Long-term survival following a single treatment
of kidney tumors with multiwalled carbon
nanotubes and near-infrared radiation
Andrew Burkea, Xuanfeng Dingb, Ravi Singha, Robert A. Kraftb, Nicole Levi-Polyachenkoc, Marissa Nichole Rylanderd,
Chris Szotd, Cara Buchanand, Jon Whitneyd, Jessica Fisherd, Heather C. Hatchera, Ralph D’Agostino, Jr.e,f, Nancy D. Kockg,
P. M. Ajayanh, David L. Carrollf,i, Steven Akmana,f, Frank M. Tortia,f, and Suzy V. Tortif,j,1
Departments ofaCancer Biology,bPhysics and Radiation Oncology,cPlastic and Reconstructive Surgery,eBiostatistical Sciences,gPathology, and
jBiochemistry, Wake Forest University School of Medicine, Winston Salem, NC 27157;dSchool of Biomedical Engineering and Science and Department of
Mechanical Engineering, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061;hDepartment of Mechanical Engineering and Materials
Science, Rice University, Houston, TX 77005;iDepartment of Physics, Wake Forest University, Winston Salem, NC 27109; andfComprehensive Cancer Center,
Wake Forest University Baptist Medical Center, Winston Salem, NC 27157
Communicated by C. N. R. Rao, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India, May 20, 2009 (received for review
February 25, 2009)
Multiwalled carbon nanotubes (MWCNTs) exhibit physical proper-
ties that render them ideal candidates for application as noninva-
sive mediators of photothermal cancer ablation. Here, we demon-
strate that use of MWCNTs to generate heat in response to
near-infrared radiation (NIR) results in thermal destruction of
of the therapy through magnetic resonance temperature-mapping
and heat shock protein-reactive immunohistochemistry. Our re-
sults demonstrate that use of MWCNTs enables ablation of tumors
with low laser powers (3 W/cm2) and very short treatment times (a
single 30-sec treatment) with minimal local toxicity and no evident
ablation of tumors and a >3.5-month durable remission in 80% of
be effective in anticancer therapy.
nanomedicine ? thermal ablation ? tumor therapy ?
photothermal therapy ? heat shock proteins
this requirement provides the selective pressure necessary to
drive the evolution of treatment-resistant cancer cell clones. In
contrast, therapies that function by exceeding physical cell
tolerances offer the advantage of eliciting a cytotoxic response
in cancer cells regardless of their phenotypic diversity.
Thermal ablation represents one such therapy. Thermal ab-
lation is achieved when cells are heated above a temperature
threshold, typically 55 °C (3). This treatment induces coagulative
necrosis, a form of cell death that involves protein denaturation
and membrane lysis (3, 4). For example, radiofrequency (RF)
ablation (RFA), a method used to treat kidney, lung, and liver
tumors (among others), employs percutaneous probes inserted
into tumors to elevate tumor temperature. Limitations of this
procedure include a single point source of thermal energy that
results in uneven tumor heating, as well as reports of tumor
‘‘seeding’’ along the needle track of RF probes that can result in
tumor recurrences (5). Thus, despite its efficacy, widespread
adoption of RFA and similar treatment modalities has been
limited by an inability to generate tumor-specific heating in a
minimally invasive manner.
Here, we explore whether multiwalled carbon nanotubes
(MWCNTs) can overcome these limitations and generate effec-
tive thermal tumor ablation. MWCNTs are nested, cylindrical
graphene structures with diameters ranging from a few to
hundreds of nanometers and lengths up to a few micrometers.
Since their discovery, carbon nanotubes have generated interest
due to their many novel properties, including their potential for
ost modern cancer treatments require participation by the
cancer cell to affect its own death (1, 2). Unfortunately,
anticancer therapy (6, 7). MWCNTs release substantial vibra-
tional energy after exposure to near-infrared radiation (NIR) (8,
9). The release of this energy within a tissue produces localized
heating, which can potentially be exploited as a tumor therapy.
Furthermore, because biological systems largely lack chro-
mophores that absorb in the NIR region, lesions can be treated
without the need for direct access to the tumor site. Although
other nanomaterials share some of these properties (10), MWC-
NTs offer an excellent combination of attributes for the devel-
opment of a noninvasive photothermal therapy. They behave as
highly efficient dipole antennae with broad absorption spectra
compared with the specific resonance absorptions of single-
walled carbon nanotubes (SWCNTs) and nanoshells, rendering
them amenable to stimulation by a range of NIR energy sources
(10, 11). Additionally, MWCNTs can be expected to absorb
significantly more NIR radiation compared with materials such
as SWCNTs, both because MWCNTs have more available
electrons for absorption per particle and because, per weight,
MWCNTs contain more metallic tubes than SWCNTs given that
the amount of NIR radiation (and consequent potential for
damage to dermal layers) needed to treat embedded cancers.
Among carbon-based nanomaterials, others (11, 13) have dem-
onstrated that SWCNTs effectively transduce heat in vitro, and
our group (9) has demonstrated the same property for nitrogen-
doped MWCNTs . However, whether carbon nanotubes can actu-
ally produce durable antitumor responses in vivo is unknown.
In this report, we demonstrate that MWCNTs are effective
thermal ablation agents that result in long-term survival of
MWCNT Suspensions Are More Efficient at Producing an NIR-
Dependent Increase in Temperature than SWCNTs. Stable suspen-
sions of MWCNTs or SWCNTs were prepared in physiologic
saline with 1%(wt/wt) Pluronic F127, a biocompatible surfactant
(see Materials and Methods), and compared for their ability to
increase temperature after exposure to a 1,064-nm Nd:YAG
(neodymium-doped yttrium aluminum garnet) laser at 3 W/cm2
X.D., R.S., R.A.K., N.L.-P., M.N.R., C.S., C.B., J.W., J.F., and H.C.H. performed research; D.L.C.
contributed new reagents/analytic tools; R.A.K., M.N.R., R.D., and N.D.K. analyzed data;
and A.B., R.S., F.M.T., and S.V.T. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
August 4, 2009 ?
vol. 106 ?
no. 31 ?
for 30 sec. MWCNTs were more efficient than SWCNTs in
inducing a temperature increase (Fig. 1). At low concentrations,
where bundling and scattering effects are minimized, MWCNTs
performed markedly better than SWCNTs: For example, after a
30-sec exposure to a Nd:YAG laser (3 W/cm2, ? ? 1,064 nm), the
23 °C to 51 ? 0.7 °C, whereas that of a 0.1 mg/ml solution of
SWCNTs increased from 23 °C to 27 ? 0.3 °C. Although SWC-
NTs were comparable with MWCNTs in inducing a temperature
increase at the upper limit of concentrations tested, it required
a 20-fold greater concentration of SWCNTs to generate a
temperature increase equivalent to that of a 0.1 mg/ml MWCNT
suspension (Fig. 1). Varying NIR laser exposure time (range:
15–60 sec) revealed that the thermal ablation temperature
threshold (50–55 °C) of a 0.1 mg/ml suspension of MWCNT
could be reached within 30 sec of laser irradiation (T30? 53.1 ?
0.1 °C) (Fig. 2). The ability of MWCNTs to induce temperature
increases compatible with thermal ablation at low concentra-
tions and short laser exposure times suggests that MWCNTs may
be useful as photothermal mediators.
MWCNTs Efficiently Kill Cancer Cells in Vitro.Wenexttestedwhether
we could identify treatment parameters that would enable
MWCNTs to be used as thermoablative agents. Cultured
RENCA kidney cancer cells were overlaid with growth media
containing 0.1 mg/ml MWCNTs and illuminated with the NIR
laser at 3 W/cm2for 0, 30, or 45 sec, and then the temperature
was measured. As shown in Fig. 2, these parameters generated
meanfinaltemperaturesof53 °Cand62.7 °C,respectively,which
were in excess of the thermal ablative temperature threshold and
significantly higher than control (P ? 0.0001). We also assessed
the effects on clonogenic survival, a rigorous assay that measures
the ability of individual tumor cells to survive, proliferate, and
form colonies. MWCNTs themselves had no statistically signif-
icant effect on colony-forming ability, and no effect was seen for
laser alone (Fig. 2). However, a 62-fold reduction in viability was
seen with the combination of MWCNTs and 30 sec of laser
exposure (P ? 0.0001), and no colonies formed from cells
treated with both MWCNTs and 45 sec of NIR laser illumina-
tion. Cell-killing depended on reaching thermoablative temper-
atures and was not a temperature-independent consequence of
NIR on MWCNTs, since modifications of the treatment condi-
tions (an increase in media fluid volume) that reduced the
temperature increase to below this threshold eliminated the
cytotoxic effect even at 10-fold higher concentrations of MWC-
NTs [supporting information (SI) Fig. S1]. The thermal sensi-
tivity of RENCA cells was similar to that of a number of other
cancer cell lines (Fig. S2), indicating that the rapid thermal death
observed in this experiment was not due to the inadvertent
selection of particularly heat-sensitive cells.
MWCNT Photothermal Therapy Is Compatible with Magnetic Reso-
nance Temperature Mapping and Produces Therapeutically Relevant
Temperature Increases in Vivo. To determine whether MWCNTs
could be used to attain thermoablative temperatures in vivo,
RENCA tumors were implanted in the flanks of 4 nude mice.
Five to 7 days after implantation, 2 mice were injected intratu-
morally with 50 ?l of a MWCNT suspension (100 ?g of total
MWCNTs), and 2 control mice were injected with 50 ?l of
diluent. Twenty-four hours later, all tumors were laser-treated (3
W/cm2, 30 sec) in the bore of 7T magnetic resonance (MR)
magnet to monitor temperature change over the tumor volume
in real time. (A diagram of the experimental set-up is shown as
Fig. S3.) Proton resonance frequency MR temperature-mapping
protocols were used to generate spatially resolved temperature
profiles (14, 15). Fig. 3A presents MR images (Fig. 3A Left) and
superimposed MR thermometry images (Fig. 3A Right) of a
control and MWCNT-containing tumor. MWCNTs induce an
increase in temperature measurable by MR thermometry: After
30 seconds of laser illumination, the average temperature of the
MWCNT-loaded tumor increased to 74 °C compared with 46 °C
in control tumors exposed to laser alone (Fig. 3B). Thus,
MWCNTs increased intratumoral temperatures to a level suffi-
cient to induce tumor ablation.
Laser-Stimulated MWCNTs Produce a Temperature Gradient That
Extends More Deeply into Tissue than Laser Treatment Alone. MR
temperature maps suggested that the combination of MWCNTs
and laser produced a deeper distribution of heat when compared
to laser alone (Fig. 3). To confirm this result, we used an
independent measure of heat generation: induction of heat
shock proteins (HSPs). HSPs are induced by elevated temper-
atures (typically in excess of 43 °C) and serve as endogenous
cellular markers of thermal stress. As seen in Fig. 4, minimal
expression exists for all HSPs in untreated tumors. In the tumor
treated with laser alone, maximal HSP27, HSP70, and HSP90
expression was induced proximal to the incident laser and then
gradually diminished. In contrast, in the tumor treated with laser
plus MWCNTs, temperature elevation at the surface was suffi-
cient to induce coagulative necrosis, thus preventing HSP in-
duction. HSP induction was seen at deeper tissue levels, at the
interface between tumor and normal tissue (Fig. 4). These
1 2 5 8 1020 50 80100
of final concentrations in saline containing 1% (wt/wt) Pluronic F127 and
illuminated at 3 W/cm2for 30 sec with a 1,064-nm continuous-wave NIR laser.
Temperature was measured by thermocouple. Shown are mean and standard
deviations of triplicate measurements. (Inset) Detail on the dilution range
from 1 to 100 ?g/ml.
MWCNTs produce a greater temperature increase than SWCNTs in
Irradia?on Time (secs)
that inhibit clonogenic survival of cultured kidney cancer cells. RENCA cells in
a final volume of 300 ?l of medium were either treated with 100 ?g/ml
MWCNT or left untreated. Approximately 15 min after the addition of
MWCNT, cells were exposed to 30 sec or 45 sec of NIR laser illumination (3
W/cm2). Temperature (two curves, right-hand scale) and clonogenic survival
(bars, left-hand scale) were measured. Shown are means and standard devi-
MWCNTs and 45 sec of laser exposure.) The combination of MWCNTs and 30
sec of laser exposure reduced viability 62-fold, and 0 colonies formed from
NIR stimulation of MWCNTs induces thermoablative temperatures
www.pnas.org?cgi?doi?10.1073?pnas.0905195106Burke et al.
results suggest that MWCNTs can be used to extend the depth
of thermal therapy.
MWCNT-Mediated Thermal Ablation Reduces Tumor Volume and En-
hances Survival. We next investigated the effectiveness of
MWCNT-based therapy in reducing tumor growth and enhanc-
ing survival in vivo. For this study, 2-mm3RENCA tumor
fragments were s.c. implanted into the flanks of 60 nude mice.
Once tumors measured ?5.5 mm (5.5 ? 0.5 mm) in greatest
dimension (?1 week), mice were randomized into one of 6
treatment groups (n ? 10 per group) as illustrated in Fig. 5.
There was no statistical difference among group mean tumor
volumes at the onset of treatment (P ? 0.44). Mice were then
injected intratumorally with 50 ?l of treatment solution con-
taining 100, 50, or 10 ?g of MWCNTs. As assessed by polarized
light microscopy and transmission electron microscopy (TEM),
MWCNTs introduced into tumors by this method appeared
well-distributed (Fig. S4). Control mice were either injected with
50 ?l of vehicle (saline with 1% Pluronic) or left untreated.
Twenty-four hours after injection, tumors were illuminated with
a 1,064-nm NIR laser for 30 sec (3 W/cm2; spot size, 5 mm). The
untreated control group received neither MWCNTs nor laser
treatment. Tumor growth was monitored, and mice were re-
moved from the study when their tumor burden reached 1,000
mm3or they were deemed moribund by veterinary consult.
Control tumors (untreated control, laser-only control, and 100
Scan Time (s)
photothermal therapy resolved by MR temperature imaging. (A) High-
resolution sagittal MR images across s.c. RENCA tumors in mice after injection
with either 100 ?g of MWCNT (i) or vehicle (iii). Bright dots in each image
(indicated by white arrows in i) correspond to tubes of Magnevist (a gadolin-
ium-based contrast agent) used in alignment of the laser aperture. Tumors
were exposed to 30 sec of NIR laser, and temperature maps were obtained by
MR temperature imaging. False-colored images depicting maximal tempera-
tures are overlaid on the MR images (ii and iv). Temperature increase after 30
sec of NIR laser exposure in a saline-injected tumor is lower in magnitude and
more superficial than that seen in the MWCNT-containing tumor. (B) Quan-
tification of temperature changes in the center of the tumors.
Intratumoral temperature distribution during MWCNT-mediated
Laser + MWCNT
Laser + MWCNT
Laser + MWCNT
Laser + MWCNT
3.7 ± 0.2
2.5 ± 0.2
1.4 ± 0.2
0.9 ± 0.1
0.1 ± 0.1
0.7 ± 0.1
1.1 ± 0.2
1.9 ± 0.2
10 ± 0.2
5.7 ± 0.2
1.3 ± 0.2
0.9 ± 0.2
0.0 ± 0.0
0.6 ± 0.1
1.3 ± 0.2
3.3 ± 0.2
3.5 ± 0.2
2.2 ± 0.2
1.3 ± 0.2
0.9 ± 0.2
0.0 ± 0.0
0.6 ± 0.2
1.2 ± 0.2
2.1 ± 0.2
*HSP expression (arbitrary units) in treated tumors normalized to HSP
expression in untreated tumors
mal therapy. Tumors that had been either untreated, treated with 30 sec of NIR
laser alone, or treated with the combination of MWCNT plus laser were serially
sectioned, and HSP expression was detected by immunofluorescent staining as
depths in a tumor treated with laser alone. Induction of HSPs proximate to the
skin surface after 30 sec of NIR laser exposure is consistent with the superficial
depict HSP expression in tumor sections taken at increasing tissue depths in a
tumor treated with the combination of MWCNT plus laser. Thermoablative
exposure prevent HSP induction at the tumor surface. (B) HSP levels were quan-
tumors (HSP expression in untreated tumors was the same throughout the
tumor). Due to changes in tissue size imposed by processing, distances from the
as ‘‘surface’’ in A) and 4 is furthest from the surface (labeled ‘‘tumor base’’ in A).
treated tumors in each group are shown.
Characterization of the HSP response to MWCNT-mediated photother-
Burke et al.PNAS ?
August 4, 2009 ?
vol. 106 ?
no. 31 ?
?g of MWCNT with no laser) grew rapidly and uniformly, and
all mice were removed from the study within the first 30 days.
There was no statistically significant difference in tumor growth
rate (P ? 0.77) or final tumor size (P ? 0.979) in any of the
control groups, indicating that tumor growth was not affected by
MWCNTs alone or by laser alone. In contrast, a statistically
significant dose-dependent attenuation of tumor growth was
observed in mice treated with MWCNT plus laser (Fig. 5).
Effects were most pronounced at the highest dose of MWCNT
1,472 ? 118 mm3in controls to 11 ? 11 mm3(P ? 0.0001). A
significant reduction in mean tumor volume was also evident at
the 50-?g dose (354 ? 176, P ? 0.0001). The lowest dose of
MWCNTs produced the most modest response, with a mean
tumor volume of 590 ? 178 mm3(P ? 0.0001).
The inhibition of tumor growth in mice treated with the
combination of MWCNTs and NIR translated into a significant
survival advantage (Fig. 6). Relative to control, overall survival
was significantly prolonged in each treatment group, with 80%
of mice alive and tumor-free ?3 months after treatment in the
100-?g treatment group (P ? 0.0001) (tumor recurrence ac-
counted for the mice that died in this group). Effect on survival
was dose-dependent: 60% remained alive in the 50-?g treatment
group (P ? 0.0004), and 20%survived in the 10-?g MWCNT
between control groups (P ? 0.775), which exhibited median
survivals of 25 days (95% C.I. 23–27) in untreated controls, 23
days (95% C.I. 21–25) in mice treated with laser alone, and
23 days (95% C.I. 21–25) in mice treated with 100 ?g of
MWCNTs alone. Median survival in the group treated with 10
?g of MWCNT plus laser was 29.5 days (95% C.I. 25–53).
Median survivals could not be calculated for the 2 highest dose
treatment groups, because too few mice died after treatment to
make the determination; however, it was extended to at least 300
days in these treatment groups.
We performed a limited survey of the long-term effects of
MWCNT plus laser treatment on mouse organs. All animals
developed a nonpermanent cutaneous surface injury that healed
over time. MWCNTs remained evident at the site of injection
after tumor regression: In all mice that were treated with
MWCNTs and laser, there was visible black material directly
under the skin, and this persisted to the conclusion of the study.
TEM analysis (performed in a subset of mice) confirmed that
this was due to the persistence of MWCNTs at the injection site
(Fig. S5). To assess effects on internal organs, 5 mice that had
remained in disease remission for 3.5 months after treatment
with 100 ?g of MWCNT plus laser were randomly selected, and
sections of the lungs, liver, spleen, skin (from MWCNT injection
site), kidneys, and brain were examined. All were essentially
normal, without evidence of nanotube-induced injury or inflam-
mation as assessed by a veterinary pathologist (Fig. S6). Fur-
thermore, the remaining 11 MWCNT-injected mice that were
not selected for this analysis continued to survive without
evidence of recurrence or notable physical or behavioral abnor-
malities for ?6 months. Thus, the treatment involving MWCNTs
produces a durable remission without evident toxicological
In this article we describe a therapeutic system using MWCNTs
stimulated by low-power NIR that results in the complete and
durable eradication of a high proportion of s.c. mouse kidney
tumors. Effects on tumor regression were dependent on the dose
of MWCNTs delivered to the tumor: At a dose of 100 ?g of
MWCNTs, complete tumor regression without recurrence for
?3 months was observed in 80% of the mice. Remarkably, this
response was attained after a single 30-sec treatment with 3
W/cm2NIR. In contrast, tumor regression was not seen in
untreated mice, mice treated with MWCNTs alone, or mice
treated with laser-generated NIR alone.
Time post-treatment (days)
Mean Tumor Volume (mm3)
100 µg MWCNTs alone
100 µg MWCNT + Laser
50 µg MWCNT + Laser
10 µg MWCNT + Laser
mice were implanted s.c. with RENCA tumors and divided into groups of 10.
Mice were either left untreated, treated with MWCNT alone, treated with
laser alone, or treated with the combination of MWCNT and laser. (A) Pho-
treated with the combination of MWCNTs and laser were injected with a
range of MWCNT doses. Tumor sizes were measured every 2 days. Means and
standard errors are shown. Control groups (untreated, treated with MWCNTs
alone, or treated with laser alone) were statistically identical. There is a
dose-dependent attenuation in tumor growth after 30 sec of NIR laser treat-
ment of MWCNT-loaded tumors (P ? 0.0001).
MWCNT-based photothermal therapy reduces tumor volume. Nu/nu
01020304050 60 300
Time post-treatment (days)
100 µg MWCNT alone
100 µg MWCNT + Laser*
50 µg MWCNT + Laser**
10 µg MWCNT + Laser***
tumor-bearing mice. Survival of mice treated as described in Fig. 5 was
assessed for ?10 months after treatment. Kaplan–Meier curves demonstrate
their tumor burden exceeded 1,000 mm3or they were deemed moribund by
veterinary consult. Survival curves for all control groups were statistically
identical (P ? 0.775).
www.pnas.org?cgi?doi?10.1073?pnas.0905195106Burke et al.
We monitored temperature changes both through MR ther-
mometry and induction of HSPs (Figs. 3 and 4). MR thermom-
etry enables monitoring of heat induction in near real-time and
can thus be used to minimize incomplete treatment of tumor
margins, a major limitation of current thermal therapies. Mea-
surement of HSPs can also be used to demarcate thermally
treated regions. Induction of HSP can increase the likelihood of
of apoptosis and enhanced resistance to chemotherapy and
radiation. Conversely, HSPs can also be used as tumor targets:
For example, anti-HSP vaccines (16) or targeting of HSP90
pathways (17) have been proposed as antitumor strategies. Thus,
therapies in the future. In our experiments, tumors treated with
due to sublethal temperature elevation. However, inclusion of
MWCNTs dramatically reduced expression of HSPs within the
tumor region due to attainment of tumor ablative temperatures
(Fig. 3). In tumors containing MWCNTs, HSP induction was
only observed at the interface between normal and tumor tissue
(Fig. 4) and did not prevent a durable response to therapy (Fig.
6). In aggregate, the combined results of MR thermometry and
HSP distribution patterns indicate that MWCNTs effectively
increase the depth of thermal tumor ablation.
The toxicity induced by carbon nanotubes is a source of debate
and may depend on nanotube type, size, shape, and surface
characteristics (18). We observed no major toxicities and only
transient local skin injury in ?6 months of follow-up after
treatment with MWCNTs plus NIR. Specifically, examination of
multiple organs did not show organ damage or inflammatory
sites. However, more definitive studies will be required to rule
out nonspecific toxicities of these materials.
A number of biomedical investigations of carbon nanotubes
have focused on their application for the treatment of cancer.
These studies include the use of carbon nanotubes as molecular
shuttles for chemotherapeutic agents (19, 20), radionuclides
(21), and nucleic acids (22), and as antitumor vaccine delivery
systems (23) and cancer diagnostic agents (24) in animal models.
In terms of tumor treatment, metallic and carbon-based nano-
materials, such as SWCNTs (11), carbon nanohorns (7), gold
nanoshells (25), and nanorods (26), have been explored. Com-
pared to these materials, MWCNTs enabled more rapid treat-
ments with reduced laser power. These properties may lessen
off-target effects and nonspecific thermal injury, which others
have reported in some applications of SWCNTs (27). In our
experiments, MWCNTs produced greater temperature increases
than SWCNTs (Fig. 1). Importantly, we observed that MWCNTs
substantially prolonged the survival of tumor-bearing mice (Fig.
6). The ability of other carbon-based nanomaterials to produce
durable remissions in tumor-bearing animals has not thus far
Although we were able to increase the temperature in
MWCNT-treated tumor tissue to 76 °C after laser irradiation,
the peak temperature measured in laser-irradiated tumors was
46 °C in the absence of MWCNTs (Fig. 3). This finding indicates
that the irradiation must be carefully monitored and controlled
to avoid injury to healthy tissue surrounding the MWCNT-
treated area. Such control might readily be achieved by reducing
Alternatively, strategies involving conjugation to cancer target-
ing moieties may be explored.
In this study, we used NIR to activate MWCNTs. An advan-
tage of NIR is that biological systems largely lack chromophores
that absorb in this region (29). However, penetration of NIR is
limited to several centimeters. Nevertheless, many tumors lie
within this distance from the surface, suggesting that the com-
bination of NIR and MWCNTs may ultimately be of benefit in
treating such tumors. An alternative approach would be the use
of tuned radiofrequency energy to enable activation of nano-
tubes with deeper tissue penetration, although this approach has
been reported to engender off-target toxicity (27).
We used direct intratumoral injection to infuse MWCNT into
tumors. MWCNTs delivered by this route appeared well-
distributed in the tumor tissue (Fig. S4). It is important to note
that this simple method of delivery is more than a convenient
experimental tool; it represents a potentially useful clinical
modality in itself. Not only are many tumors in reach of NIR, but
many that represent difficult clinical problems are accessible to
direct injection with MWCNTs, including cutaneous, s.c., and
muscle tumors, as well as prostate cancer, superficial bladder
cancers, lung cancers accessible by bronchoscopy, and some
breast, oral, and kidney tumors, among others.
MWCNTs also have some important advantages relative to
RF ablation, the most common modality in current clinical use
for thermal ablation. Unlike RF treatment, which is largely
incompatible with simultaneous MR imaging, the compatibility
of nanotubes and fiberoptic laser materials with MR thermom-
etry can enable precise delivery of heat to the tumor volume, a
critical tool in limiting injury of adjacent normal tissues while
ensuring complete ablation of the target lesion. In addition, the
laser beam delivering the NIR is not a point source, as it is in RF
treatment, but can be expanded and shaped to provide relatively
even distribution of heat to the tumor volume. Finally, once the
nanotubes are delivered, the possibility of multiple, fractionated
laser treatments of the tumor volume exists, which provides
substantial advantage over RF, which requires direct insertion of
the RF probe with each treatment.
Our results suggest that the combination of MWCNTs and
NIR for photothermal treatment of cancer may be a viable
approach for cancer therapy. We also note that thermal effects
generated by MWCNTs may have benefits in addition to direct
thermal ablation of cancer cells. For example, hyperthermia can
increase the permeability of tumor vasculature, which can en-
hance the delivery of drugs into tumors, as well as synergistically
enhance tumor cytotoxicity when combined with chemotherapy
or radiotherapy (30). When this advantage is considered within
the context of other previously elucidated MWCNT capabilities,
such as the ability to function as carriers for chemotherapeutic
compounds and MRI contrast agents, MWCNTs have the
potential to become multifunctional platforms for the treatment
Materials and Methods
Forest University Center for Nanotechnology and Molecular Materials and
characterized by TEM (Phillips 400, Phillips) (see Fig. S7). MWCNTs were
sonication . HiPCo-generated SWCNTs were purchased from Carbon Nano-
SI Materials and Methods.
Cell Culture. RENCA murine kidney cancer cells were a gift from Robert
Wiltrout (National Cancer Institute). MCF-7 breast cancer, PC-3 prostate can-
cer, Caki-1 kidney cancer, and HeLa cervical cancer cell lines were obtained
from the American Type Culture Collection and cultured as described in SI
Materials and Methods.
TEM Imaging. Stock solutions of 2 mg/ml MWCNT and SWCNT were deposited
on formvar-coated grids and imaged with a TEM 400 at either 55 or 80 KeV.
Comparison of Heating by SWCNT and MWCNT. MWCNT and SWCNT were
suspended at a range of final concentrations (wt/vol) in saline containing 1%
(wt/wt) Pluronic F127. Three hundred microliters of each suspension was
illuminated at 3 W/cm2for 30 sec by using a 1,064-nm continuous-wave NIR
laser. Temperature was measured by thermocouple.
NIR-Heating and Cell Killing of MWCNTs in Solution. MWCNT were diluted in
growth medium, illuminated with a 1,064-nm continuous-wave NIR laser
Burke et al.PNAS ?
August 4, 2009 ?
vol. 106 ?
no. 31 ?
(power density, 3 W/cm2; spot size, 5 mm; exposure duration, 15–60 sec).
Temperatures were measured by thermocouple (Fluke). Effects on clonoge-
nicity were assessed by diluting and replating cells immediately after treat-
ment and measuring colony formation after 13 days.
Thermal Sensitivity of Cultured Cancer Cells. Cells were trypsinized, suspended
in growth medium, and incubated for 30 min in a heat block equilibrated to
the desired temperature. Aliquots measuring 100 ?l were transferred in
triplicate to 96-well plates and incubated overnight at 37 °C in a humidified
MTT assay (Sigma).
Animal Handling. All animal studies were performed in compliance with the
institutional guidelines on animal use and welfare (Animal Care and Use
Committee of Wake Forest University Health Sciences) under an approved
protocol. Female nu/nu athymic mice were obtained from Charles River Lab-
oratories (5–8 wk old). Mice were housed 5 per cage in standard plastic cages,
provided food and water ad libitum, and maintained on a 12-h light/dark
Tumor Regression and Survival Studies. RENCA tumor fragments measuring 2
mm3were transplanted into the flanks of 60 female athymic mice. Once
of 6 treatment groups. For groups receiving MWCNTs, 2 mg/ml MWCNT stock
solution was diluted with vehicle (1% Pluronic in saline) to provide the
appropriate dosages of nanotubes (MWCNT control: 100 ?g; MWCNT plus
laser groups: 100, 50, and 10 ?g, respectively). In all cases, solutions were
injected directly into the center of the tumor mass. Twenty-four hours after
injection, the following groups were laser treated: vehicle control and all 3
MWCNT plus laser cohorts. Laser treatment consisted of illuminating the
tumor with a 1,064-nm continuous-wave NIR laser beam (IPG Photonics) at 3
W/cm2(spot size, 5 mm) for 30 sec. After treatment, tumor volumes were
tracked every 2 days by digital caliper, and tumor volumes were calculated
according to the formula (4/3)???(x/2)?(y/2)?(z/2). For the survival study, mice
were removed once their tumor volume reached ?1,000 mm3or when
deemed moribund by veterinary consult.
MR Temperature Mapping. Temperature-mapping experiments were per-
was acquired by using a gradient-echo sequence across the mouse tumor
followed by a low-resolution sagittal scan to detect the temperature-induced
phase difference across the treated mouse tumor. During the first scan the
power density, 3 W/cm2) for 30 sec. All image reconstruction and analysis was
performed in Matlab (Mathworks).
HSP Expression Measurement. Tumors from mice that were untreated (basal
control), treated with laser alone, or treated with the combination of laser
cryomatrix (Sakura Finetek). Sections were prepared at various tumor depths
and analyzed by fluorescent immunostaining for expression of HSP27, HSP70,
and HSP90. Fluorescence was quantified by using Leica Microsystems AF6000
Statistical Analysis. All analyses were performed by a statistician (R.D.) in the
statistical core facility of the Comprehensive Cancer Center of Wake Forest
University with SPSS software (SPSS).
ACKNOWLEDGMENTS. We thank the Ben Mynatt family for support and Ken
Grant and the Microscopy Core of the Comprehensive Cancer Center at Wake
Forest University for assistance with TEM and light microscopy. This work was
supported in part by National Institutes of Health Grant RO1CA12842 (to
S.V.T.). A.B., H.C.H. and R.S. were supported in part by National Institutes of
Health Training Grant T32CA079448.
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