DNA Origami Uptake in Y‐79 Retinoblastoma Cells Driven by Oligolysine Coating

DNA origami nanoparticles (DONs) are attractive nanocarriers of controllable size, shape, and addressability that have potential for treating eye diseases by overcoming ocular barriers. However, suboptimal physiological stability and poor cell uptake due to the negative charge may limit their use. Previous reports show that electrostatic complexation of DONs with cationic PEG‐oligolysine block copolymers like PEG5K‐K10 can improve structural integrity and promote cell internalization. Here, we investigated a dual approach of PEG5K‐K10 coatings and PL3‐targeting peptides to improve uptake of 24‐helix bundle (24HB) DONs into Y‐79 retinoblastoma cells. Uptake studies revealed that PEG5K‐K10 was essential for DON uptake in Y‐79 cells, as uptake only occurred upon exceeding a distinct PEG5K‐K10 amount. Longer exposure times or increased polymer amounts improved cell association. However, no beneficial effect of PL3 was observed. While free PEG5K‐K10 reduced cell viability at higher concentrations (IC 50 36.8 µM), coated DONs were well‐tolerated. Furthermore, single particle tracking in ex vivo porcine eyes revealed comparable vitreal mobility for uncoated and coated 24HB, with a slight decrease at higher coating amounts. Our findings highlight that PEG5K‐K10 can enhance ocular cell uptake without limiting nanoparticle diffusivity in the vitreous and support further optimization of DONs for ocular drug delivery.