Disclosure Information
Sunil Krishnan
Converging physical and biological
strategies for radiation sensitization of
tumors using nanoparticles
I have the following financial relationships to disclose:
Grant or research support from:
Genentech, Merck, Hitachi
Honoraria from:
Carestream Molecular Imaging
I WILL include discussion of investigational or off-label use
of a product in my presentation.
Sunil Krishnan, MD
Radiation Oncology
Nanoparticles
Gold nanoshells
• Dielectric silica core
• Thin gold coating
• Light absorbed by the
free electrons on the
gold is converted to
heat
• Core-shell ratio
determines the
optical characteristics
1
Electromagnetic spectrum
Light – non-ionizing, safe,
affordable, non-invasive
Penetration depth in tissues
depends on the wavelength
and tissue type
Near infrared region
Clinical optical window
Tissue penetration up to 5 cm
Accumulation in tumors
Why gold nanoshells?
Robust structure
less susceptible to chemical/thermal denaturation
Biocompatiblity (silica, noble metal surface)
acceptable toxicity at high concentrations (up to 3% of
body weight) of gold in the body
Very high absorption cross section
~ 3.8 x 10 –14 m2 vs. 1.66 x 10-20 m2 for ICG
L.R.Hirsch et al. PNAS, 100 (23), 13549-13554.
Ease of surface modification for bioconjugation and
PEGylation
less uptake in liver
longer biological half-life in blood due to slower clearance
from the body
Gold nanoshells
Enhanced Permeability and Retention (EPR) effect through leaky
vasculature and inefficient lymphatic drainage of tumors
(size : 60 to 400 nm size)
Brigger et al, Adv. Drug Deliv. Rev. 54, 2002
Wide interendothelial junctions, incomplete or absent basement
membrane, a dysfunctional lymphatic system and large number of
transendothelial channels.
O’Neal P et al. Cancer Lett 209(2):171-6, 2004
2
Hyperthermia
Is thermoradiotherapy underutilized ?
Invasive techniques
No real-time temperature monitoring or dosimetry
No uniform description of dose, time attributes
Gold nanoshell mediated hyperthermia
Laser

 em
P max
Diomed – 15 plus
Delivery
Fiber optic cable
collimating lens
Beam Dia
Exp time
Aiming beam
1 cm
15 to 20 minutes
632 HeNe laser
Temperature measurements
Invasive method
Non-invasive method
Needle thermocouple
Magnetic Resonance Thermal imaging
(MRTI)
808 nm
15 W
Class 3b or 4
Diagaradjane et al. Nano Lett. 2008 8(5):1492-500
3
Thermocouple measurements
MRTI
15
14
T (in tumor center)
12
Laser power
10
T(C)
0.6 W/
cm2
0.4 W/ cm2
~ 13 to 15 ºC (n=2)
0 min
5 min
10
8
~ 10  1.5 ºC (n=4)
6
T (ºC)
0.8 W/ cm2
~ 4 to 5 ºC (n=2)
4
Tumor-bottom
2
5
Tumor-core
0
0
300
600
900
1200
1500
10 min
1800
20 min
Time (sec)
MRTI & thermocouple measurements demonstrated a T ~ 11 °C (from a baseline of ~ 30 °C )
Irradiation with laser alone (no nanoshells) demonstrated a T ~ 2 to 3 °C
Temperature profiles
Real time MRTI
Thermocouple
15ºC
MRTI
14
14
12
12
10
8
T (C)
T (ºC)
10ºC
T(C)
10
6
4
Tumor Temperature
2
Tumor-core
0
0
0
(0.6 W/cm2 for 20 min at 808-nm)
6
4
Tumor-bottom
2
5ºC
8
300
600
900
1200
1500
Time (sec)
= 808 nm;
Power = 0.6 W (75 % duty cycle);
Power density = 350 mW/cm2
1800
0
300
600
900
1200
1500
1800
Time (sec)
Exp time = 20 min
Beam Dia = 1 cm
4
Dynamic contrast enhanced MRI
Post-Hyperthermia
Pre-Contrast
Image
DCMRI
Pre-hyperthermia
DCMRI
Post-hyperthermia
Pixel intensity
Distribution in ROI
T2-weighted
image
Pre-Hyperthermia
Dynamic contrast enhanced MRI
Increased perfusion with ~ 2-fold increase in the contrast enhancement was observed
immediately (3 to 5 min) after gold nanoshell mediated hyperthermia.
Contrast uptake
Experimental groups
Tumor Center
Control
Hyperthermia
Radiation
Hyp + Rad
Whole Tumor
(n=7)
(n=7)
(n=7)
(n=7)
Radiation
350
250
300
C o n tra s t u p ta k e (a .u )
C o n tra s t u p ta k e (a .u )
200
150
100
50
0
50
100
150
200
T im e (s e c )
250
300
200
Phillips RT-250 Orthovoltage X-ray Unit
150
100
50
P re h y p e rth e rm ia
P o s t h y p e rth e rm ia
0
Radiation Dose
250
125 Kv; 20 mA ; 2 mm Al filter
P re h yp e rth e rm ia
P o st h yp e rth e rm ia
0
350
0
50
100
150
200
250
300
350
Skin cone – 1.5 cm diameter
T im e (s e c )
Total delivered dose = 10 Gy
5
Normalized tumor volume
Tumor doubling time
5.0
Control
*
35
Hyp
4.0
Average tumor doubling time (days)
Normalized tumor volume (mm 3)
40
Rad
4.5
Rad+Hyp
3.5
3.0
2.5
2.0
1.5
30
25
20
15
10
5
1.0
~5 days
0.5
0
5
~8 days
~13 days
10
~14days
15
20
0
25
Hyperthermia
Radiation
Hyp+Rad
* P < 0.005
Days
H&E
Hypoxia, cell proliferation, perfusion
Hyperthermia
Radiation
Thermoradiotherapy
Core
Periphery
Control
Control
30
6
Microvessel staining – CD31
Microvessel Density
Raw Image
450
Tumor periphery
Processed Image
Average number of blood vessels
400
Tumor core
350
300
250
200
150
100
50
0
Control
Scanning Electron Microscopy
Hyp
Rad
Hyp+Rad
Conclusions

Optically activated gold nanoshells serve as a novel means to noninvasively generate hyperthermia.

Temperature profiles can be monitored regionally and globally within
tumors using MRTI.

Combining low-dose hyperthermia with radiation therapy leads to
potent radiosensitization that is characterized by the dual effect of:
(a) an initial increase in vascular perfusion of the hypoxic core of
the tumor resulting in tumor cell radiosensitization, and
(a) a subsequent disruption of vasculature that results in a
profound increase in the size of the necrotic core of the
tumor.
7
Enhancing physical dose enhancement
Conclusions
Early effects
T2 MRI image
Late effects
Necrosis
Pixel intensity
Prehyperthermia
Pimonidazole = green
Hoechst = blue
h
h
on the order of 10 m
Radiation
e-
ee-
Anti-hypoxic effect
Vascular disrupting effect?
Posthyperthermia
250
Hyperthermia +
Radiation
Contrast enhancement in tumor center
nanoparticles
nanoparticles
+ peptides
Passive targeting
Active targeting
C o n tra s t u p ta k e (a .u )
200
150
100
50
P re h y p e rth e rm ia
P o s t h y p e rth e rm ia
0
0
50
100
150
200
250
300
350
T im e (s e c )
Nanoparticles
Nanoparticles
Emission spectrum of blue 12nm nanocrystal excited at 360nm
8
Nanoparticles
Nanoparticles
Emission spectra of nanocrystals of varying sizes
all being excited at the same wavelength (360nm)
532 nm
488 nm
405 nm
Peptide-nanoparticle bioconjugates
1. Flexible excitation but very discrete emission
Comparison to other fluorescent dyes:
Black – Alexa488
Blue – Cy3
Red – Alexa568
Green – Nanocrystal
Quantitative, can be multiplexed
2. Lower limits of detection (dye curves were blown up
10-1000 times to be seen on this graph)
Nano- or pico-molar concentrations enough
3. Less photobleaching
Photostable, permits repetitive imaging
In vivo imaging and spectral unmixing
Raw image
Unmixed Autofluorescence/
Background
NH2
ZnS
NH2
S
O
CdTe
NH
SH
HS
N
SH
SH
SH
O
O
NH2
~21 nm
~0.83 nm
~5 nm
EGF-conjugated Quantum Dot
Diagaradjane et al. Clin Cancer Res. 2008, 14(3):731-41.
Unmixed Quantum dot
fluorescence
Remixed background
and Quantum dot
fluorescence
9
In vivo quantification
In vivo testing
After Cetuximab
EGF-QD-800
: 10 pmol
HCT 116 colorectal xenografts
Ex vivo characterization
Ex vivo characterization
EGF-QD-800
CD31
Brain
Heart
Lung
Liver
Spleen
Kidney
QD-800
24 hr
QD-800
4 hr
24 hr
EGF-QD-800
4 hr
EGFR
Tumor
10
Concept
Concept
CONJUGATED
UNCONJUGATED
Uptake
Clearance
Accumul.
5 min
1 hr
4-6 hrs
Resid.
24 hrs
Uptake
5 min
Clearance
1 hr
Accumul.
4-6 hrs
CONJUGATED
UNCONJUGATED
Resid.
24 hrs
Uptake
Clearance
Accumul.
5 min
1 hr
4-6 hrs
Concept
Resid.
24 hrs
Uptake
5 min
Clearance
1 hr
Accumul.
4-6 hrs
Resid.
24 hrs
Concept
CONJUGATED
UNCONJUGATED
Uptake
Clearance
Accumul.
5 min
1 hr
4-6 hrs
Resid.
24 hrs
Uptake
5 min
Clearance
1 hr
Accumul.
4-6 hrs
CONJUGATED
UNCONJUGATED
Resid.
24 hrs
Uptake
Clearance
Accumul.
5 min
1 hr
4-6 hrs
Resid.
24 hrs
Uptake
5 min
Clearance
1 hr
Accumul.
4-6 hrs
Resid.
24 hrs
11
A unique particle
Conjugated gold nanorod
Gold nanorod
Tumor regrowth delay
4.0
4 hrs
24 hrs
Normalized Tumor Volume
30 min
C225-GNR
PEG-GNR
Control
Control
3.8
PEG-GNR
3.6
C-GNR
3.4
Cetuximab
Rad
3.2
Cetuximab + Rad
3.0
PEG-GNR + Rad
2.8
C-GNR + Rad
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0
5
10
15
20
25
30
Days after treatment
Krishnan lab, unpublished data
12
Biodistribution
Clonogenic survival
30 min
S u rv iv in g F ra c tio n
35
30
25
20
15
10
S u rv iv in g F ra c tio n
PEG-GNR
C225-GNR
40
0.100
0.010
C o n tro l
P E G -G N R
C 225-G N R
0.001
5
0.100
0.010
C ontrol
P E G -G N R
C 225-G N R
0.001
0
Brain
Heart
Lung
Liver
Spleen Kidney Tumor
0.000
Blood
0.000
0
2
4
6
8
0
2
4
D o se (G y)
Cho SH. Phys Med Biol 2005; 50: N163-73
DNA damage
6
8
D ose (G y)
DEF 10%
DEF 15%
DNA damage
70
No Radiation
60
Average Number of Foci per cell
% ID
24 hrs
1.000
1.000
45
Radiation (4 Gy)
GNR + Rad (4 Gy)
C225-GNR + Rad (4 Gy)
50
40
30
20
10
0
0.05
0.5
1
2
Time after irradiation (hrs)
R4
C
0
1
R4 + GNR
4
C
0
1
4
24
R4 + cGNR
4
C
0
1
4
 γ H2AX
 H2AX
 PARP
13
Total oxidative stress
Tissue effects
Post irradiation time
Rad
1.2
GNR
1.1
CGNR
PEG-GNR
+
Radiation
1.1
1.0
1.0
0.9
0.9
0.8
0.8
Control
Immediate
1 hr
4 hrs
C225-GNR
+
Radiation
Normalized protein carbonyl content
Radiation
1.3
1.2
4 days
4 hrs
Protein carbonyl assay
Time after 4 Gy radiation
Tissue effects
Intracellular distribution
Time
350
Average microvessel density
per field of view with 10X objective
Radiation (10 Gy)
300
GNR + Rad (10Gy)
*
C225-GNR + Rad (10 Gy)
250
200
150
100
50
0
4 hrs
4 days
Post irradiation period
14
Tissue distribution
Tissue distribution
Modeling dose
Summary
• Targeted payload delivery feasible with smaller
nanoparticles bioconjugated to peptides/antibodies
• While the tumor accumulation does not increase
dramatically, the distribution is altered at the cellular
(internalized) and tissue (more perivascular) levels
• Both the intracellular localization and the perivascular
sequestration result in greater radiosensitization at a
biological level, mediated primarily by:
•Increased DNA damage and downstream signaling
•Increased oxidate stress
•Increased vascular disruption
15
Other nanoparticles
Translational issues
• Biocompatibility – toxicity, stability
Cooling
device
Capacitor
bank
Inductor
• Biodistribution/kinetics
- renal filtration
- RES capture
- internalization
- modeling
• Quantify, Visualize, Predictive Dosimetry
• Clinical applicability
Krishnan et al. Intl. J Hyperthermia. 2010
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
Translational issues
Translational issues
• Biocompatibility – toxicity, stability
• Biocompatibility – toxicity, stability
• Biodistribution/kinetics
• Biodistribution/kinetics
- renal filtration
- RES capture
- internalization
- modeling
- renal filtration
- RES capture
- internalization
- modeling
• Quantify, Visualize, Predictive Dosimetry
• Quantify, Visualize, Predictive Dosimetry
• Clinical applicability
• Clinical applicability
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
16
Renal filtration
Translational issues
• Biocompatibility – toxicity, stability
• Biodistribution/kinetics
- renal filtration
- RES capture
- internalization
- modeling
• Quantify, Visualize, Predictive Dosimetry
• Clinical applicability
Choi HS et al. Nature Biotech 2007, 25: 1165 - 1170
RES capture
EGF-QD
Translational issues
GdCl3 + EGF-QD
(a)
• Biocompatibility – toxicity, stability
Background
QD
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
3 min
• Biodistribution/kinetics
5µ
1 hr
(b)
- renal filtration
- RES capture
- internalization
- modeling
• Quantify, Visualize, Predictive Dosimetry
4 hrs
• Clinical applicability
5µ
Diagaradjane et al. ACS Nano, 2010
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
17
Translational issues
Quantifying gold nanoparticles in tumor
• Biocompatibility – toxicity, stability
• Biodistribution/kinetics
- renal filtration
- RES capture
- internalization
- modeling
• Quantify, Visualize, Predictive Dosimetry
• Clinical applicability
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
Quantifying gold nanoparticles in tumor
Zaman et al IEEE J Sel Top Quant Elec 13(6):1715-20, 2007.
Zaman et al IEEE J Sel Top Quant Elec 13(6):1715-20, 2007.
Imaging gold nanoparticles in tumors
Puvanakrishnan P et al. J Biomed Optics 14(2):024044, 2009.
18
Thermal dosimetry
Cheong S-K et al. Med Phys 36(10):4664-71, 2009
Puvanakrishnan P et al. J Biomed Optics 14(2):024044, 2009.
Translational issues
- renal filtration
- RES capture
- internalization
- modeling
• Quantify, Visualize, Predictive Dosimetry
Immediately
after surgery
3 weeks
after surgery
While light photograph
• Biodistribution/kinetics
Bioluminescence
• Biocompatibility – toxicity, stability
Before Surgery
X 10 6 Ph/Sec/cm2/sr
Imaging gold nanoparticles in tumors
• Clinical applicability
- Light: depth of penetration - ?intraop
- Magnetic: focusing, AMF field strength
19
Summary
• Larger particles for vascular-targeted applications (thermoablation, hyperthermia, vascular imaging)
• Smaller particles for parenchymal applications (imaging,
targeted payload delivery)
• Radiation dose enhancement
• Combinations of above
• Unresolved issues related to clinical translation
Atkinson RA, et al. Sci Translat Med, 2010; 2(55):55ra79
Acknowledgements
Krishnan lab
UT Austin
Baylor
Parmesh Diagaradjane
Amit Deorukhkar
Dev Chatterjee
Nga Ta
Krystina Sang
Jacobo Orenstein Cardona
Norman Colon
Hee Chul Park
Brook Walter
James Tunnell
Raiyan Zaman
Priya Puvanakrishnan
Jaesook Park
Jeffrey Rosen
Rachel Atkinson
Georgia Tech
Sang Cho
Seong-Kyun Cheong
Bernard Jones
Nanospectra
Don Payne
Jon Schwartz
Glenn Goodrich
James Wang
Imaging Physics
John Hazle
Jason Stafford
Anil Shetty
Andrew Elliott
Exp Diag Imaging
Juri Gelovani
Funding
NIH - KL2, R21, R01, U01
DOD – pre-center grant
UT Cntr Biomed Engg, Hitachi, MDACC
20