DNA-Conjugated Gold Nanoparticles as Ultrasound- Responsive Drug Delivery Systems

1. DNA-Conjugated Gold Nanoparticles as Ultrasound-Responsive Drug Delivery Systems • Shih-Tsung Kang, Yun-Ling Luo, Yu-Fen Huang, Chih-Kuang Yeh Department of Biomedical Engineering and Environmental Sciences, National TsingHua University Hsinchu, Taiwan. 2012.8.14

2. HIFU Excitation Introduction • Nanoparticles as drug delivery systems: • High stability • High carrier capacity • Specific targeting capability • Controlled release capability • Gold nanoparticles (Au NPs): • Easy synthesis and bioconjugation • Plasmonic resonance oscillations • Tunable optical characteristics • Strong photothermalresponse • Photoacoustic imaging contrast Au 13 nm Au NP Doxorubicin (Dox)-DNA Complexes

3. Dox-DNA Complex Targeting • Sgc8c aptamer • CCRF-CEM cells • PTK7 proteins with a Kd ≈ 1 nM • 5’-TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3’ • Extended double strand DNA for Dox intercalation • 5’-thiol-TTT TTT TTT TCC CTA ACC CTA ACC CTA ACC C-3’ • 5’-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTG GGT TAG GGT TAG GGT TAG GG-3’ Drug loading Dox:ds(sgc8c)

4. Dox:ds(sgc8c)-Au NPs • Incubation of ds(sgc8c)-Au NPs (9.7 nM) withDox (4 μM) for 2 h yields the nanoconjugates loaded with 2.7 μMDox  68% loading efficiency • For a single 13-nm Au NP • 61 ± 10 Dox-DNA complexes • 280 ± 23 Dox molecules • 4-5 Dox molecules per DNA • Hydrodynamic diameter change: • 19.9 ±0.6 nm of citrate-stabilized Au NPs • 42.1 ±4.0 nm of ds(sgc8c)-Au NPs Au Gold-Thiol Bonding

5. Sgc8c aptamer • Parameters: • Frequency: 10 MHz • Acoustic pressure: 4 MPa, • Duty cycle: 10% • Treatment time: 10 min Dox-DNA Complex Au Au Cells (2×105 cells/well) were incubated with Dox-loaded nanoconjugates (4.9 nM) loadedwith1.35-μM Dox in RPMI-1640 medium for 2 h. PTK7 CEM Cell HIFU Treatment Nucleus http://oncozine.ning.com/profiles/blogs/new-drug-delivery-system-gold

6. Experimental Setup Power Amplifier • Flow cytometry: Mean Fluorescence Intensity (MFI) • MTT assay: Cell Viability Immediately Waveform Generator 2 Agar Phantom Waveform Generator 1 10-MHz HIFU Transducer 24 h later Trigger

7. Cellular Uptake Enhanced by Sgc8c Dark field images of cells processed for silver enhancement. Dox:ds(control)-Au NPs Dox:ds(sgc8c)-Au NPs ~9000 Dox:ds(sgc8c)-Au NPs per cell determined by atomic absorption spectra.

8. Dox Release from Gold Nanoparticles 10-MHz HIFU, 4 MPa, duty cycle 10% (focal intensity ~140 W/cm2) Pre-HIFU Treatment Post-HIFU Treatment Significant increase in fluorescence intensity indicates the release of Dox.

9. Dox Release from Gold Nanoparticles 10-MHz HIFU, 4 MPa, duty cycle 10% (focal intensity ~140 W/cm2) Flow cytometric analysis MTT Assay ** ** **: p < 0.01 15% No cytotoxicity was observed for Dox:ds(control)-Au NPs (withoutspecificbindingaffinity) ds(sgc8c)-Au NPs (without Dox payload)

10. Comparisons of Different Cases Dox release was only achieved when both Dox-DNA complexes and Au NPs were present.

11. Varying HIFU Parameters Release of Dox can be achieved by ultrasound excitation at low acoustic pressures and duty cycles Duty cycle: 10% Pressure: 4 MPa.

12. Possible mechanisms Au ** • Thermal effects? • Mechanical effects? Microcavitation Temperature (˚C) ΔMFI (a.u.) ΔMFI (a.u.) Normal Degassed Focal intensity: ~140 W/cm2 Sonication Time (s)

13. Intracellular Reactive Oxygen Species Cells were treated with DCFH-DA before HIFU treatments ROS production was observed in Cells with ds(sgc8c)-Au NPs after HIFU treatments. s

14. Summary • Au NPs conjugated with Dox-DNA complexes have shown to be ultrasound-responsive drug delivery systems. The drug release mechanism was found to relate to microcavitation. • The presence of Au NPs might provide nucleation sites for facilitating gas nuclei on the surface of Au NPs to grow into cavitation bubbles under HIFU excitation. • Further investigation is required to clarify which phenomena during microcavitation responsible for the Dox release. • ROS scavenger treatment. • DNA integrity evaluation. • Different HIFU parameters. • Different types of nanoparticles.

15. Thanks for your attention.

16. Enhanced Dox LeakageAfter HIFU Treatment 2.6-fold increase

17. Varying Concentration of Au and Gas Content (ROS measurement)

18. Cellular uptake of ds(sgc8c)-Au NPs determined by atomic absorption spectra.

19. TEM image of 13-nm Au NPs 20 μm

20. MTT Assay nontreatedcells, and cells incubated with Dox Dox:ds(sgc8c), Dox:ds(sgc8c)-Au NPs Dox:ds(control)-Au NPs ds(sgc8c)-Au NPs

21. ROS Measurement • Superoxide anion (,Lifetime: 3 us) • Hydroxyl radical (OH ·,Lifetime: <1 us) • Singlet oxygen (1O2, Lifetime: <1 us) • Hydrogen peroxide (H2O2, Lifetime: ~ms)

22. Possible Mechanisms Au • Pyrolysis in bubble interior. • Dissociation due to high temperature. • Hydroxylation at bubble interface. • Reaction with Reactive Oxygen Species. • Super-critical water at bubble interface. I. Hua, R. H. Hoechemer, and M. R. Hoffmann, “Sonolytic Hydrolysis of p-Nitrophenyl Acetate: The Role of Supercritical Water,” The Journal of Physical Chemistry, vol. 99, no. 8, pp. 2335-2342, Feb. 1995.