Biodistribution of DNA-origami nanostructures in live zebrafish embryos with single-cell resolution

DNA origami-based nanotechnology is a versatile tool for exploring fundamental biological questions and holds significant promise for future biomedical applications. However, the development of DNA origami-based therapeutic agents is hindered by the challenge of translating in vitro performance into effective applications in vivo. Here, we exploit the optical transparency of the embryonic zebrafish to track intravenously injected, fluorescently labelled wireframe DNA origami nanostructures. Our approach integrated long-term, high-resolution imaging of transgenic live embryos with single-cell RNA sequencing, to elucidate the biodistribution of DNA nanostructures over time, up to 3 days post-injection (dpi). Notably, we observed rapid accumulation of nanostructures in the caudal hematopoietic tissue (CHT), akin to the fetal liver in mammals. We tested the effects of coating the nanostructures with an oligolysine PEG copolymer (K-PEG), a widely used strategy to enhance their stability. The K-PEG coating mitigated the accumulation rate in CHT, enabling higher percentages of the nanostructures to engage with other tissues. Additionally, our findings highlighted the pivotal role of scavenger endothelial cells in DNA origami clearance, with K-PEG offering sustained protection for the nanostructures at the CHT. Furthermore, by monitoring DNA origami in a transgenic zebrafish line designed for targeted macrophage ablation, we found that macrophages contribute to nanostructure clearance at later time points. This study introduces a framework for the analyses of the biodistribution and clearance of DNA origami nanostructures in vivo with single cell resolution and establishes a foundation for the investigation of DNA origami-based nanomedicines in animal models. ### Competing Interest Statement The authors have declared no competing interest.