Gold Nanorods as Absorption Contrast Agents for Noninvasive Detection of Cancer and Arterial Vascular Disorders

Dr. Rinat Ankri and Dr. Dror Fixler

Faculty of Engineering and the Institute of Nanotechnology and

Advanced Materials, Bar-Ilan University, Israel

Input Diffuse

Reflection

Absorption

Diffuse Transmission

Scattering

Specular

Reflection

Direct Transmission

Light Interaction with a Turbid Medium

Light Path in Irradiated Tissues- The Diffusion Theory

1

3( ')a s

D

2a

JD J J

t

Where: ρ= light source-detector distance

Thus, the diffusion reflection (DR) intensity depends on µa and µs’:

12( ) exp( )

m

cc

Where:

2 3 's ac

Schmitt et al., 1990m = 1 or 2

m= ?

250 µm per step650 nm PD Output

Digital ScopePD InputOptical fiber

PD

 

The experimental set-up

Sample

Fiber Optic

Detector1mm

250 µm per step

1 2 2 4 5 6-2.5

-2

-1.5

-1

-0.5

0

(mm)

ln(

((

))

0.0137 mm-1

0.0182 mm-1

0.0227 mm-1

The Diffusion Theory; m=1

0.01 0.012 0.014 0.016 0.018 0.02 0.022 0.0240

0.05

0.1

0.15

0.2

0.25

f(x) = 8.61054770891312 x − 0.0143171977361507

f(x) = 8.22666502644852 x + 0.0205806933202116

m=1SimulatedLinear (Simulated)Exprimental

Absorption coefficient (mm-1)

slop

e2

Expected µs’=1.6 mm-1*

* Cubeddu et.al., 1993

Experimental µs’~2.8 mm-1

Simulation

Experiments

1 2ln( ( ))m c c

2 3 's ac

1 2 3 4 5 6-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

(mm)

ln(

2 ((

))

0.0182 mm-1

0.0137 mm-1

0.0227 mm-1

0.01 0.012 0.014 0.016 0.018 0.02 0.022 0.0240

0.05

0.1

f(x) = 4.44673158121791 x − 0.0393530535243912

f(x) = 4.75326397465863 x − 0.0268147770882027

m=2SimulatedLinear (Simulated)Exprimen-tal

absorption coefficient (mm-1)Sl

ope2

The Diffusion Theory; m=2

Expected µs’=1.6 mm-1*

Experimental µs’~1.5 mm-1

* Cubeddu et.al., 1993

Simulation

Experiments

Ankri et.al, The Open Optics Journal, 2011

m= 2 !

Diffusion Reflection and Gold Nano Particles

Structural Imaging Molecular Imaging

The Vision“Fantastic Voyage” Isaac Asimov (1966)

Why Gold?

Subcellular Cellular Whole body

TEM FAAS CT

The Concept

Hand with Ring (Wilhelm Röntgen,1895)

Surface plasmon resonance

Figure: Schematic of plasmon oscillation for a sphere, showing

the displacement of the conduction electron charge cloud

relative to the nuclei.

When a nanoparticle is much smaller than the wave length of light, coherent oscillation of the conduction

band electrons induced by interaction with an electromagnetic field. This resonance is called Surface

Plasmon Resonance (SPR).

Optical Properties of Gold Nanorods

15

Gold nanorods have shape and size-dependent optical properties originating from anisotropic shape and tunable aspect ratio.

Aspect Ratio (AR) = L/W

L

W

Nikoobakht et al. Chem Mater. 2003, 15, 1957-1962.

Gold Nanorods

16

Under electromagnetic field of light, the conduction band electrons undergo a collective coherent oscillation in resonance with

the frequency of the incident light. This is known as the localized surface plasmon resonance (LSPR) .

Due to their anisotropic shape, 2 extinction peaks can be observed from gold

nanorods.Huang et al. Adv Mater. 2009, 21, 4880-4910.

A new method for cancer detection based on diffusion reflection measurements of targeted

gold nanorods

400 450 500 550 600 650 700 750 800 850 9000

20

40

60

80

100

Wavelength [nm]

Nor

mal

ized

Inte

nsity

(A.U

)

GNR (25 x 65 nm) UV-Vis absorption spectra (normalized) and TEM image

Au Au

Head & Neck Cancer Cell

Tumor detection based on DR measurements of targeted gold nanorods (GNR)

Enhancing the tumor absorption

coefficient by EFGR labeled GNR

injection.

650 nm

400 450 500 550 600 650 700 750 8000.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

Bare GNR

PEG coated GNR

Anti-EGFR coated GNR

Wavelength (nm)

Nor

mal

ized

abs

orpti

on (a

.u.)

Tumor µa 650 nm

Diffusion Reflection measurements

Before t =10 h0.5

1Cancerous

Normal

Time [hours]Sl

ope

of ln

(Г(ρ

))

Diffusion reflection decreases faster due to GNRs accumulation

6

6

-6 6

6

-6

GNRs injection

Before t=0 t >10 h0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1CancerousNormal

Time [hours]

Slop

e of

ln(Γ

(ρ))

1 2 3 4 5 6-3.5

-2.5

-1.5

-0.5

0

(mm)

ln(

())

Normal before injection

Tumor before injection

Normal, t=10 h

Tumor, t=10 h

Tumor detection based on DR of targeted gold nanorods (GNR)

The tumor presents a slope different from the normal

tissue’s slope even more than 20 hours post injection.

Ankri et.al; A new method for cancer detection based on diffusion reflection measurements of targeted gold nanorods, International journal of Nanomedicine, 2012

2

( )

ln( ( )) 3 '

' 0a s

s GNR

Expected µa (mm-1) Slope2 Experimental µa (mm-1)

0.0123 0.011±0.006 0.012±0.0007

0.0132 0.0143±0.003 0.0131±0.0031

0.0152 0.0256±0.003 0.0146±0.0007

0.0216 0.0625±0.004 0.0229±0.001

0.0312 0.137±0.0016 0.0397±0.0004

0.0986 0.42±0.001 0.104±0.0002

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.090

0.2

0.4

0.6

0.8

1

GNR concentration (mg/ml)

Abs

orpti

on (O

.D)

GNR exntinction coefficient

1 ml/mg*mm

2 3 's ac Different absorption coefficients

Ankri et.al; In-vivo tumor detection using diffusion reflection measurements of targeted gold nanorods- a quantitative study, Journal of Biophotonics, 2012

GNR conc. in tumor FAA spectroscopy

=

Calculating tumor GNR concentration from the DR profile in-vivo

EGFR- targeted

Time (∆Slope)2 GNR concentration (mg/ml)

Before GNR injection 0 0

t=15 min post GNR injection ------------------ ------------------

t= 5 h post GNR injection 0.0036 0.0096

t= 10 h post GNR injection 0.0275 0.015

1 2 3 4 5 60

0.2

0.4

0.6

0.8

1

1.2

1.41.4

(mm)

ln(

2 ((

))

Tumor before

t = 15 min

t=5 h

t=10 h

slopes= GNR concentration

DR measurements of high concentrations of GNR-

a Red shift is observed

Dark field Microscopy:

GNR with different densities

450 500 550 600 650 700 750 800 850 9000

0.2

0.4

0.6

0.8

1

1.2

Wavelength (nm)

No

rmal

ized

ab

sorp

tio

n (

a.u

)

0.01 0.02 0.05 0.1 0.15 0.20

0.2

0.4

0.6

0.8

1

1.2

1.4650 nm780 nm

Concentration (mg/ml)

∆Slo

pe

450 500 550 600 650 700 750 800 850 9000

0.2

0.4

0.6

0.8

1

1.2

Wavelength (nm)

Ab

sorp

tio

n (

a.u

)

λ0 ∆λ

DR measurements of tumor bearing mice with different

concentrations of targeted GNR-

a spectral broadening is observed

1 2 3 4 5 6

-3

-2.5

-2

-1.5

-1

-0.5

0

(mm)

ln(

())

Before, 650 nmBefore,780 nmt>10,650 nmt>10, 780 nm

1 2 3 4 5 6

-6

-5

-4

-3

-2

-1

0

(mm)

ln(

())

Before, 650 nmBefore, 780 nmt>10 h, 650 nmt>10 h, 780 nm

Ankri et.al; Intercoupling surface plasmon resonance and diffusion reflection measurements for real-time cancer detection, Journal of Biophotonics, 2012

NEJM Dec 2013

The Aim: To develop a new, easy to use, and non-invasive method at low cost, to locate atherosclerotic vascular disease at its early

stages, particularly unstable plaques with ongoing inflammation prone to rupture

Stable vs. Unstable (vulnerable) Plaques

Rich in extracellular matrixsmooth muscle cells, thick cap

Rich in macrophages, foam cells,inflammatory cells, thin fibrous cap

Macrophages before incubation with GNRs

Macrophages+ GNRs 0.02 mg/ml Macrophages+ GNRs 0.2 mg/ml

450 470 490 510 530 550 570 590 610 630 65010

12

14

16

18

20

Wavelength (nm)

Refle

ction

(a.u

)

Phantom+GNRs Phantom no GNRs Phantom+ macrophages+GNRs

Phantom+ macrophages

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Slop

e of

DR

profi

le

1 1.5 2 2.5 3 3.5 4 4.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

Source-detector seperation (mm)

Nor

mal

ized

refle

ctan

ce in

tens

ity

(a.u

)

Macrophages +/-GNRs

Homogenous and GNRs

A cross-section of a rat balloon-injured carotid artery 2 weeks post-injury stained withhematoxylin and eosin (H&E) clearly depicts cellular-rich neointima development.Magnification is 100x for the large photomicrograph. A right carotid artery B left injured carotid artery

IN VIVO ; RAT INJURY MODEL

First in vivo DR measurements of atherosclerosis with GNR

Before GNR injection

24h post GNR injection

Control0

2

4

6

8

10

12

3D CT ARTERY IMAGING