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 (CK ₃₀PEG) 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 CK ₃₀PEG DNPs made using two different molecular weights of PEG, CK ₃₀PEG _10k and CK ₃₀PEG _5k. We found that PEG MW did not have a major effect on particle morphology nor nanoparticle intracellular transport. CK ₃₀PEG _10k and CK ₃₀PEG _5k 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 CK ₃₀PEG _10k and CK ₃₀PEG _5k, respectively. CK ₃₀PEG _10k and CK ₃₀PEG _5k 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.