Use of Nanoshells for Combined Two-Photon Imaging and Therapy of Cancer

1. Use of Nanoshells for Combined Two-Photon Imaging and Therapy of Cancer Emily S. Day1, Lissett R. Bickford1, Jason H. Hafner2, Rebekah A. Drezek1, and Jennifer L. West1 1Department of Bioengineering 2Department of Physics and Astronomy Rice University Houston, Texas, USA

2. Outline • Motivation for research • Properties and synthesis of nanoshells • Nanoshells in cancer management • Use of nanoshells with two-photon microscopy

3. Strategies for Cancer Treatment • Conventional treatment includes surgery, radiation, and chemotherapy • Limited by invasiveness; residual disease; nonspecific toxicity • Future treatments with nanoparticles are promising • Minimally invasive • Cell-specific targeting • Multi-functionality

4. Nanoshells • Nanoshells are strong near-infrared (NIR) absorbers • Absorption Heat Cancer cell death Dielectric Core Gold Shell of Desired Thickness

5. Nanoshell-Assisted Photothermal Therapy Nanoshells • Nanoshells accumulate within tumor • Irradiate nanoshells with NIR laser • Absorption causes heating of nanoshells and necrosis of tumor tissue NIR Laser Cancer cells Tumor capillary • Extravasation of Nanoshells • Application of NIR Laser • Necrosis

6. 20 nm shell 5 nmshell 20 nm 10 nm 7 nm 5 nm Extinction (Arb. Units) 600 800 500 700 900 1000 1100 1200 Optical Properties of Nanoshells 120 nm silica core Oldenburg, et al. Chemical Physics Letters. 1998. Wavelength (nm)

7. Absorption coefficient (cm-1) Wavelength (nm) Laser-Tissue Interactions • Wavelength dependent absorption by many native chromophores • Near infrared window • 650–900 nm • Low absorption • High transmission • Maximum absorption by nanoshells • Absorption  Heat  Cell Death Weissleder. Nature Biotechnology. 2001.

8. Gold-Silica Nanoshell Fabrication • Grow monodisperse SiO2 cores Stöber Method • Surface aminationAPTES (3-aminopropyltriethoxysilane) • Adsorb gold colloid onto surface2-4 nm diameter • Shell growthGold Reduction—HAuCl4 and formaldehyde Oldenburg SJ, et al. Chemical Physics Letters. 1998.

9. Gold-Silica Nanoshell Fabrication • Shell growth on a silica core (TEM) Oldenburg SJ, et al. Chemical Physics Letters. 1998.

10. Gold-Gold Sulfide Nanoshell Fabrication • Prepare reagents • 2 mM HAuCl4 and 1 mM Na2S • Age 40-48 hours • Mix volumetrically • 1:2 Na2S to HAuCl4 • Gold-sulfide core forms, followed by completion of gold shell 40 nm TEM of Au2S Nanoshells Averitt, et al. Phys Rev Lett. 1997.

11. Extinction of Nanoshells

12. Nanoshell-Assisted Photothermal Therapy • Deliver nanoshells to tumor • Irradiate with NIR laser • Nanoshells convert energy into heat, causing necrosis of tumor tissue The idea: Results of in vitro and in vivo studies have been promising

13. Nanoshell Therapy In Vitro Nanoshells Only Nanoshells + Laser Laser Only Calcein AM Live Stain Phase contrast Hirsch, et al. PNAS. 2003.

14. Antibody Conjugation onto Nanoshells • OPSS-PEG-NHS (2000 Da) • N-hydroxysuccinimide Strong leaving group • Poly(ethylene glycol) Improves antibody mobility • Orthopyridyl disulfide Binds gold surface • PEG-thiol (5000 Da) • Occupies remaining adsorption sites • Eliminates non-specific protein adsorption • Provides steric stabilization NH-PEG-OPSS- Nanoshell NH2 + NHS-PEG-OPSS NH-PEG-OPSS + NHS PEG-SH

15. Antibody Targeting Provides Specific Therapy • Co-culture two cell lines adjacently • SK-BR-3—over-express the HER2 receptor • Human Dermal Fibroblasts (HDF)—control cell type • Incubate with anti-HER2 nanoshells (for cell-specific targeting) or PEG nanoshells (no cellular interactions expected) • Rinse and irradiate at cell interface (820 nm, 88 W/cm2, 7 min) • Assess viability Anti-HER2 Nanoshells PEG Nanoshells Laser spot outlined in white Red Dead Green Alive Lowery, et al. Int J Nanomedicine. 2006.

16. Diode Laser 820 nm Nanoshell Therapy In Vivo Wait 6 hr • CT-26 colon carcinoma tumors grown on BALB/c mice • SiO2 nanoshells (PEG-coated, 3E6 particles) injected into tail vein • Tumors irradiated at 4 W/cm2 for 3 min • Resultant tumor growth/regression monitored O’Neal, et al. Cancer Letters. 2004.

17. Nanoshell Therapy Induced Tumor Regression and Improved Survival Nanoshell treatment group Complete tumor regression 100% survival White Nanoshells + Laser Light gray Laser only Dark gray No treatment O’Neal, et al. Cancer Letters. 2004.

18. Combined Imaging and Therapy • Nanoshells can be used for both imaging and therapy • Culture SK-BR-3 Expose to nanoshells Image via dark-field microscopy Perform laser irradiation Loo, et al. Nano Letters. 2005.

19. Imaging In Vivo • Grow CT-26 tumors in mice Intravenously deliver nanoshells Perform OCT (optical coherence tomography) • Normal tissue shows no difference in contrast between PBS or nanoshell groups • Nanoshell accumulation in tumor tissue dramatically increased the OCT contrast

20. Improving Combined Imaging and Therapy • Ideal scenario • Locate tumor with wide-field imaging • Pinpoint precise treatment sites with high-resolution imaging Two-photon microscopy offers the possibility to use one system to “see and treat”

21. Two-Photon Excitation of Fluorophores Visible excitation NIR excitation P α I2 Single-photon and two-photon illumination of fluorescein Soeller and Cannell. Microsc Res Tech. 1999.

22. Two-Photon Excitation of Metals • Two-photon excitation of metals is a different process than described for fluorophores • Absorption of two photons results in: • Excitation Relaxation Emission • NIR-absorbing nanoparticles are ideal contrast agents for two-photon microscopy • Low background signal • Depth of penetration • Plasmonic materials exhibit enhanced two-photon luminescence Mooradian. Phys Rev Lett. 1969.; Boyd, et al. Phys Rev B. 1986.

23. Nanoshells Display Two-Photon Luminescence • Silica nanoshells have been used for TPL both in vitro and in vivo • Coat silica nanoshells with anti-HER2 antibody • Incubate with SK-BR-3 cells (HER2+) or MCF10A cells (control) • Perform two-photon microscopy SK-BR-3 cells MCF10A cells Scale bar= 20 µm Bickford, et al. Nanotechnology. 2008.

24. Two-Photon Microscopy In Vivo • CT26 tumor-bearing BALB/c mice receiving intravenous delivery of silica nanoshells exhibit increased contrast compared to mice without nanoshells White light Two-photon With Nanoshells Without Nanoshells Park, et al. OptExpress. 2008.

25. Combined Two-Photon Imaging and Therapy • SiO2 nanoshells have been successful as a two-photon contrast agent and as a tool for photothermal therapy • Build upon this work by combining imaging and therapy • “See and Treat” • Low laser power Imaging • High laser power Heating, cell death • New data focuses on Au2S nanoshells since they are more efficient NIR-absorbers

26. Two-Photon System Configuration META Detector IR-Blocking Filter Plate Dichroic Mirror Ti: Sapphire Laser 20X Objective Sample

27. Proof of Two-Photon Excitation • Quadratic dependence of luminescence on power verifies two-photon excitation of Au2S and SiO2 nanoshells

28. Two-Photon Imaging and Therapy In Vitro • SK-BR-3; HER2+ breast carcinoma • Three different coatings: • Anti-HER2 + PEG-SH • Anti-IgG + PEG-SH • PEG-SH only • Mix in suspension for 30 minutes • 600,000 cells + 1 ml nanoshells at OD=1 • Rinse to remove unbound particles • Let cells adhere overnight at 37°C • Imaging—10 mW; 15 sec • Therapy—50 mW; 15 sec

29. Two-photon microscopy performed with Au2S nanoshells provides enhanced contrast of cancer cells Anti-HER2 Anti-IgG PEG-SH Next step: Repeat exposure at 50 mW and perform calcein AM viability staining. Excitation: 800 nm; 10 mW

30. Cell death induced only in the presence of targeted Au2S nanoshells and 50 mW laser exposure Anti-HER2 Anti-IgG PEG-SH 10 mW 50 mW Green= Calcein AM viability stain

31. Summary of Results • Au2S nanoshells strongly absorb near-infrared light, rendering them useful for two-photon applications • Verified two-photon induced photoluminescence • Absorption Light & Heat • Low laser power Imaging • High laser power Therapy • Effective imaging and therapy only when targeted nanoshells are used

32. Advantages of Nanoshells + Two-Photon Microscopy • Localized therapy only where nanoshells and NIR light are combined • Minimally invasive and highly effective • Ability to “see and treat” with one setup • Potential to provide very specific therapy after initial imaging with wide-field modalities

33. Acknowledgments West Lab Members National Science Foundation National Institutes of Health Center for Biological and Environmental Nanotechnology

34. Extra Content

35. Photoluminescence of Metals • Single-photon luminescence of metals first described in 1969 Excitation Relaxation Emission • Metal luminescence further explored in 1980s by Boyd, et al. • Two-photon luminescence only observed on roughened metal surfaces Mooradian. Phys Rev Lett. 1969 • Conclusion: Two-photon luminescence is amplified by local field enhancements due to local plasmon resonance Boyd, et al., Phys Rev B, 1986.