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Use of Nanoshells for Combined Two-Photon Imaging and Therapy of Cancer. Emily S. Day 1 , Lissett R. Bickford 1 , Jason H. Hafner 2 , Rebekah A. Drezek 1 , and Jennifer L. West 1. 1 Department of Bioengineering 2 Department of Physics and Astronomy Rice University Houston, Texas, USA.

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use of nanoshells for combined two photon imaging and therapy of cancer

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

  • Motivation for research
  • Properties and synthesis of nanoshells
  • Nanoshells in cancer management
  • Use of nanoshells with two-photon microscopy
strategies for cancer treatment
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
  • Nanoshells are strong near-infrared (NIR) absorbers
    • Absorption Heat Cancer cell death

Dielectric Core

Gold Shell of Desired Thickness

nanoshell assisted photothermal therapy
Nanoshell-Assisted Photothermal Therapy


  • 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
optical properties of nanoshells

20 nm shell

5 nmshell

20 nm

10 nm

7 nm

5 nm

Extinction (Arb. Units)









Optical Properties of Nanoshells

120 nm silica core

Oldenburg, et al. Chemical Physics Letters. 1998.

Wavelength (nm)

laser tissue interactions

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.

gold silica nanoshell fabrication
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.

gold silica nanoshell fabrication1
Gold-Silica Nanoshell Fabrication
  • Shell growth on a silica core (TEM)

Oldenburg SJ, et al. Chemical Physics Letters. 1998.

gold gold sulfide nanoshell fabrication
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.

nanoshell assisted photothermal therapy1
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

nanoshell therapy in vitro
Nanoshell Therapy In Vitro

Nanoshells Only

Nanoshells + Laser

Laser Only

Calcein AM Live Stain

Phase contrast

Hirsch, et al. PNAS. 2003.

antibody conjugation onto nanoshells
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







antibody targeting provides specific therapy
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.

nanoshell therapy in vivo

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.

nanoshell therapy induced tumor regression and improved survival
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.

combined imaging and therapy
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.

imaging in vivo
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
improving combined imaging and therapy
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”

two photon excitation of fluorophores
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.

two photon excitation of metals
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.

nanoshells display two photon luminescence
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.

two photon microscopy in vivo
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






Park, et al. OptExpress. 2008.

combined two photon imaging and therapy
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
two photon system configuration
Two-Photon System Configuration

META Detector

IR-Blocking Filter


Dichroic Mirror

Ti: Sapphire Laser

20X Objective


proof of two photon excitation
Proof of Two-Photon Excitation
  • Quadratic dependence of luminescence on power verifies two-photon excitation of Au2S and SiO2 nanoshells
two photon imaging and therapy in vitro
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

Two-photon microscopy performed with Au2S nanoshells provides enhanced contrast of cancer cells




Next step: Repeat exposure at 50 mW and perform calcein AM viability staining.

Excitation: 800 nm; 10 mW


Cell death induced only in the presence of targeted Au2S nanoshells and 50 mW laser exposure




10 mW

50 mW

Green= Calcein AM viability stain

summary of results
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
advantages of nanoshells two photon microscopy
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

West Lab Members

National Science Foundation

National Institutes of Health

Center for Biological and Environmental Nanotechnology

photoluminescence of metals
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.