DNA nanoparticles for cancer therapy: characterization of octahedral DNA cages as drug delivery vehicles for Doxorubicin

In the last years, a great variety of materials have been used in nanotechnology for building nanoparticles with different size and shape. In particular, DNA possesses intrinsic properties of biocompatibility and high stability that makes it suitable for the generation of nanostructures. DNA-based nanostructures can be functionalized with different molecules, such as small ligands or antibodies, in order to achieve specific cellular targeting to be used for a wide variety of applications, including drug delivery. More studies about DNA nanostructures stability in biological fluids, mechanism of internalization and behavior as drug delivery vehicles are necessary for their use in biomedicine. For my doctoral thesis, covalently bound truncated DNA octahedral cages were assembled and functionalized with folic acid for targeting cancer cells overexpressing the alpha isoform of the folate receptor (αFR). In the first part of my Ph.D. work, the influence of two distinctive receptor-mediated internalization pathways on the intracellular fate and degradation rate of DNA cages inside cells was investigated. In particular, two cellular systems, one expressing the oxidized low-density lipoprotein receptor-1 (LOX-1) and the other expressing the α isoform of the folate receptor (αFR) were compared. The results indicate that DNA cages are stable in biological fluids and are very efficiently internalized in vesicular structures by both receptors. LOX-1 and αFR receptormediated mechanisms internalize an amount of DNA cages 30 and 40 times higher, respectively, compared to cells not expressing the receptors. Furthermore, when the internalization is mediated by αFR, DNA cages show high stability inside cells. On the contrary, when internalized by LOX1 DNA cages traffic to lysosomes and are rapidly degraded, demonstrating that the selection of the cellular receptor is crucial for modulating the DNA cage intracellular half-life. Starting from these data, folate-functionalized DNA cages were loaded with Doxorubicin (Dox), an anticancer drug that intercalates the double helices of DNA. Folate-DNA cages divert the Dox natural traffic and accumulate in the cytoplasm where Dox is released inducing a toxic mechanism that degrades DNA cages, avoiding the problem of nanostructures accumulation in vivo. Moreover, Dox delivered through folate-DNA cages shows a cytotoxic effect at a lower concentration compared to Dox in the free state that selectively kills αFR-positive cancer cells. Taken together these results indicate that functionalization with folate can be pursued to selectively target αFR overexpressing cells in cancer therapy. The use of nanostructures as drug vehicles can potentially improve the pharmacological effects of drugs, reducing their dosage and minimizing unwanted side effects.