nanocapsules (NANs) are enzyme-responsiveDNA-functionalizedmicelles built for the controlled release of DNA-surfactant conjugates(DSCs) that present sequences with demonstrated therapeutic potential.Here, we investigate the mechanisms by which DSCs gain access to intracellularspace in vitro and determine the effects of serumon the overall uptake and internalization mechanism of NANs. Usingpharmacological inhibitors to selectively block certain pathways,we show, through confocal visualization of cellular distribution andflow cytometry quantification of total cellular association, thatscavenger receptor-mediated, caveolae-dependent endocytosis is themajor cellular uptake pathway of NANs in the presence and absenceof serum. Furthermore, as NANs can be triggered to release DSCs byexternal stimuli such as enzymes, we sought to examine the uptakeprofile of particles degraded by enzymes prior to cell-based assays.We found that while scavenger receptor-mediated, caveolae-dependentendocytosis is still at play, energy-independent pathways as wellas clathrin-mediated endocytosis are also involved. Overall, thisstudy has helped to elucidate early steps in the cytosolic deliveryand therapeutic activity of DSCs packaged into a micellular NAN platformwhile shedding light on the way in which DNA functionalized nanomaterialsin general can be trafficked into cells both as nanostructures andas molecular entities. Importantly, our study also shows that theNAN design in particular is able to stabilize nucleic acids when deliveredin the presence of serum, a critical step for effective therapeuticnucleic acid delivery.