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DOI: 10.1002/adma.200800941
Ultra-Low Energy Threshold for Cancer PhotothermalTherapy Using Transferrin-Conjugated Gold Nanorods**
By Jing Liang Li,* Daniel Day, and Min Gu*
Gold nanoparticles have shown potential applications for
cancer detection and localized photothermal therapy (PTT).[15]
Among the gold nanoparticles that have been studied, gold
nanorods are of particular interest because of their tuneable
near-infrared (NIR) absorption and the recent success in their
size-controlled, large-scale synthesis.[6,7] The strong two-photon
photoluminescence (TPL) of gold nanorods render them good
contrast agents for cancer cell imaging under two-photon
excitation,[8,9] which makes it more suitable to three-dimensional
nonlinear optical imaging of biological samples.[10,11] Despite the
successful application of gold nanorods in PTT and in vitrocancer imaging, this technique still has a long way to go before it
can be implemented clinically. For clinical applications, the
energy input should be as low as possible to avoid damage to
healthy tissues, which is always a concern for laser-based
applications. The energy threshold can be reduced by optimizing
tumor targeting of the particles, choosing a suitable laser mode,
and improving the light absorption efficiency of the nanorods.
It has been observed that a pulsed femtosecond laser beam
in PTT was more effective than a continuous wave (CW) laser
beam.[12,13] However, most of the gold nanorods are randomly
oriented in cells, making it impossible to achieve the maximum
energy efficiency due to the limited fraction of excitation of the
total nanorods under linearly polarized light. To increase the
light absorption efficiency and hence the photothermal effect
of the nanorods, a circularly polarized laser beam, which can
emit a light beam of all polarization angles within one optical
period, can be used to activate as many nanorods as possible, as
illustrated by Figure 1a. To demonstrate the circular polariza-
tion effect on nanorod excitation, nanorods from a dilute
solution were deposited on a cover slide and the same
population of nanorods was subject to a linearly and circularly
polarized light illumination in sequence. The corresponding
images of the nanorods are given in Figure 1b and 1c, respec-
tively. Both single gold nanorods and aggregated nanorods
(larger and brighter spots as circled in Fig. 1b) canbe identified.
It shows that under illumination of circularly polarized light at
the same incident power, more nanorods can be excited and be
imaged clearly. To demonstrate the circular polarization effect
for cancer therapy, we have developed biofunctional nanorods
for the specific targeting of cancer cells. Transferrin has been
shown to be effective as a ligand for actively targeting
malignant cells for the delivery of drugs, proteins and
genes.[14,15] Although it has been used to enhance the cellular
uptake of gold nanoparticles[16] and quantum dots/rods,[17]
conjugating transferrin to gold nanrods for combined imaging
and therapy of cancer cells has not been reported. Our work
shows the successful development of transferrin-conjugatedgold nanorods that can be used for efficient targeting and
imaging of cancer cells. More interestingly, the use of circularly
polarized femtosecond light for two-photon-induced cancer
PTT with gold nanorods lowers the damage energy threshold
to one order of magnitude below the medical laser safety
standard, making it potentially viable for medically safe
applications.
In this work, an inverted scanning optical microscope
coupled with a femtosecond laser beam (see Experimental
section for details) was used for simultaneous TPL imaging and
PTT of human cervical cancer cells, HeLa, at a wavelength of
800 nm. A quarter-wave plate was used to convert the linearly
polarized beam into a circularly polarized beam to increase the
light absorption efficiency of the nanorods. To increase the
cellular uptake of gold nanorods, the surface of gold nanorods
was conjugated with transferrin, which can target the
transferrin receptors on HeLa cells. To stabilize the conjugated
nanorods in the culture medium, the surface of the nanorods
was also conjugated with polyethylene glycol (PEG).
The structure of a conjugated gold nanorod with a mixed
layer of PEG and transferrin molecules is schematically shown
in Figure 2a. Figure 2b is a transmission electron microscopy
(TEM) image of the as-synthesized gold nanorods with an
average length of 45nm and an aspect ratio of 4. These
surfactant cetyltrimethylammonium bromide (CTAB)-coated
nanorods (CTAB-NRs) display a maximum longitudinal
absorbance at a wavelength of 787 nm in water (Fig. 2c).
When dispersed in the culture medium RPMI, the spectra
became broader and the maximum absorbance wavelength
shifted to 795 nm. This indicates that slight aggregation might
occur because of a decrease in surfactant coverage on the
nanorods. Precipitation occurred after the solution was left static
at room temperature for a few hours. The PEG-conjugated
(PEG-NRs) and transferrin-conjugated nanorods (PEG-Tf-
NRs) showed good stability in the culture medium, as indicated
by their absorbance spectra. The maximum absorption of the
[*] Dr. J. L. Li, Prof. M. Gu, Dr. D. DayCenter for Micro-PhotonicsFaculty of Engineering and Industrial SciencesSwinburne University of TechnologyHawthorn, VIC 3122 (Australia)E-mail: [email protected]; [email protected]
[**] The authors made equal contributions to this work. The authorswould like to thank Mr. Peter Zijlstra for help with the TEMmeasurements and the Australia Research Council for financialsupport.
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protein-coated gold nanorod solution shifted to a wavelength
of 790 nm due to the change in refractive index of the particle
surface. Slight precipitation did occur in the solution of the
PEG-NRs and PEG-Tf-NRs. However, the particles could be
redispersed simply by hand shaking, which was in contrast to
the irreversible aggregation of the CTAB-NRs. Broadening of
the spectra was also observed for PEG-Tf-NRs, which
indicates that the size distribution of the particles might
become broader in the presence of a protein coating. We
observed that the PEG-NRs and PEG-Tf-NRs could be used
after long storage periods, i.e., of a few months, without causing
obvious changes in their cellular uptake. Figure 2d shows the
dependence of the TPL intensity I on the laser excitation
power P on a double logarithmic scale; the slope of the curve
was 2.04, indicating that the photoluminescence from the gold
nanorods resulted from two-photon excitation.
Figure 3a and 3b show the combined TPL images of PEG-
Tf-NRs nanorods, transmission images of cells and combined
TPL, and cell transmission images after
being incubated with the nanorods for 4 and
6 h, respectively. After six hours of incuba-
tion, the cellular uptake of the nanorods was
so significant that the shape of thecells could
be identified. In contrast, due to the
nonspecific binding of PEG to cells, thecellular uptake of PEG-NRs nanorods was
not significant even after six hours of incuba-
tion (Fig. 3c). The pronounced difference of
the cellular uptakes between PEG-NRs and
PEG-Tf- NRs indicates that the chemical
surface properties of the nanorods play a
significant role in controlling the uptake of
the nanorods by the cells. When the nanorods
were conjugated with transferrin molecules,
the particles display a much greater affinity
to the cells due to the interaction of the
transferrin molecules with the transferrin
receptors on the membrane of the cells. Thetransferrin receptor on HeLa cells has been
well characterized and it has been reported
that that transferrin receptor was over-
expressed on HeLa cells.[18] The transfer-
rin-mediated uptake of gold nanorods was
successfully demonstrated in a recent
work,[19] in which transferrin molecules
were physically attached to the surface of
gold nanorods for dark-field scattering
imaging of HeLa cells. To further prove
that the uptake of nanorods by the cells is
primarily mediated by the transferrin-
transferrin receptor interaction, the cells
were preblocked with free transferrin mole-
cules for one hour before the PEG-Tf-NRs
were supplied. The results showed that the
cellular uptake was significantly reduced
due to the presence of the blocking
molecules (Fig. 3d), which proved the importance of the
transferrin-transferrin receptor interaction in controlling the
cellular uptake of thenanorods. The uptake of PEG-Tf-NRs by
blocked cells is similar to that of PEG-NRs (Fig. 3c) because of
the non-specific uptake in these two cases. Due to the abundant
transferrin receptors on malignant cells, the transferrin-
transferrin receptor interaction should be an effective pathway
for enhancing the cellular uptake of nanoparticles. The above
results also indicate that two-photon excitation is a good
technique for in vitro imaging of gold nanorods and thus for
cancer diagnostics as long as the nanorods are functionalized
with suitable targeting molecules. Due to the severe aggregation
and precipitation of the CTAB-coated gold nanorods in culture
medium, negligible uptake of these particles by cells was
observed (image not shown).
Figure 4 shows the PTT results. Ethidium bromide (EB) was
used to stain the nuclei of dead cells. EB is membrane-
impermeable to live cells and thus can be used to examine the
Figure 1. a) A schematic description of enhanced plasmon absorption of gold nanorods in cellsby converting linearly polarized light into circularlypolarized light. The circularly polarized light canactivate a larger number of randomly oriented nanorods (red) in cells than the linearly polarizedbeam along the vertical direction. b) Image of nanorods excited by linearly polarized light and
c) image of the same nanorods excited by a circularly polarized light. The circled area in(b) indicates aggregated gold nanorods. To prevent the possible damage of the nanorods byrepeated scanning, the incident power for excitation was kept at a very low value of 100mW forboth linearly and circularly polarized illumination. The scale bars are 5 mm.
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membrane integrity of cells. It was
observed that in the absence of gold
nanorods, the cells could not be killed at
a laser power of 20mW after 60 scans
(1.05 s per scan, scanning area
60mm60mm) (Fig. 4a) (linear polar-
ization). Clear cell death was observed at25mW after 20 scans (Fig. 4b). In the
absence of gold nanorods, the thermal
effect of the laser on cells should not be
significant due to the low absorption of
the cells of NIR light. However, the
highly focused laser could induce the
disruption of membrane structures, com-
promising membrane integrity.[20,21]
However, in the presence of nanorods,
the laser power effective for therapy was
observed to be 0.5mW, indicating that
the photothermal effect of nanorods
contributes largely to the cell death.Figure 4c shows the cells after being
scanned 150 times (threshold). Interest-
ingly, when the linearly polarized light
was converted into circularly polarized
light, the number of scans at the same
power needed to damage cells was
reduced to 30, indicating the higher
efficiencyof the nanorods undercircularly
polarized illumination (Fig. 4d). When
the laser power was increased to 1.0 mW,
the cells were killed after 40 and 10 scans
under linearly and circularly polarized
light, respectively (Fig. 4e and 4f).
The minimum number of scans
required as a function of the incident
power is shown in Figure 4g. The results
indicate that the scanning duration could
be reduced by converting the linearly
polarized light into the circularly polar-
ized light. For example, when the laser
power was 1.5 mW, cells could be killed
after 10 and 5 scans for linearly and
circularly polarized beams, respectively.
A lower power of 0.2mW under circular
polarization illumination was also effec-
tive after 150 scans. No cell death was
observed at 0.1 mW after 400 scans. This
means that 0.2 mW might be the thresh-
old power for the circularly polarized
light illumination, which is two orders of
magnitude lower than the threshold
observed in the absence of goldnanorods.
The reduced exposure time required
for circularly polarized light indicates
that this illumination method is more
energy-efficient compared to the linearly
Figure 3. Combined TPLof nanorodsand transmissionimagesof HeLacells incubated withgoldnanorods: a) PEG-Tf-NRs, 4 h; b) PEG-Tf-NRs, 6 h; c) PEG-NRs, 6 h; and d) PEG-Tf-NRs, 6 h,blocked with free transferrin molecules. Scale bars: 10 mm.
Figure 2. The structure and properties of biofunctionalized gold nanorods. a) The structure of aPEG-Tf-NR. b) TEM image of nanorods. c) UV-visible absorption of CTAB-NRs in water, PEG-NRs andPEG-Tf-NRs in culture medium RPMI. d) Dependence of fluorescence intensity of gold nanorods onexcitation power.
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polarized light at the same power. The energy fluences were
calculated and given in Table 1. Under either linear or circular
polarization illumination, the energy fluences at the laser
powers used in this work are below the medical safety level of
100 mJ cm2.[22] The circularly polarized light reduces the
fluence from half to one fifth of those required for linearly
polarized light for the laser power from
1.5mW to 0.5 mW. The above results indi-
cate that a longer laser exposure and higher
energy fluence are needed when the laser
power is reduced, which means that a higher
power is more effective to destruct cells.
When a laser beam operating at a high poweris irradiated on cells labeled with gold
nanorods, heat could be built up quickly in
the nanorods and released to cells. This quick
energy transfer could destroy the cell
membrane integrity more efficiently. This
is verified by the more intense membrane
blebbing shown in Figure 4e and f at the
higher laser power. Membrane blebbing has
been proven a mechanism of cell death in the
presence of gold nanorods.[13] The thresholds
of energy densities observed in this work are
comparable to those reported in the litera-
ture when CW-mode laser was used.[1,4,23]
However, in this work, the effective illumi-
nation time per scan for nanorods is only
about 38.0ms (see experimental section for
calculation). Therefore, the total illumina-
tion time ranges from 0.19 (5 scans) to 5.70
(150 scans) milliseconds, significantly less
than the exposure durations of a few minutes
reported in literature. At the same level of the
power density, these short exposures result in
energy fluence a few orders of magnitude
lower. In case of a femtosecond laser, the
energy can be more efficiently confined by
gold nanoparticles.[12] This together with the
efficient targeting of the cancer cells by the
particles contribute to the low energy level
required for cell therapy. With the implemen-
tation of circularly polarized light, the light
absorption and conversion by the gold
nanorods were further improved.
In summary, this work reports the efficiency of transferrin-
conjugated gold nanorods in targeting, two-photon imaging and
photothermal therapy of HeLa cancer cells. The results
showed that the cancer cells can be clearly imaged after a
few hours incubation with the nanorods. The significant
cellular uptake could be attributed to the transferrin-
transferrin receptor interaction. Due to the general upregula-
tion of transferrin receptors on cancer cells, the biofunctional
gold nanorods can potentially be used for the imaging of a
number of cancer cells. Under both linearly and circularly
polarized femtosecond illumination, the energy thresholds for
cell therapy were observed to fall below the medical safety level.
The low energy thresholds are attributable to the high cellular
uptake of the gold nanorods. A circularly polarized light was
found to be more efficient in cancer cell therapy. By converting a
linearly polarized light into a circularly polarized light, the
energy threshold was significantly reduced, making the photo-
Figure 4. Photothermal therapy of cancer cells in the absence and presence of gold nanorods:a) no nanorods, 20 mW, 60 scans; b) no nanorods, 25mW, 20 scans; c) with PEG-Tf-NRs, linearpolarization (LP), 0.5 mW, 150 scans; d) with PEG-Tf-NRs, circular polarization (CP), 0.5 mW,30 scans; e) with PEG-Tf-NRs, LP, 1.0 mW, 40 scans; f) with PEG-Tf-NRs, CP, 1.0 mW, 10 scans.Scale bars: 10mm. g) Dependence of theminimum number of scans on theincidentmean powerof linearly and circularly polarized light.
Table 1. Energy fluence for cell therapy.
Mean Power
[mW]
Power density
[W cm2]
Energy fluence
[mJ cm2]
linear circular
1.5 41.7 15.8 7.9
1.0 27.8 31.7 10.6
0.7 19.4 59.1 11.1
0.5 13.9 79.2 15.8
0.2 5.6 ineffective 31.7
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thermal therapy medically safer. Due to the large difference in
the threshold power for unlabeled cells and gold nanorod-
targeted cells, simultaneous imaging and highly localized PTT
can be achieved without harming the healthy tissue exposed to a
laser beam.
Experimental
Materials: Gold(III) chloride trihydrate (!99.9%), sodium bor-ohydride (99.99%), L-ascorbic acid (!99.0%), CTAB (!99.0%),O-(2-Aminoethyl)-O-Methylpolyethylene Glycol 5000 (NH3-PEG,MW5000), transferrin, N-ethyl-N0-(3-dimethylaminopropyl) carbodii-mide hydrochloride (EDAC) (!99.0%) and N-Hydroxysuccinimide(NHS) (98%) were obtained from Sigma. All the chemicals were usedas received. A cancerous cell line HeLa (cervical cancer) was obtainedfrom American Type Culture Collection (ATCC).
Nanorods Preparation: Gold nanorods were prepared following areported method, but scaled up to 400mL. [6] Briefly, gold seedswere prepared by adding freshly prepared ice-cold NaBH (10mM,0.6 mL) into a solution containing CTAB (100 mM, 7.5 mL) and
HAuCl4 3H2O (10 mM, 0.25 mL), which were mixed for 2 minutes.The color of the seed solution was pale brown. To prepared goldnanorods, seed solution (2 mL) was added into a nanorods growthsolution, mixed gently and left still for ten hours. The growth solutionwas prepared by adding the following reagents into a 500 mL conicalglass flask in the following order with gentle mixing: CTAB (100 mM,400mL), HAuCl4 3H2O(10mM, 17mL),AgNO3 (10mM, 2.5 mL),andascorbic acid (100 mM, 2.7 mL).
Bioconjugation of Gold Nanorods: The conjugation of goldnanorods with PEG and transferrin, the CTAB molecules on thesurface of gold nanorods were replaced with thioglycolic acid molecules.To linkthe particles with thioglycolic acid, the nanorodswere centrifugedand washed twice to reduce the CTAB concentration and dispersed in2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 5.5, 10 mL).Then, thioglycolic acidaqueous solution(1 mM, 50mL)wasaddedandthe
solution was magnetically stirred for three hours. Then, EDAC (100 mM
,100mL) and NHS (100mM, 250mL) solutions were added into theglycolic acid-coated nanorod solution. The carboxylic groups on theparticle surface were activated to formreactive NHS ester intermediates.After 30 min, the activated particle solution was centrifuged and theparticles were dispersed in phosphate buffered saline (PBS) solution(pH7.5, 10mL) containing NH3-PEG (1.5mM) and transferrin(0.65mM). The solution was stirred for another two hours. The aminegroups on the protein and PEG molecules reacted with the active estergroups on the surface of the gold nanorods to form stable amide bonds.The excess reactants and by-products were removed by centrifuge. Theconjugated nanorods were dispersed in RPMI culture medium for cellculture.
Cell Culture: The cells were cultured in a RPMI medium suppliedwith fetal bovine serum (10%) at 37 8C under 10% CO2 atmosphere.For two-photon imaging and therapy of the cells, the cells were
incubated with the gold nanorods for a desired time in 24-well cultureplates with thin glass cover slides on the bottom of the wells. Cellsincubated with PEG-coated gold nanorods were used as controls.To verify that the cellular uptake was primarily mediated byinteractions between transferrin and transferrin receptors, the cellswere preblocked with free transferrin molecules for 1.5h. Theconcentration of the free transferrin molecules was a thousand timesof that for conjugation.
Two-Photon in-vitro Imaging and Photothermal Therapy: The invitro two-photon imaging was carried out on a Fluoview invertedscanning microscope (FV300). A femtosecond Ti:sapphire laser(MaiTai, Spectra Physics) with a repetition rate of 80 MHz was usedfor the two-photon excitation and photothermal therapy of cancercells. The wavelength was fixed at 800 nm and a water immersion 60
objective lens (N.A. 1.20) was used. For imaging nanorods on a coverslide, the surfactant CTAB-coated nanorods were centrifuged, washedthree times, and redispersed in water. A small volume of dilutenanorod solution was deposited on a cover slide. The sample was driednaturally under room temperature. The same area of the slide wasilluminated by a linearly and circularly polarized light in sequence. Thelinearly polarized laser beam was converted into a circularly polarized
beam with a quarter-wave plate. For photothermal therapy, cells wereincubated with gold nanorods for a few hours and washed with freshmedium, and then fresh medium (0.3 mL) and ethidium bromide (EB)(2mg mL1) in PBS (0.6 mL) were added into each well. The incidentpower was measured at the focus point. EB is a fluorescence dyecommonly used to stain nucleic acid and membrane impermeable forliving cells. It can be used to examine the membrane integrity andviability ofcells.In this work,EB stained on thenucleiof dead cells wasimaged using two-photon excitation at a wavelength of 740 nm. Themean power density was calculatedby dividing the average laser powerwith the scanning area (60mm 60mm). The energy fluence wascalculated by multiplying the mean power density (mean power/scanning area) by the total exposure time t (exposure time perscannumber of scans) of a nanorod. The exposure time of thenanorod was calculated following a known method. [13] For each scanwith a duration of 1.05 s, the area covered was 60mm 60mm
(512 pixels 512 pixels, pixel area 117nm 117 nm). The exposuretime per pixel was 4.0ms. The focal spot area was calculated as pd2/4,where d0.61 l/NA is the half width at half maximum of the beamwaist. In one scan, the exposure time of an individual nanorod equal to(focal spot area/pixel area)1.738.0ms. The total irradiation timet38.0msnumber of scans.
Received: April 7, 2008
Revised: May 23, 2008
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