TPL LASER FLUENCE CANCER GOLD NANOROD

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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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TPL LASER FLUENCE CANCER GOLD NANOROD