Fluorescence Anisotropy for Detailed Analysis of Doxorubicin Loading into DNA Origami Nanocarriers for Drug Delivery

Owing to doxorubicin's high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing nondestructively the binding between DOX and DONs, and the purity of the formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs >0.5, homo-Förster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios >1. For loading ratios >0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios >1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to the regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.