Nanoscale robots have potential as intelligent drug delivery systems that respond to molecular triggers. Using DNA origami we constructed an autonomous DNA robot programmed to transport payloads and present them specifically in tumors. Our nanorobot is functionalized on the outside with a DNA aptamer that binds nucleolin, a protein specifically expressed on tumor-associated endothelial cells, and the blood coagulation protease thrombin within its inner cavity. The nucleolin-targeting aptamer serves both as a targeting domain and as a molecular trigger for the mechanical opening of the DNA nanorobot. The thrombin inside is thus exposed and activates coagulation at the tumor site. Using tumor-bearing mouse models, we demonstrate that intravenously injected DNA nanorobots deliver thrombin specifically to tumor-associated blood vessels and induce intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth. The nanorobot proved safe and immunologically inert in mice and Bama miniature pigs. Our data show that DNA nanorobots represent a promising strategy for precise drug delivery in cancer therapy.

Although a multitude of promising anti-cancer drugs have been developed over the past 50 years, effective delivery of the drugs to diseased cells remains a challenge. Recently, nanoparticles have been used as drug delivery vehicles due to their high delivery efficiencies and the possibility to circumvent cellular drug resistance. However, the lack of biocompatibility and inability to engineer spatially addressable surfaces for multi-functional activity remains an obstacle to their widespread use. Here we present a novel drug carrier system based on self-assembled, spatially addressable DNA origami nanostructures that confronts these limitations. Doxorubicin, a well-known anti-cancer drug, was noncovalently attached to DNA origami nanostructures through intercalation. A high level of drug loading efficiency was achieved, and the complex exhibited prominent cytotoxicity not only to regular human breast adenocarcinoma cancer cells (MCF 7), but more importantly to doxorubicin-resistant cancer cells, inducing a remarkable reversal of phenotype resistance. With the DNA origami drug delivery vehicles, the cellular internalization of doxorubicin was increased, which contributed to the significant enhancement of cell-killing activity to doxorubicin-resistant MCF 7 cells. Presumably, the activity of doxorubicin-loaded DNA origami inhibits lysosomal acidification, resulting in cellular redistribution of the drug to action sites. Our results suggest that DNA origami has immense potential as an efficient, biocompatible drug carrier and delivery vehicle in the treatment of cancer.

Many chemotherapeutics used for cancer treatments encounter issues during delivery to tumors in vivo and may have high levels of systemic toxicity due to their nonspecific distribution. Various materials have been explored to fabricate nanoparticles as drug carriers to improve delivery efficiency. However, most of these materials suffer from multiple drawbacks, such as limited biocompatibility and inability to engineer spatially addressable surfaces that can be utilized for multifunctional activity. Here, we demonstrate that DNA origami possessed enhanced tumor passive targeting and long-lasting properties at the tumor region. Particularly, the triangle-shaped DNA origami exhibits optimal tumor passive targeting accumulation. The delivery of the known anticancer drug doxorubicin into tumors by self-assembled DNA origami nanostructures was performed, and this approach showed prominent therapeutic efficacy in vivo. The DNA origami carriers were prepared through the self-assembly of M13mp18 phage DNA and hundreds of complementary DNA helper strands; the doxorubicin was subsequently noncovalently intercalated into these nanostructures. After conducting fluorescence imaging and safety evaluation, the doxorubicin-containing DNA origami exhibited remarkable antitumor efficacy without observable systemic toxicity in nude mice bearing orthotopic breast tumors labeled with green fluorescent protein. Our results demonstrated the potential of DNA origami nanostructures as innovative platforms for the efficient and safe drug delivery of cancer therapeutics in vivo.

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Charge-reversal functional gold nanoparticles first prepared by layer-by-layer technique were employed to deliver small interfering RNA (siRNA) and plasmid DNA into cancer cells. Polyacrylamide gel electrophoresis measurements of siRNA confirmed the occurrence of the charge-reversal property of functional gold nanoparticles. The expression efficiency of enhanced green fluorescent protein (EGFP) was improved by adjuvant transfection with charge-reversal functional gold nanoparticles, which also had much lower toxicity to cell proliferation. Lamin A/ C, an important nuclear envelope protein, was effectively silenced by lamin A/C-siRNA delivered by charge-reversal functional gold nanoparticles, whose knockdown efficiency was better than that of commercial Lipofectamine 2000. Confocal laser scanning microscopic images indicated that there was more cy5-siRNA distributed throughout the cytoplasm for cyanine 5-siRNA/ polyethyleneimine/cis-aconitic anhydride-functionalized poly(allylamine)/polyethyleneimine/11-mercaptoundecanoic acid-gold nanoparticle (cy5-siRNA/PEI/PAH-Cit/PEI/MUA-AuNP) complexes. These results demonstrate the feasibility of using charge-reversal functional gold nanoparticles as a means of improving the nucleic acid delivery efficiency.Keywords gold nanoparticles; charge-reversal polyelectrolyte; drug delivery; layer-by-layer assembly; siRNA delivery Over the past decade, due to good biocompatibility, easy synthesis, monodispersity, and ready functionalization, gold nanoparticles have emerged as an attractive candidate for delivery of various payloads into cells, such as small drug molecules or large biomolecules, 1-5 such as DNA and siRNA. [6][7][8][9][10][11][12][13] The intracellular release could be triggered by glutathione (GSH), 3 pH, or external (e.g., light) stimuli. 1,4,[14][15][16][17] siRNA has emerged recently as a promising method for biological research and holds great potential for treatment of human * Address correspondence to liangxj@nanoctr.cn. ⊥ These authors contributed equally to this work. Supporting Information Available:Preparation process of charge-reversal polyelectrolyte-coated gold nanoparticles using layer-bylayer technique; TEM images of colloidal AuNPs after the coating steps; fluorescence microscope image of 293T cells transfected with DNA/PEI/PAH-Cit/PEI/MUA-AuNP complexes; and cell viability of HeLa cells treated with PEI/PAH-Cit/PEI/MUA-AuNPs and nucleic acid complexes by MTT assay. This material is available free of charge via the Internet at http://pubs.acs.org. [18][19][20] Nucleic acid was mostly loaded by gold nanoparticles through thiol linkages or electrostatic interaction with cationic gold nanoparticles. [6][7][8][9][10][11][12][13]21,22 Elbakry et al. first developed the PEI/siRNA/PEI-AuNP system to deliver siRNA into cells and knockdown the expression of target gene based on the self-assembly layer-by-layer technology. 11 PEI, which has strong escape capacity from the endosome due to its so-called "proton sponge" effect and is usually a gold standa...