Abstract 303: Thiol-mediated rapid cytosolic uptake of tumor-targeted DNA nanorobot for siRNA delivery

Abstract

Abstract Most FDA approved anticancer drugs rely on passive delivery for cellular uptake, which will compromise therapeutic efficacy and lead to off-target-associated toxicity. DNA nanostructures are promising drug delivery platforms for targeted cancer therapy due to their highly programmable molecular interactions, precisely defined geometry, stoichiometry, as well as outstanding biocompatibility. However, most DNA nanostructures are taken up by cells through endocytosis pathway, resulting in the subsequent endo- and lysosomal trapping and degradation. Here we design a smart DNA nanorobot that will go through endocytosis-independent pathway with tumor target specificity and therapeutic effects for the treatment of breast cancer. It has been reported that disulfide units are able to mediate cytosolic uptake of DNA and RNA molecules by disulfide exchange reactions with thiol groups on the cell membrane protein. Therefore, we site-specifically modify disulfide units onto the inside surface of DNA nanorobot. 24-helix DNA origami nanotube is used as drug delivery vehicle. To achieve target specificity, we use HER2 affibody, a short peptide that has high binding affinity [KD=22pM] to HER2 protein that overexpressed on the cell membrane of breast cancer cells, and truncated HER2 protein as a temporary lock-and-key to close DNA nanorobot. In this way, DNA nanorobot can only be opened when it reaches tumor cells that overexpress HER2 protein which has a higher binding affinity to HER2 affibody. The inner disulfide units will be exposed to mediate cytosolic uptake of DNA nanorobot. To achieve potent therapeutic effect, we use therapeutic siRNAs/shRNAs as drugs due to their efficient target gene silencing, splice modulation and gene activation potentials. Cytosolic uptake of siRNAs/shRNAs by DNA nanorobot could maximize their target knockdown effect since siRNAs/shRNAs silencing occurs primarily in the cytoplasm. We use atomic force microscope (AFM), transmission electron microscope (TEM), agarose gel electrophoresis and fluorimeter to characterize the formation of the DNA nanorobot. The cellular uptake of DNA nanorobot is evaluated by confocal laser scanning microscope (CLSM) and flow cytometry. Use RT-qPCR and western blot to test the gene and protein knockdown effect. In the future, we will do in vivo experiment on tumor xenograft mice and assess the antitumor effect of DNA nanorobot. Our current results demonstrated that DNA origami modified with disulfide units can directly get internalized into the cytoplasm of cells. In addition, protecting disulfide units inside the DNA origami nanotube can block the cytosolic uptake. Therefore, our DNA nanorobot has the potential to be an efficient and safe drug delivery platform for cancer therapeutics. Citation Format: Lu Yu. Thiol-mediated rapid cytosolic uptake of tumor-targeted DNA nanorobot for siRNA delivery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 303.