Magnesium-Free Assembly of Cationic Peptide/DNA Nanostructures with Defined Geometries for Anticancer Drug Delivery

DNA nanostructures are extensively utilized in synthetic biology, biosensing, in vivo bioimaging, and therapeutic delivery because of their programmability and biocompatibility. Although DNA nanostructure-based drug delivery systems have shown significant potential, the clinical application of nucleic acid drugs has been hindered by their biological instability and low delivery efficiency. This study proposes utilizing a cationic targeting peptide by combining a cell-penetrating peptide (TAT) with a cancer cell-targeting peptide (RGD) instead of conventional magnesium ions to facilitate the self-assembly of defined DNA nanostructures. In vitro study results show that the peptide/DNA nanostructures exhibited high membrane penetration, integrin targeting, and lysosome escape properties due to the RGD-TAT peptide, leading to improved cellular uptake efficiency. Furthermore, the structural and serum stabilities of DNA nanostructures were significantly enhanced by using protease-resistant D-type peptides for assembly, which will further amplify the application potential of DNA nanostructures in physiological conditions. As a proof of concept, a doxorubicin and KRAS siRNA-loaded RGD-TAT/DNA nanotube (NT^D-RGD-TAT-DOX-siKRAS) was utilized as a nanomedicine platform for anticancer therapy on an immunodeficient mouse tumor model. The NT^D-RGD-TAT-DOX-siKRAS demonstrated excellent tumor accumulation efficiency and anticancer effects in vivo. Mechanistically, the NT^D-RGD-TAT-DOX-siKRAS suppressed KRAS expression and improved the therapeutic effects of chemo drug doxorubicin by the higher drug delivery efficiency endowed by peptides. These findings suggest that the introduction of peptides can greatly enhance the functionality and performance of DNA nanostructures as drug delivery systems. The diversity and potent function of peptides will further expand the significant potential of DNA nanomedicine for future biomedical applications.