Effects of DNA Origami-Based Nanoagent Design on Apoptosis Induction in a Large 3D Spheroid Model

The extrinsic activation of programmed cell death by FasR/CD95 is a promising minimally invasive strategy for cancer treatment. This can be leveraged using high-precision nanoscale therapeutics: Utilizing DNA origami for precise Fas ligand (FasL) presentation resulted in over 100 times more potent apoptosis induction in single cells. However, treating large, solid tumors poses challenges for DNA origami-based therapeutics, including drug distribution and altered cellular behavior. Here, we addressed these challenges using a 3D spheroid model. First, we assessed DNA origamis ability to penetrate tumor tissue, finding that penetration is influenced by the DNA origami size rather its structural flexibility. Second, we evaluated the apoptosis induction efficacy by DNA origami-FasL nanoagents within the spheroid model. The most potent nanoagents were able to completely eradicate all cells in the spheroid. Results indicated that apoptosis induction depended strongly on FasL attachment strategy rather than DNA origami design. Notably, only a rigid neutravidin linker for FasL attachment, rather than a flexible dsDNA linker, halted spheroid growth and fully eradicated all cancer cells. This study offers critical insights into designing DNA-based therapeutics for complex cellular environments and significantly advances DNA origami nanotherapeutic development, highlighting the impact of nanoagent design on cell fate decisions.