Hypoxia Induced Modulation of Cellular and in vivo uptake of DNA nanocage 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; in HeLa, MDA-MB-231, and MCF-7 cells. Hypoxia was confirmed by HIF-1; nuclear translocation. Confocal microscopy revealed significantly reduced DNA Td uptake under hypoxia, 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 and galectin/glycan-dependent pathways toward lipid raft/cholesterol-dependent uptake. 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. These findings highlight surface charge engineering as a strategy for improving DNA nanostructure delivery under hypoxic conditions.