Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release

Doxorubicin (DOX) is a commonly employed drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing programmable DOX-loaded DNA nanostructures that can be further tailored for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of promising DOX-loaded DNA nanocarriers remains limited and incoherent. A number of reports have overlooked the fundamentals of the DOX-DNA interaction, let alone the peculiarities arising from the complexity of the system as a whole. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures. In our experimental conditions, all of our DNA origami designs show rather similar DOX binding capacities, which are, however, remarkably lower than previously reported. To simulate the possible physiological degradation pathways, we examine the stability and DOX release properties of the complexes upon DNase I digestion, which reveals that they disintegrate and release DOX into the surroundings at characteristic rates related to the DNA origami superstructure and the loaded DOX content. In addition, we identify DOX self-aggregation and precipitation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration that have been largely ignored in experimenting with DNA nanostructures and in spectroscopic analysis performed with routine UV-Vis and fluorescence techniques. Nevertheless, we demonstrate the possibility of customizing drug release profiles through rational DNA origami design. Therefore, we believe this work can be used as a guide to tailor the release profiles and develop better drug delivery systems based on DNA nanostructures.