Anna Klose

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.

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.

Owing to doxorubicin’s high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing nondestructively the binding between DOX and DONs, and the purity of the formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs >0.5, homo-Förster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios >1. For loading ratios >0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios >1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to the regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.

Doxorubicin-loaded DNA origami required additional Mg²⁺ to be stable in cell medium and vitreous at 37 °C. In retinal epithelial cells (ARPE-19), they were well-tolerated, but limited in their uptake.

Owing to doxorubicin’s high DNA binding affinity, doxorubicin-loaded DNA origami nanostructures (DOX-DONs) are promising nanocarriers against cancer. However, understanding the interactions between doxorubicin (DOX) and DNA origami nanostructures (DONs) is important to ensure the quality of DOX-DONs. This interaction is often taken for granted and the influence of DOX loading conditions is poorly characterized. Exploiting the inherent fluorescence of DOX, steady-state and time-resolved fluorescence anisotropy spectroscopy techniques are used for characterizing non-destructively the binding between DOX and DONs, and the purity of formed complexes. The difference in fluorescence anisotropy between free DOX and DOX-DONs confirms the DOX-DON complex formation. Further, at loading ratios of DOX to DNA base pairs > 0.5, homo-Förster resonance energy transfer (homo-FRET) between closely packed DOX molecules is observed. Moreover, time-resolved anisotropy reveals DOX aggregation on DONs at high loading ratios > 1. For loading ratios > 0.1, spin-filtration to remove excess free DOX is efficient and necessary, though at loading ratios > 1 some DOX aggregates remain attached to the DONs. In summary, fluorescence anisotropy analysis provides more detailed information and insight into DOX-DONs compared to regularly used fluorescence intensity-based characterization methods, and these results can help designing more efficient and safer DNA intercalator-based nanocarriers.