Hypoxia-Induced Modulation of Cellular and in vivo uptake of DNA nanocages – implications in therapeutics

DNA tetrahedra (DNA Td) are promising nanocarriers for drug delivery, but how hypoxia affects their cellular internalisation remains poorly understood. We synthesised and characterised Cy5-labelled DNA Td and established chemical hypoxia using 100 µM CoCl□ in HeLa, MDA-MB-231, and MCF-7 cells. Hypoxia was confirmed by HIF-1α nuclear translocation with >92% cell viability. Confocal microscopy revealed significantly reduced DNA Td uptake under hypoxia (P < 0.01), whereas transferrin and cholera toxin B uptake increased, indicating cargo-selective regulation. Temperature-arrest experiments confirmed reduced energy-dependent internalisation. Pharmacological profiling showed a shift from clathrin-mediated (42% → 29%) and galectin/glycan-dependent (43% → 35%) pathways toward lipid raft/cholesterol-dependent uptake (36% → 56%). Hypoxia increased plasma membrane electronegativity, suggesting a biophysical barrier to DNA Td uptake. Importantly, DOTMA complexation restored uptake to normoxic levels, identifying electrostatic repulsion as a key determinant. In zebrafish larvae, hypoxia significantly enhanced whole-larva DNA Td accumulation (P < 0.001). These findings highlight surface charge engineering as a strategy for improving DNA nanostructure delivery under hypoxic conditions.