1

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

2

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

Electromagnetic spectrum

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

Why gold nanoshells?

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.

3

Gold nanoshells

O’Neal P et al. Cancer Lett 209(2):171-6, 2004

Invasive techniques

No real-time temperature monitoring or dosimetry

No uniform description of dose, time attributes

Is thermoradiotherapy underutilized ?

Gold nanoshell mediated hyperthermia

Laser Diomed – 15 plus

em 808 nm

P max 15 W

Delivery Fiber optic cable

collimating lens

Beam Dia 1 cm

Exp time 15 to 20 minutes

Aiming beam 632 HeNe laser

Class 3b or 4

Diagaradjane et al. Nano Lett. 2008 8(5):1492-500

4

Temperature measurements

Invasive method Non-invasive method

Needle thermocouple Magnetic Resonance Thermal imaging

(MRTI)

Thermocouple measurements

0

2

4

6

8

10

12

14

0 300 600 900 1200 1500 1800

Time (sec)

DT(

C)

Tumor-bottom

Tumor-core

Laser power DT (in tumor center)

0.8 W/ cm2 ~ 13 to 15 ºC (n=2)

0.6 W/ cm2 ~ 10 1.5 ºC (n=4)

0.4 W/ cm2 ~ 4 to 5 ºC (n=2)

MRTI

15

10

5

DT (º

C)

0 min

10 min

5 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

5

Real time MRTI

15ºC

10ºC

5ºC

DT

(ºC)

(0.6 W/cm2 for 20 min at 808-nm)

Temperature profiles

Thermocouple MRTI

= 808 nm; Exp time = 20 min

Power = 0.6 W (75 % duty cycle); Beam Dia = 1 cm

Power density = 350 mW/cm2

0

2

4

6

8

10

12

14

0 300 600 900 1200 1500 1800

Time (sec)

DT

(C

)

Tumor Temperature

0

2

4

6

8

10

12

14

0 300 600 900 1200 1500 1800

Time (sec)

DT(

C)

Tumor-bottom

Tumor-core

Dynamic contrast enhanced MRI

Pre-Hyperthermia Post-Hyperthermia

6

Dynamic contrast enhanced MRI

DCMRI

Pre-hyperthermia

DCMRI

Post-hyperthermia

Pre-Contrast

Image

T2-w

eig

hte

d

imag

e

Pix

el

inte

nsit

y

Dis

trib

uti

on

in

RO

I

Increased perfusion with ~ 2-fold increase in the contrast enhancement was observed

immediately (3 to 5 min) after gold nanoshell mediated hyperthermia.

Contrast uptake

Time (sec)

0 50 100 150 200 250 300 350

Co

ntr

as

t u

pta

ke

(a

.u)

0

50

100

150

200

250

Pre hyperthermia

Post hyperthermia

Time (sec)

0 50 100 150 200 250 300 350

Co

ntr

as

t u

pta

ke

(a

.u)

0

50

100

150

200

250

300

350

Pre hyperthermia

Post hyperthermia

Tumor Center Whole Tumor

Experimental groups

Control (n=7)

Hyperthermia (n=7)

Radiation (n=7)

Hyp + Rad (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

7

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 5 10 15 20 25 30

Days

No

rma

lize

d t

um

or

vo

lum

e (

mm

3)

Control

Rad

Hyp

Rad+Hyp

Normalized tumor volume

~5 days ~8 days

~13 days ~14days

Tumor doubling time

*

* P < 0.005

0

5

10

15

20

25

30

35

40

Control Hyperthermia Radiation Hyp+Rad

Avera

ge t

um

or

do

ub

lin

g t

ime (

days)

H&E

Pe

rip

he

ry

Co

re

Hyperthermia Radiation Thermoradiotherapy Control

8

Hypoxia, cell proliferation, perfusion

Microvessel staining – CD31

Microvessel Density

Raw Image

Processed Image

0

50

100

150

200

250

300

350

400

450

Control Hyp Rad Hyp+Rad

Avera

ge n

um

ber

of

blo

od

vessels

Tumor periphery

Tumor core

9

Scanning Electron Microscopy

Conclusions

Optically activated gold nanoshells serve as a novel means to non-

invasively 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

Pre- hyperthermia

Post- hyperthermia

T2 MRI image Pixel intensity

Radiation

Hyperthermia + Radiation

Pimonidazole = green Hoechst = blue

Necrosis

Early effects Late effects

Time (sec)

0 50 100 150 200 250 300 350

Co

ntr

as

t u

pta

ke

(a

.u)

0

50

100

150

200

250

Pre hyperthermia

Post hyperthermia

Contrast enhancement in tumor center

Anti-hypoxic effect

Vascular disrupting effect?

10

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

11

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

12

Physical dose enhancement

Enhancing physical dose enhancement

hn

nanoparticles

+ peptides

Active targeting

on the order of 10 m

e- e-

e-

nanoparticles

hn

Passive targeting

Peptide-nanoparticle bioconjugates

NH2

NH2

NH2

NH2

CdTe

ZnS O

O O

O O

O

N N

O

O

O

N

NH2

NH2

NH

NH2

CdTe

ZnS

+

Amino functionalized Quantum Dot

Bifunctional crosslinker- SMCC

1hr, 21 °C

Maleimide-activated Quantum Dot

Step-1

+ SH

SH SH

SH

HS

HS

S-S

OH

OH

SH HS

EGF Dithiothretiol (DTT)

30 min, 21 °C

Reduced- EGF

S

O

O

O

N

NH2

NH2

NH

NH2

CdTe

ZnS

SH

SH SH

SH HS

~21 nm ~0.83 nm ~5 nm

EGF-conjugated Quantum Dot

Diagaradjane et al. Clin Cancer Res. 2008, 14(3):731-41.

13

In vivo quantification

Conjugated gold nanorod

Gold nanorod

Control 30 min 4 hrs 24 hrs

PE

G-G

NR

C

22

5-G

NR

Krishnan lab, unpublished data

14

Tumor regrowth delay

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

0 5 10 15 20 25 30

No

rma

lize

d T

um

or

Vo

lum

e

Days after treatment

Control

PEG-GNR

C-GNR

Cetuximab

Rad

Cetuximab + Rad

PEG-GNR + Rad

C-GNR + Rad

Biodistribution

0

5

10

15

20

25

30

35

40

45

Brain Heart Lung Liver Spleen Kidney Tumor Blood

% ID

PEG-GNR

C225-GNR

Cho SH. Phys Med Biol 2005; 50: N163-73

Clonogenic survival

Dose (Gy)

0 2 4 6 8

Su

rviv

ing

Fra

cti

on

0.000

0.001

0.010

0.100

1.000

Control

PEG-GNR

C225-GNR

Dose (Gy)

0 2 4 6 8

Su

rviv

ing

Fracti

on

0.000

0.001

0.010

0.100

1.000

Control

PEG-GNR

C225-GNR

DEF 10% DEF 15%

30 min 24 hrs

15

DNA damage

DNA damage

0

10

20

30

40

50

60

70

0.05 0.5 1 2 4 24

Av

era

ge

Nu

mb

er

of

Fo

ci p

er

ce

ll

Time after irradiation (hrs)

No Radiation

Radiation (4 Gy)

GNR + Rad (4 Gy)

C225-GNR + Rad (4 Gy)

γ H2AX

H2AX

PARP

C 0 1 4

R4 + GNR R4 + cGNR

C 0 1 4 C 0 1 4

R4

Apoptotic markers

0

0.5

1

1.5

Rad pGNRcGNR

-N

TP

ra

tio

G H

1 hr

Ra

d

-20.0 -15.0 -10.0 -5.0 ppm (t1) -20.0 -15.0 -10.0 -5.0 -20.0 -15.0 -10.0 -5.0

24

hr

Contro

l

pGNR

cGNR

NTP

NTP

NTP

Procaspase 3

Procaspase 9

Procaspase 8

Actin

pGNR + Rad cGNR + Rad Rad (4 Gy)

C 0 1 4 24 C 0 1 4 24 C 0 1 4 24 hr post IR

Caspase mediated apoptotic markers Mitochondrial mediated apoptotic markers E

Bcl-2

PUMA

Bax

pGNR + Rad cGNR + Rad Rad (4 Gy)

C 0 1 4 24 C 0 1 4 24 C 0 1 4 24 hr post IR

Actin

16

Total oxidative stress

0.8

0.8

0.9

0.9

1.0

1.0

1.1

1.1

1.2

1.2

1.3

Control Immediate 1 hr 4 hrs

No

rma

lize

d p

rote

in c

arb

on

yl c

on

ten

t

Time after 4 Gy radiation

Rad

GNR

CGNR

Protein carbonyl assay

Tissue effects

4 hrs 4 days

Post irradiation time

Ra

dia

tio

n

PE

G-G

NR

+

Ra

dia

tio

n

C2

25

-GN

R

+

Ra

dia

tio

n

Tissue effects

0

50

100

150

200

250

300

350

4 hrs 4 days

Post irradiation period

Avera

ge m

icro

vessel d

en

sit

y

per

field

of

vie

w w

ith

10X

ob

jecti

ve

Radiation (10 Gy)

GNR + Rad (10Gy)

C225-GNR + Rad (10 Gy)

*

17

Intracellular distribution

Time

Tissue distribution

Tissue distribution

18

Modeling dose

• 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

Summary

Another approach

19

Thermosensitive liposome

PEG

AuNp

Aqueous core

Phospholipid

bilayer

Hydrophobic region

12

0-1

30

nm

26 28 30 32 34 36 38 40 42 44 46 48 50 52

0

20

40

60

80

100

41.5 o

C

TSLAuNps

NTSLAuNps

Perc

en

t o

f A

uN

ps r

ele

ased

Temperature ( o

C )

38.5 oC

Focused ultrasound

Radiosensitization

0 3 6 9 12 15 18 21 24 27 300

50

100

150

200

250

300

350

400

Control

HT

TSLAuNp

Rad

TSLAuNp + Rad

TSLAuNp + HT+ Rad

Days

No

rmali

zed

tu

mo

r vo

lum

e

20

Radiosensitization

0 3 6 9 12 15 18 21 24 27 300

50

100

150

200

250

300

350

400Control

HT

TSLAuNp

TSLAuNp + HT

Rad

TSLAuNp + Rad

TSLAuNp + HT+ Rad

AuNp+Rad

NTSLAuNp + Rad

NTSLAuNp + HT

NTSLAuNp + HT + Rad

HT+ Rad

Days after intravenous injection

No

rmalized

tu

mo

r vo

lum

e

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

21

QD EGF-QD GdCl3 + EGF-QD

Backg

rou

nd

3 m

in

1 h

r 4 h

rs

(a)

5 µ

5 µ

(b)

Diagaradjane et al. ACS Nano, 2010

RES capture

Thermal dosimetry

Cheong S-K et al. Med Phys 36(10):4664-71, 2009

Zaman et al IEEE J Sel Top Quant Elec 13(6):1715-20, 2007.

Quantifying gold nanoparticles in tumor

22

Puvanakrishnan P et al. J Biomed Optics 14(2):024044, 2009.

Imaging gold nanoparticles in tumors

Imaging gold nanoparticles in tumors

Puvanakrishnan P et al. J Biomed Optics 14(2):024044, 2009.

23

24

Photoacoustic imaging

(A)

(B)

(C)

(A1)

(A2)

(A3)

(A4)

• Larger particles for vascular-targeted applications (thermo-

ablation, 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

Summary

25

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 5 10 15 20 25 30

Days

No

rma

lize

d t

um

or

vo

lum

e (

mm

3)

Control

Rad

Hyp

Rad+Hyp150nm

NIR laser XRT

Diagaradjane et al. Nano Lett. 2008 8(5):1492-500

Atkinson RA, et al. Sci Translat Med, 2010; 2(55):55ra79

• Perfusion

• Vascular

disruption

• Stem cell

sensitization

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

0 5 10 15 20 25 30

No

rma

lize

d T

um

or

Vo

lum

e

Days after treatment

Control

PEG-GNR

C-GNR

Cetuximab

Rad

Cetuximab + Rad

PEG-GNR + Rad

C-GNR + Rad

~25nm

conjugated

nanorod

Progressive

intracellular

accumulation

Inc’d oxidative stress

Inc’d DNA damage

XRT

~120nm

Thermo-

sensitive

liposome

with gold

XRT FUS PEG

AuNp

Aqueous core

Phospholipid

bilayer

Hydrophobic region

12

0-1

30

nm

0 3 6 9 12 15 18 21 24 27 300

50

100

150

200

250

300

350

400

Control

HT

TSLAuNp

Rad

TSLAuNp + Rad

TSLAuNp + HT+ Rad

Days

No

rmali

zed

tu

mo

r vo

lum

e

Deep penetration

of nanoparticles

26

0

200

400

600

800

-2 0 2 4 6 8 10 12 14 16 18 20 22

Norm

aliz

ed t

um

or

volu

me

(as p

erc

en

tag

e o

f post-

su

rgic

al

marg

in)

Days post-surgery

PBS -> Surgery

RGDyk-GNS -> Surgery

PEG-RGD -> Surgery -> Laser

Pre

-su

rgic

al

tum

or

Post-

su

rgic

al

marg

in

Laser

*

Vascular

targeted

nanoshell

NIR laser

NBI imaging Surgery Ablate

+ve margin

Krishnan lab 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

Imaging Physics John Hazle

Jason Stafford

Andrew Elliott

UT Austin James Tunnell

Raiyan Zaman

Priya Puvanakrishnan

Jaesook Park

Georgia Tech Sang Cho

Seong-Kyun Cheong

Bernard Jones

Nanospectra Glenn Goodrich

Don Payne

Jon Schwartz

James Wang

Texas Southern Univ Huan Xie

Funding NIH - KL2, R21, R01 x 2, U01

DOD PCRP, ANH pre-center grant, Shell

UT Cntr Biomed Engg, Hitachi, FUSF, MDACC

Acknowledgements

Baylor Jeffrey Rosen

Rachel Atkinson

Amit Joshi

Rice

Naomi Halas

Thank you

27

X 5

K

X 2

5K

X

50

K

X 1

00

K

cGNR

Chlorpromazine + cGNR

Bafilomycin + cGNR

Lactacystin + cGNR

Chloroquine + cGNR

28

Radiation Dose (Gy)

0 2 4 6 8

Su

rviv

ing

Fra

cti

on

0.00

0.01

0.10

1.00

PBS

cGNR

Bafilomycin + cGNR

Lactacystin + cGNR

CPZ + cGNR

pGNR

0

10

20

30

40

50

60

70

80

15 m 30 m 1 h 2 h 4 h 8 h 16 h 24 h 48 h 72 h

Nu

mb

er o

f g

am

ma

-H2

AX

fo

ci p

er

ce

lls

Time after radiation treatment

PBS

pGNR

Clorpromazin + cGNR

cGNR

Bafilomycin + cGNR

Lactacystin + cGNR

6000

6500

7000

7500

8000

8500

9000

9500

10000

Me

an

sig

na

l in

ten

sit

y a

t 5

20

nm

2 Gy cGNR + 2 Gy

29

Delivery of Proton Radiation to

Prostate Tumors in Mice (xenograft

subcutaneous)

Request mice to

be transported

and housed

over the PTC

Cells implantation

(5x106 PC3 cells)

Select tumors with

longer axis 9-11mm

gAuNP injection

(24h prior irradiation) Proton

Irradiation

Request mice to

be transported

back to BSRB

for follow up

Experimental Arms

No radiation

Proton

radiation

No NPs

Goserelin alone

gAuNR

pAuNR

SOBP

Beam Entrance

No NPs

gAuNR

pAuNR

No NPs

gAuNR

pAuNR

30

Acknowledgment