Combining nanoparticles with radiation rationally –
are we there yet?
Sunil Krishnan, MD
Director, Center for Radiation Oncology Research
MD Anderson Cancer Center
Disclosure Information
Sunil Krishnan
I have the following financial relationships to disclose:
Grant or research support from:
Genentech, Merck, Hitachi, Shell, MPOB, FUSF
Honoraria from:
Carestream Molecular Imaging
I WILL include discussion of investigational or off-label use
of a product in my presentation.
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 2-3
cm
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
Accumulation in tumors
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.
2
Gold nanoshells
O’Neal P et al. Cancer Lett 209(2):171-6, 2004
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
808 nm
15 W
Class 3b or 4
Diagaradjane et al. Nano Lett. 2008 8(5):1492-500
3
Temperature measurements
Invasive method
Non-invasive method
Needle thermocouple
Magnetic Resonance Thermal imaging
(MRTI)
Thermocouple measurements
14
DT (in tumor center)
12
Laser power
10
DT(C)
0.8 W/ cm2
0.6 W/ cm2
0.4 W/
cm2
~ 13 to 15 ºC (n=2)
8
~ 10  1.5 ºC (n=4)
6
~ 4 to 5 ºC (n=2)
4
Tumor-bottom
2
Tumor-core
0
0
300
600
900
1200
1500
1800
Time (sec)
MRTI
15
5 min
10
DT (ºC)
0 min
5
10 min
20 min
MRTI & thermocouple measurements demonstrated a DT ~ 11 °C (from a baseline of ~ 30 °C )
Irradiation with laser alone (no nanoshells) demonstrated a DT ~ 2 to 3 °C
4
Real time MRTI
15ºC
DT (ºC)
10ºC
5ºC
(0.6 W/cm2 for 20 min at 808-nm)
Temperature profiles
Thermocouple
MRTI
14
14
12
12
10
8
DT (C)
DT(C)
10
6
4
8
6
4
Tumor-bottom
2
Tumor Temperature
2
Tumor-core
0
0
0
300
600
900
1200
1500
1800
0
300
= 808 nm;
Power = 0.6 W (75 % duty cycle);
Power density = 350 mW/cm2
600
900
1200
1500
1800
Time (sec)
Time (sec)
Exp time = 20 min
Beam Dia = 1 cm
Dynamic contrast enhanced MRI
Pre-Hyperthermia
Post-Hyperthermia
5
Dynamic contrast enhanced MRI
DCMRI
Pre-hyperthermia
DCMRI
Post-hyperthermia
Pixel intensity
Distribution in ROI
T2-weighted
image
Pre-Contrast
Image
Increased perfusion with ~ 2-fold increase in the contrast enhancement was observed
immediately (3 to 5 min) after gold nanoshell mediated hyperthermia.
Contrast uptake
Tumor Center
Whole Tumor
350
250
300
Contrast uptake (a.u)
Contrast uptake (a.u)
200
150
100
50
0
50
100
150
200
250
300
200
150
100
50
Pre hyperthermia
Post hyperthermia
0
250
Pre hyperthermia
Post hyperthermia
0
350
0
50
100
150
200
250
300
350
Time (sec)
Time (sec)
Experimental groups
Control
Hyperthermia
Radiation
Hyp + Rad
(n=7)
(n=7)
(n=7)
(n=7)
Radiation
Radiation Dose
Phillips RT-250 Orthovoltage X-ray Unit
125 Kv; 20 mA ; 2 mm Al filter
Skin cone – 1.5 cm diameter
Total delivered dose = 10 Gy
6
Normalized tumor volume
5.0
Control
Rad
Normalized tumor volume (mm 3)
4.5
Hyp
4.0
Rad+Hyp
3.5
3.0
2.5
2.0
1.5
1.0
~5 days
0.5
0
5
~8 days
~13 days
10
~14days
15
20
25
30
Days
Tumor doubling time
40
*
Average tumor doubling time (days)
35
30
25
20
15
10
5
0
Control
Hyperthermia
Radiation
Hyp+Rad
* P < 0.005
H&E
Hyperthermia
Radiation
Thermoradiotherapy
Core
Periphery
Control
7
Hypoxia, cell proliferation, perfusion
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
Hyp
Rad
Hyp+Rad
8
Scanning Electron Microscopy
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.
Conclusions
Early effects
T2 MRI image
Late effects
Necrosis
Pixel intensity
Prehyperthermia
Pimonidazole = green
Hoechst = blue
Radiation
Anti-hypoxic effect
Vascular disrupting effect?
Posthyperthermia
250
Hyperthermia +
Radiation
Contrast enhancement in tumor center
Contrast uptake (a.u)
200
150
100
50
Pre hyperthermia
Post hyperthermia
0
0
50
100
150
200
250
300
350
Time (sec)
9
Atkinson RA, et al. Sci Translat Med, 2010; 2(55):55ra79
Physical dose enhancement
Hainfeld et al. Phys Med Biol 2004; 49: N309-15
Physical dose enhancement
Cho et al. Phys Med Biol 2009
10
Physical dose enhancement
Cho et al. Phys Med Biol 2009
Physical dose enhancement
Cho et al. Phys Med Biol 2009, 54(16):4889-905.
Physical dose enhancement
Cho, Krishnan Med Phys 2010
11
Physical dose enhancement
Enhancing physical dose enhancement
hn
on the order of 10 m
hn
e-
ee-
nanoparticles
nanoparticles
+ peptides
Passive targeting
Active targeting
Peptide-nanoparticle bioconjugates
NH2

NH2 S-S
ZnS
NH2
NH2
NH2
ZnS
1hr, 21 °C
O

O
CdTe
NH2
+
+
CdTe
N O
HS
OH
N
NH
O
O O
SH
N
S
O
SH
SH
30 min, 21HS
°C CdTeHSSH

NH2
AminoEGF
functionalized
Quantum Dot
NH2
ZnS
O
O
SH
NH
SH
HS
O
NH2
O
SH N
SH
O
SH
OH
NH2 crosslinkerDithiothretiol
(DTT)
Bifunctional
SMCC
Reduced- EGF
Maleimide-activated
Quantum Dot
Step-1
~21 nm
~0.83 nm
~5 nm
EGF-conjugated Quantum Dot
Diagaradjane et al. Clin Cancer Res. 2008, 14(3):731-41.
12
In vivo quantification
Conjugated gold nanorod
Gold nanorod
30 min
4 hrs
24 hrs
C225-GNR
PEG-GNR
Control
Krishnan lab, unpublished data
13
Tumor regrowth delay
4.0
Control
3.8
PEG-GNR
3.6
C-GNR
3.4
Cetuximab
Rad
Normalized Tumor Volume
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
Biodistribution
45
PEG-GNR
C225-GNR
40
35
% ID
30
25
20
15
10
5
0
Brain
Heart
Lung
Liver
Spleen Kidney Tumor
Blood
Cho SH. Phys Med Biol 2005; 50: N163-73
Clonogenic survival
30 min
24 hrs
1.000
Surviving Fraction
Surviving Fraction
1.000
0.100
0.010
Control
PEG-GNR
C225-GNR
0.001
0.100
0.010
Control
PEG-GNR
C225-GNR
0.001
0.000
0.000
0
2
4
Dose (Gy)
DEF 10%
6
8
0
2
4
6
8
Dose (Gy)
DEF 15%
14
DNA damage
DNA damage
70
No Radiation
Average Number of Foci per cell
60
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
R4 + GNR
1
4
C
0
1
4
24
R4 + cGNR
C
4
0
1
4
 γ H2AX
 H2AX
 PARP
Apoptotic markers
Mitochondrial mediated apoptotic markers
Caspase mediated apoptotic markers
E
Rad (4 Gy)
C
0
1
4
pGNR + Rad
24
C
0
1
cGNR + Rad
4 24
C
0
1
4 24
Rad (4 Gy)
 hr post IR
C
 Procaspase 3
0
1
4 24
pGNR + Rad
C
0
1
cGNR + Rad
4 24
C
 Procaspase 9
0
1
4 24
 hr post IR
 Bcl-2
 Bax
 Procaspase 8
 PUMA
 Actin
 Actin

H
NTP
NTP


NTP






Ra
pGNRd
cGNR
-NTP ratio
G
1.5
1
0.5
0
-5.0
-10.0
-15.0
Contro
l
-20.0
-5.0
-10.0
1 hr
-15.0
-20.0
-5.0
-10.0
-15.0
-20.0
ppm (t1)
Rad pGNRcGNR
24
hr
15
Total oxidative stress
Protein carbonyl assay
1.3
Rad
Normalized protein carbonyl content
1.2
1.2
GNR
1.1
CGNR
1.1
1.0
1.0
0.9
0.9
0.8
0.8
Control
Immediate
1 hr
4 hrs
Time after 4 Gy radiation
Tissue effects
Post irradiation time
4 days
C225-GNR
+
Radiation
PEG-GNR
+
Radiation
Radiation
4 hrs
Tissue effects
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
16
Intracellular distribution
Time
Tissue distribution
Tissue distribution
17
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
Another approach
18
Thermosensitive liposome
PEG
Hydrophobic region
AuNp
120-130 nm
Aqueous core
100
TSLAuNps
NTSLAuNps
Percent of AuNps released
80
Phospholipid
bilayer
o
41.5 C
60
40
o
38.5 C
20
0
26
28
30
32
34
36
38
40
42
44
46
48
50
52
o
Temperature ( C )
Focused ultrasound
Radiosensitization
400
Normalized tumor volume
350
300
250
Control
TSLAuNp
200
HT
150
TSLAuNp + Rad
Rad
TSLAuNp + HT+ Rad
100
50
0
0
3
6
9
12
15
18
21
24
27
30
Days
19
Radiosensitization
400
Control
HT
TSLAuNp
Normalized tumor volume
350
300
TSLAuNp + HT
250
NTSLAuNp + HT
Rad
AuNp+Rad
TSLAuNp + Rad
NTSLAuNp + Rad
HT+ Rad
NTSLAuNp + HT + Rad
200
150
100
TSLAuNp + HT+ Rad
50
0
0
3
6
9
12
15
18
21
24
27
30
Days after intravenous injection
Deep penetration of tumors
Summary
• Delivery of nanoparticles using thermosensitive
liposomes enhances deep penetration of nanoparticles
when triggered by hyperthermia
• Deep penetration of gold nanoparticles improves
radiosensitization independent of the effect of
hyperthermic radiosensitization
• In principle, this could be a class solution for a variety
of tumors accessible by ultrasound
20
RES capture
EGF-QD
GdCl3 + EGF-QD
(a)
3 min
Background
QD
5µ
4 hrs
1 hr
(b)
5µ
Diagaradjane et al. ACS Nano, 2010
Thermal dosimetry
Cheong S-K et al. Med Phys 36(10):4664-71, 2009
Quantifying gold nanoparticles in tumor
Zaman et al IEEE J Sel Top Quant Elec 13(6):1715-20, 2007.
21
Imaging gold nanoparticles in tumors
Puvanakrishnan P et al. J Biomed Optics 14(2):024044, 2009.
Imaging gold nanoparticles in tumors
Puvanakrishnan P et al. J Biomed Optics 14(2):024044, 2009.
22
23
Photoacoustic imaging
(A)
(A 1 )
(A 2 )
(B)
(A 3)
(C)
(A 4)
Summary
• Larger particles for vascular-targeted applications (thermoablation, hyperthermia, vascular imaging)
• Smaller particles for parenchymal applications (imaging,
targeted payload delivery)
• Combinations of above
• Unresolved issues related to clinical translation
Summary
• Overcome radioresistance via
• increased perfusion, reduced hypoxia
• stem cell sensitization
• vascular disruption
• physical radiation dose enhancement
• oxidative stress
• DNA damage
• triggering these effects deep within tumor core
24
NIR laser
XRT
5.0
Control
Rad
150nm
• Perfusion
• Vascular
disruption
• Stem cell
sensitization
Normalized tumor volume (mm 3)
4.5
Hyp
4.0
Rad+Hyp
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
5
10
15
20
25
30
Days
Diagaradjane et al. Nano Lett. 2008 8(5):1492-500
Atkinson RA, et al. Sci Translat Med, 2010; 2(55):55ra79
XRT
4.0
PEG-GNR
3.6
C-GNR
3.4
Cetuximab
Rad
3.2
Normalized Tumor Volume
~25nm
conjugated
nanorod
Control
3.8
Cetuximab + Rad
3.0
PEG-GNR + Rad
2.8
C-GNR + Rad
2.6
2.4
2.2
Progressive
2.0
1.8
intracellular
1.6
1.4
accumulation
1.2
Inc’d1.0oxidative stress
0.8
Inc’d
DNA 5damage
0
10
15
20
25
30
Days after treatment
FUS
PEG
XRT
Hydrophobic region
AuNp
120-130 nm
Aqueous core
Phospholipid
bilayer
400
350
Normalized tumor volume
~120nm
Thermosensitive
liposome
with gold
300
250
Control
TSLAuNp
200
HT
150
TSLAuNp + Rad
Rad
TSLAuNp + HT+ Rad
100
50
0
0
3
6
9
12
15
Deep penetration
Days
of nanoparticles
18
21
24
27
30
25
NIR laser
Surgery
NBI imaging
600
400
200
Pre-surgical tumor
Post-surgical margin
Laser
Vascular
targeted
nanoshell
Normalized tumor volume
(as percentage of post-surgical margin)
800
Ablate
+ve margin
PBS -> Surgery
RGDyk-GNS -> Surgery
PEG-RGD -> Surgery -> Laser
*
0
-2
0
2
4
6 8 10 12 14 16 18 20 22
Days post-surgery
Acknowledgements
Krishnan lab
UT Austin
Baylor
Parmesh Diagaradjane
Amit Deorukhkar
Edward Agyare
Dev Chatterjee
Shanta Bhattarai
Tatiana Marques Pinto
Jihyoun Lee
Aaron Brown
Kevin Kotamarti
Nga Diep
Krystina Sang
Jacobo Orenstein Cardona
Norman Colon
Hee Chul Park
Brook Walter
James Tunnell
Raiyan Zaman
Priya Puvanakrishnan
Jaesook Park
Jeffrey Rosen
Rachel Atkinson
Amit Joshi
Georgia Tech
Glenn Goodrich
Don Payne
Jon Schwartz
James Wang
Texas Southern Univ
Huan Xie
Sang Cho
Seong-Kyun Cheong
Bernard Jones
Imaging Physics
John Hazle
Jason Stafford
Andrew Elliott
Nanospectra
Rice
Naomi Halas
Funding
NIH - KL2, R21, R01 x 2, U01
DOD PCRP, ANH pre-center grant, Shell
UT Cntr Biomed Engg, Hitachi, FUSF, MDACC
Thank you
26
Chloroquine
+ cGNR
Lactacystin
+ cGNR
Bafilomycin
+ cGNR
Chlorpromazine
+ cGNR
cGNR
X 5K
X 25K
X 50K
X 100K
27
Surviving Fraction
1.00
0.10
PBS
cGNR
Bafilomycin + cGNR
Lactacystin + cGNR
CPZ + cGNR
pGNR
0.01
0.00
0
2
4
6
8
Number of gamma-H2AX foci per cells
Radiation Dose (Gy)
PBS
80
pGNR
70
Clorpromazin + cGNR
cGNR
60
Bafilomycin + cGNR
Lactacystin + cGNR
50
40
30
20
10
0
15 m
30 m
1h
2h
4h
8h
16 h
24 h
48 h
72 h
Mean signal intensity at 520 nm
Time after radiation treatment
10000
2 Gy
cGNR + 2 Gy
9500
9000
8500
8000
7500
7000
6500
6000
28
Delivery of Proton Radiation to
Prostate Tumors in Mice (xenograft
subcutaneous)
gAuNP injection
Cells implantation
Select tumors with
Proton
(24h prior irradiation) Irradiation
(5x106 PC3 cells)
longer axis 9-11mm
Request mice to
be transported
and housed
over the PTC
Request mice to
be transported
back to BSRB
for follow up
Experimental Arms
No NPs
gAuNR
No radiation
pAuNR
Goserelin alone
No NPs
SOBP
Proton
radiation
gAuNR
pAuNR
No NPs
Beam Entrance
gAuNR
pAuNR
29
Acknowledgment
30