Non-degradative intracellular trafficking of highly compacted polymeric DNA nanoparticles

Highly compacted DNA nanoparticles (DNPs) composed of polyethylene glycol linked to a 30-mer of poly-l-lysine via a single cysteine residue (CK30PEG) have previously been shown to provide efficient gene delivery to the brain, eyes and lungs. In this study, we used a combination of flow cytometry, high-resolution live-cell confocal microscopy, and multiple particle tracking (MPT) to investigate the intracellular trafficking of highly compacted CK30PEG DNPs made using two different molecular weights of PEG, CK30PEG10k and CK30PEG5k. We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK30PEG10k and CK30PEG5k DNPs both entered human bronchial epithelial (BEAS-2B) cells via a caveolae-mediated pathway, bypassing degradative endolysosomal trafficking. Both nanoparticle formulations were found to rapidly accumulate in the perinuclear region of cells within 2 h, 37 ± 19% and 47 ± 8% for CK30PEG10k and CK30PEG5k, respectively. CK30PEG10k and CK30PEG5k DNPs moved within live cells at average velocities of 0.09 ± 0.04 μm/s and 0.11 ± 0.04 μm/s, respectively, in good agreement with reported values for caveolae. These findings show that highly compacted DNPs employ highly regulated trafficking mechanisms similar to biological pathogens to target specific intracellular compartments.

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