Doxorubicin-loaded DNA origami nanostructures: stability in vitreous and their uptake and toxicity in ocular cells

Abstract

Biocompatibility and precise control over their size and shape make DNA origami nanostructures (DONs) promising for drug delivery applications. Whilst many investigations have focused on cancer treatment, this might not be the best fit for DONs that get degraded by nucleases in blood. In comparison, an eye is a uniquely isolated target organ, which could benefit from DONs to achieve and maintain therapeutic concentrations in diseases that threaten the eyesight of millions of patients every year. We investigated the loading of doxorubicin (DOX) as a model drug into three distinct DONs and tested their stability upon storage. Further, we chose one structure (24HB) to probe its stability under physiological conditions in cell media and porcine vitreous, before examining the uptake and effect of DOX-loaded 24HB (24HB-DOX) on the cell viability in a retinal cell line (ARPE-19). Similar to previous reports, the tested low μM loading concentrations of DOX resulted in high drug loadings of up to 34% (m/m), and remained mostly intact in water for at least 2 months at 4 °C. In cell media and porcine vitreous at 37 °C, however, 24HB required additional Mg²⁺ supplementation to avoid degradation and the loss of the attached fluorophores. With added Mg²⁺, 24HB remained stable in vitreous for 7 days at 37 °C. The treatment with 24HB-DOX was well tolerated by ARPE-19 cells, compared to the observed higher toxicity of free DOX. Uptake studies revealed, however, that in contrast to free DOX, very little 24HB-DOX was taken up by the cells. Instead, the particles were observed to attach around the cells. Hence, our results suggest that since the uptake seems to be the bottleneck for therapies using DONs, further strategies such as adding ocular targeting moieties are necessary to increase the uptake and efficacy of doxorubicin-loaded DONs.