DNA origami offers highly accurate control over shape and addressability on the nanoscale. This precise control makes it highly valuable in various fields, particularly precision nanotherapeutics. For cancer treatment, the extrinsic activation of programmed cell death by Fas receptor (FasR)/CD95-based nanoagents is a promising, minimally invasive strategy. However, treating large, solid tumors poses challenges for the design of DNA origami-based therapeutics, including drug distribution and altered cellular behavior. Here, these challenges are addressed by establishing design principles for nanoagents and testing them in a 3D cancer spheroid model. First, the ability of DNA origami nanostructures are assessed to penetrate large cancer spheroids, finding that penetration is influenced by the DNA origami size rather than its structural flexibility. Second, the capability of FasL-DNA origami-based nanoagents are evaluated to induce apoptosis in cancer spheroids, representing a more biologically relevant environment, compared to 2D studies. It is found that apoptosis induction is primarily determined by the FasL attachment strategy rather than the underlying DNA origami structure. The most effective nanoagents constructed in this study halted spheroid growth and eradicated all cancer cells within the spheroids. This study offers important insights into critical design considerations for DNA-based therapeutics for complex cellular environments, advancing DNA origami nanotherapeutic development.
Keywords: DNA origami; FasR/FasL; cancer spheroids; nanotherapeutics; penetration.
© 2025 The Author(s). Small published by Wiley‐VCH GmbH.