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aState Key Laboratory for Modication of

College of Materials Science and Engin

201620, China. E-mail: hu.junqing@dhu.e

21-6779-2947bDepartment of Orthopaedics, Shanghai Fi

University, Shanghai 200080, China

† Electronic supplementary information10.1039/c3nr06242b

Cite this: Nanoscale, 2014, 6, 3274

Received 25th November 2013Accepted 2nd January 2014

DOI: 10.1039/c3nr06242b

www.rsc.org/nanoscale

3274 | Nanoscale, 2014, 6, 3274–3282

Cu7.2S4 nanocrystals: a novel photothermal agentwith a 56.7% photothermal conversion efficiencyfor photothermal therapy of cancer cells†

Bo Li,a Qian Wang,ab Rujia Zou,a Xijian Liu,a Kaibing Xu,a Wenyao Lia and Junqing Hu*a

Copper sulphides, as a novel kind of photothermal agent for photothermal therapy (PTT) of cancer cells,

have attracted increasing attention in recent years due to good photostability, synthetic simplicity, low

toxicity and low cost. However, the unsatisfactory photothermal conversion efficiency of copper

sulphides limits their bioapplication as PTT agents. Herein, Cu7.2S4 NCs with a mean size of �20 nm as a

novel photothermal agent have been prepared by a simple thermal decomposition route. Moreover,

these NCs exhibit strong near-infrared (NIR) absorption, good photostability and significant photothermal

conversion efficiency up to 56.7% due to strong NIR absorption, good dispersity and suitable size.

Importantly, these NCs can be very compatibly used as a 980 nm laser-driven PTT agent for the efficient

PTT of cancer cells in vitro and in vivo.

1. Introduction

Photothermal therapy (PTT), employing hyperthermia gener-ated by photoabsorbers from near-infrared (NIR) laser energy to“cook” cancer cells, has gained increasing attention in recentyears as a potentially effective way to target cancerous cell deathwithout damaging surrounding healthy tissue.1–13 Currently,nanoparticles with unique optical properties which are exten-sively used as photothermal agents mainly include four classes,i.e., noble metal nanostructures (such as Au nanorods,1 Aunanoshells,2 Au nanocages,3 Pd-based nanosheets,4 and Genanoparticles5), organic compounds (indocyanine green (ICG)dye,6 polyaniline,7 and polypyrrole8), carbon-based materials(e.g., carbon nanotubes (CNTs)9 and graphene10), and semi-conductor nanostructures (Cu2�xS nanocrystals (NCs),11

Cu2�xSe NCs,12 and W18O49 nanowires13). Among these photo-thermal agents, Au nanostructures are the most studied pho-tothermal agent, which have an excellent photothermalconversion effect, but poor photostability aer a long period oflaser irradiation.14,15 To improve the photostability, several newphotothermal agents have been developed, however, thereremains a key issue about the promotion of their photothermalconversion efficiency, which is essential in practically realizing

Chemical Fibers and Polymer Materials,

eering, Donghua University, Shanghai

du.cn; Fax: +86-21-6779-2947; Tel: +86-

rst People's Hospital, Shanghai Jiaotong

(ESI) available: Figures. See DOI:

the application of PTT.16 For instance, the photothermalconversion efficiency of polypyrrole,17 Cu9S5 NCs,11 and Cu2�xSeNCs12 is 44.7% from an 808 nm laser, 25.7% from a 980 nm laserand 22% from an 808 nm laser, respectively, and thus is still lowfor PTT. Generally, with a higher photothermal conversionefficiency, photothermal agents could cause equally cancerouscell death with a lower concentration of nanoparticles, a shorterirradiation time, or a lower power density of the NIR laser,which is safer for healthy tissues of the body. Comparatively,with a lower photothermal conversion efficiency, a higherconcentration of nanoparticles or a higher power density of theNIR laser is needed. Due to the lower photothermal conversionefficiency, for example, the power density of the NIR laser forCu2�xSe NCs reaches 30W cm�2 under an 808 nm laser,12 whichis greatly beyond the safe power density limit according tothe American National Standard for the Safe Use of Lasers(�0.33 W cm�2 for the 808 nm laser9 and �0.726 W cm�2 forthe 980 nm laser18). Moreover, the diameter of some photo-thermal agents deviates the right size range, as the optimumintravenously administered nanoparticles with a diameterbetween 10 and 50 nm could increase bloodstream circulationtime.19,20 Au nanoshells,2 polyaniline,7 CuS superstructures,18

W18O49 nanowires,13 and hollow CuS nanoparticles21 can beconsiderably large with a size of more than 100 nm, while Genanoparticles5 and Fe3O4@Cu2�xS15 can be considerably smallwith a size of less than 10 nm, which limits their biologicalapplications as larger nanoparticles could be removed by thereticuloendothelial system, primarily by the liver and spleen,and smaller particles by the renal system.22,23 To meet thesevere demands of PTT in future, it is still necessary to developnew photothermal agents with high photothermal conversionefficiency, good photostability, small size (between 10 and

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50 nm), lack of toxicity and immunogenicity for the efficientPTT of cancer cells.

It has been revealed that highly self-doped semiconductorcopper sulphide NCs show strong NIR localized surface plas-mon resonances (LSPRs) of free holes in the valence band.24

Additionally, the relatively high photothermal conversion effi-ciency, good photostability, synthetic simplicity, low toxicityand low cost make copper sulphide NCs promising platforms asphotothermal agents.11,15,18,21,25,26 Also, the photothermalconversion efficiency could be promoted when the plasmonresonance wavelength of as-synthesized NCs is tuned by self-doping to be equal to the illumination laser wavelength.16 Thesefeatures trigger our interest in developing novel coppersulphide NCs with a suitable size, highly self-doping, andmaximum absorption wavelength close to the illumination laserwavelength. To the best of our knowledge, this work is the rstone that reports the preparation of Cu7.2S4 NCs by a thermaldecomposition reaction with a mean size of �20 nm, exhibitingstrong NIR absorption, a 56.7% photothermal conversion effi-ciency, and good photostability when excited by NIR light at980 nm. Importantly, these NCs can be very compatibly used asa 980 nm laser-driven PTT agent at a safe power density(0.72 W cm�2) for the efficient PTT of cancer cells in vitro andin vivo.

2. Experimental section2.1 Synthesis of copper diethyldithiocarbamate[Cu(DEDTC)2] precursor

Diethyldithiocarbamate (DEDTC) and CuCl2$2H2O were rstdissolved in deionized water (40 mL and 20 mL, respectively).Then, the two solutions were mixed with stirring for 1 h,forming a dark brown turbid solution. And the resulting darkbrown precipitate was ltered, washed several times withdeionized water, and dried under vacuum at 50 �C before use.

2.2 Synthesis of hydrophobic Cu7.2S4 nanocrystals

In a typical procedure, 10 mL of oleylamine (OLA, 80–90%,purchased from Aladdin) and 5 mL of oleic acid (OC, AR,purchased from Aladdin) were mixed and quickly heated to130 �C in a 100 mL 3-neck ask under magnetic stirring for 15min to remove residual water and oxygen under a dry nitrogengas ow. The ask was then slowly heated to 280 �C undernitrogen gas for 40 min. Thereaer, another 6 mL of OLA (4 mL)and OC (2 mL) containing 0.18 g of Cu (DEDTC)2 was injectedinto the above hot solution; the solution boiled vigorouslyfollowing the injection. The solution became dark brown uponinjection and was held at 240–260 �C for 4–10 minutes. Theask was removed from the heating mantle and allowed to coolto 60 �C. The solution became dark green during the coolingprocedure and was allowed to cool to 40 �C by the addition ofethanol. Green precipitates were collected by centrifugation andwashed with ethanol twice at 10 000 rpm for 10 min with aAllegra 64R Centrifuge purchased from Shanghai SincerelandScience-Instrument Co., Ltd., P. R. China. The precipitates werethen dispersed in 10mL of chloroform and the dispersions were

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centrifuged at 4000 rpm for 2 min to remove bigger andaggregated poorly capped nanocrystals. The green supernatantwas stored in a glass vial under ambient conditions before use.A typical reaction yields about 60 mg of nanocrystal material.

2.3 Synthesis of polymer-modied Cu7.2S4 nanocrystals

The as-prepared Cu7.2S4 nanoparticles were coated with anamphiphilic hydrolyzed polymaleic anhydride premodied witholeylamine according to a modied literature procedure.15,27,28

To a 100 mL round-bottom ask was added 0.15 mM ofmonomer units (dissolved in 5 mL of chloroform and 5 mL ofethanol) and 10 mL of the oleylamine and oleic acid passivatednanocrystals (6.0 mg mL�1 in anhydrous CHCl3). The resultingmixture was stirred for 30 min. Subsequent rotary evaporationof the solvent resulted in a dark-green lm of polymer coatednanocrystals attached to the inner wall of the ask. 10 mL ofaqueous sodium borate buffer (SBB, pH¼ 12) was then added tothe ask and subject to ultrasonication for 15 min. Aer phasetransfer from chloroform to aqueous solution, the hydrophilicnanocrystals were puried by centrifugation at 10 000 rpm for20 min.

2.4 Synthesis of cetyltrimethyl-ammoniumbromide (CTAB)stabilized gold nanorods

CTAB stabilized gold nanorods were synthesized using the silverion-assisted seed-mediated method, by referring to the previousliterature.16 Briey, 0.25 mL of HAuCl4 solution (0.01 mM) wasrst mixed with 10 mL of CTAB solution (0.1 M) with gentlemixing. Then 0.60 mL of a freshly prepared, ice-cold NaBH4

solution (0.01 M) was then injected into the mixture solutionand vigorously stirred for 2 min. The seed solution was kept atroom temperature for 2 h before use. To grow Au nanorods,2.0 mL of HAuCl4 (0.01 M) and 0.4 mL of AgNO3 (0.01 M) weremixed with 40 mL of CTAB (0.1 M). 0.4 mL of HCl (1.0 M) wasthen added, followed by the addition of 0.32 mL of ascorbic acid(0.1 M). Finally, 96 mL of the seed solution was injected into thegrowth solution. The solution was gently mixed for 10 s and leundisturbed at room temperature for at least 6 h before beingcentrifuged at 10 000 rpm for 10 min, three times. The collectedCTAB gold nanorods were re-dispersed in deionized water.

2.5 Characterization

The size, morphology, and microstructure of the Cu7.2S4 NCswere determined by HRTEM (JEOL JEM-2010F). XRD measure-ments were performed on a Bruker D4 X-ray diffractometerusing Cu Ka radiation (l ¼ 0.15418 nm). UV-vis absorptionspectra were recorded on a Shimadzu UV-2550 UV-visible-NIRspectrophotometer using quartz cuvettes with an optical path of1 cm. The content of copper ions released (from Cu7.2S4 NCs)and gold ions released (from Au nanorods) in the solution wasdetermined using a Leeman Laboratories Prodigy high-disper-sion inductively coupled plasma atomic emission spectrometer(ICP-AES).

To measure the photothermal conversion performances ofthe Cu7.2S4 NCs and Au nanorods, radiation from a 980 nm laserwas sent through a quartz cuvette containing an aqueous

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dispersion (0.3 mL) with different concentrations (0–40 ppm);the light source was a 980 nm wavelength semiconductor laserdevice (Xi'an Tours Radium Hirsh Laser Technology Co., Ltd.,P. R. of China) whose power could be adjusted externally (0–0.3W). The output power was independently calibrated using ahand-held optical power meter (Newport model 1918-C, CA,USA) and was found to be �0.29 W for a spot size of �0.40 cm2.A thermocouple with an accuracy of �0.1 �C was inserted intothe aqueous dispersion at such a position that the direct irra-diation of the laser on the probe was avoided. The temperaturewas recorded by an online type thermocouple thermometer (DT-8891E Shenzhen Everbest Machinery Industry Co., Ltd., China)every 5 s. To measure the photothermal conversion perfor-mances of the Au nanorods under the irradiation from an 808nm laser, an 808 nm laser was chosen instead of a 980 nm laserwithout changing other conditions.

To compare the photostability between the Cu7.2S4 NCs andAu nanorods, 32 ppm Cu7.2S4 NC solution was chosen due to thesame absorbance at 980 nm as 40 ppm CTAB capped Au nano-rods. The samples (3 mL) were irradiated with a 980 nm laser(SFOLT Co., Ltd, P. R. of China, 0–2 W) and an output of 2 W for10 min (LASER ON), followed by naturally cooling to roomtemperature without irradiation for 20 min (LASER OFF). Thetemperature was measured every 10 s. This cycle was repeatedfour times and then UV-vis absorption spectra and TEM imagesof the irradiated samples were obtained for characterizing theabsorption and morphology properties, respectively.

2.6 In vitro photothermal therapy of cancer cells withCu7.2S4 nanocrystals

HeLa cells were seeded into a 24 well plate at a density of 10 000cells mL�1 in RPMI-1640 culture medium at 37 �C in the pres-ence of 5% CO2 for 24 h prior to treatment. Aer incubation, thecell medium was removed, and the cells were washed with PBSbuffer solution three times. 100 mL of the polymer-modiedCu7.2S4 NCs dispersed in a PBS solution was then added into thewells at gradient concentrations (0, 20, and 40 ppm). Aerincubation for another 12 h, the cells were irradiated for 0 minand 7 min, respectively, using a 980 nm laser with an outputpower density of 0.72 W cm�2 (�0.29 W for a spot size of �0.40cm2). The cell viability was measured using the MTT assayaccording to the procedures suggested by the manufacturer. Allof the tests were independently performed twice.

2.7 In vitro quantitative analysis of Cu7.2S4 NC uptake bycancer cells

The cellular uptake of Cu7.2S4 NCs by HeLa cells was evaluatedby ICP-AES. In brief, HeLa cells were seeded in a 24-well plate ata density of 5 � 105 cells per well in RPMI-1640 culture mediumat 37 �C in the presence of 5% CO2 for 24 h prior to treatment.200 mL of the polymer-modied Cu7.2S4 NCs dispersed in a PBSsolution was then added into the wells at gradient concentra-tions (0, 20, and 40 ppm). Aer 12 hour incubation, the mediawere removed. The cells were carefully washed 5 times with PBSbefore being digested by aqua regia and diluted by ultrapurewater for the ICP analysis.

3276 | Nanoscale, 2014, 6, 3274–3282

2.8 In vivo photothermal therapy of cancer cells with Cu7.2S4nanocrystals

Severe combined immunodeciency (SCID) mice were inocu-lated subcutaneously with 2� 106 K7M2 cells for 20 days. Whenthe tumors inside the mice had grown to 5–10 mm in diameter,the SCID nude mice were randomly allocated into treatmentand control groups. The SCID nudemice in the treatment groupwere injected with 100 mL of phosphate-buffered saline (PBS)solution containing 40 ppm Cu7.2S4 NCs via the hypodermicinjection to the central region of the tumor with a depth of�4 mm, while the SCID nude mice in the control group wereinjected with 100 mL of saline solution. Aer 1 h, mice from boththe control and treatment groups were simultaneously irradi-ated with a 980 nm laser at 0.72W cm�2 power density (�0.29Wfor a spot size of �0.40 cm2) for 7 min. During the laser treat-ment, full-body infrared thermal images were captured in realtime using a photothermal medical device (GX-300; ShanghaiInfratest Electronics Co., Ltd, P. R. China) with an infraredcamera.

Aer the laser treatment, the SCID mice were killed, andtumors were removed, embedded in paraffin, and cryosectionedinto 4 mm slices. The slides were stained with H&E. The sliceswere examined under a Zeiss Axiovert 40 CFL inverted uores-cence microscope, and images were captured with a ZeissAxioCam MRc5 digital camera.

3. Results and discussion

Hydrophobic Cu7.2S4 NCs capped with oleylamine and oleicacid ligands were prepared by a simple thermal decompositionroute in the presence of a mixture of oleylamine and oleic acidat 280 �C. As-synthesized Cu7.2S4 NCs that were dispersed inchloroform and kept at room temperature for two monthsretained their strong NIR absorption, indicating that the parti-cles are not undergoing aggregation (Fig. S1, see ESI†). Thetransmission electron microscopy (TEM) image of Cu7.2S4 NCsaer two months gives further evidence towards lack of aggre-gation (Fig. S2, see ESI†). The TEM image shows good mono-dispersity of the resulting Cu7.2S4 NCs with a mean size of �20nm (Fig. 1a, and S3, see ESI†). Further microstructure infor-mation of the as-synthesized Cu7.2S4 NCs is obtained from thehigh-resolution transmission electron microscope (HRTEM)image and electron diffraction (ED) pattern. The HRTEM image(Fig. 1b) shows a single crystal with an interplanar spacing of0.196 nm, which corresponds to the d-spacing for (220) planesof the cubic structured Cu7.2S4 crystal. The diffraction pattern(Fig. 1c) of the fast Fourier transform (FFT) from the HRTEMimage in Fig. 1b can be indexed to the [220] zone axis of thecubic structure of the Cu7.2S4 crystal. All of the X-ray diffraction(XRD) peaks of the Cu7.2S4 NCs (Fig. 1d) could be well indexed tocubic Cu7.2S4 with lattice parameters similar to those on JCPDSle (card no.: 24-0061), indicating the formation of a pure cubicphase of Cu7.2S4 with high crystallinity. The cell constants ofCu7.2S4 are calculated to be a ¼ b ¼ c ¼ 5.57 A, which agree wellwith the data obtained from JCPDS card. In addition, theinjection temperature plays an important role in the formation

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of pure cubic Cu7.2S4 NCs. A mixed crystal structure of cubicphase and hexagonal phase was obtained when the injectiontemperature was below 240 �C (Fig. S4, see ESI†).

The most striking feature of the as-obtained cubic Cu7.2S4NCs is that they exhibit broad and very strong peaks in the NIRregion. To make them well-suited for PTT applications, the NCswere coated with an amphiphilic polymer (Fig. S5, seeESI†).15,27,28 Fig. 2a shows the room temperature UV-vis absor-bance spectra for Cu7.2S4 NCs dispersed in water with varioussolution concentrations of Cu2+. It exhibits a short-wavelengthabsorption edge at approximately 560 nm and reaches aminimum at around 570 nm, conrming the effect of quantumsize connement,25 which can be further demonstrated byFig. S6 (see ESI†). There is a blue-shi from 580 nm to 560 nmwith the decrease of the size of the NCs from 25 nm to 20 nm.Obviously, Cu7.2S4 NCs show an increased and strong absorp-tion in the NIR region, which originated from a localized

Fig. 1 (a) Low-magnification and (b) HRTEM images of as-synthesizedCu7.2S4 NCs via 4 min. (c) Corresponding FFT diffraction pattern fromthe image in (b). (d) XRD patterns of the as-prepared product (red line)and the standard Cu7.2S4 powders (black bar) on a JCPDS card (no. 24-0061).

Fig. 2 (a) UV-vis absorbance spectra for the aqueous dispersion ofhydrophilic Cu7.2S4 NCs with various concentrations of Cu2+ (i.e., 2.5,5, 10, 20, 30 and 40 ppm). (b) Plots of linear fitting absorbance at 980nm versus concentration for the aqueous dispersion of hydrophilicCu7.2S4 NCs.

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surface plasmon resonance of the free holes in the p-typeCu7.2S4 NCs.24 The strong absorption intensity should be mainlyattributed to many Cu deciencies and high monodispersity aswell as the lack of inter-particle aggregation.24 The spectralposition of Cu7.2S4 NCs is different from that of Cu9S5 NCspossibly because the spectral position depends on the crystalphase.29 Luther’s study can give further evidence that LSPRspectroscopy can be tuned by the degree of crystal phase.24

Moreover, the absorbance increases linearly at 980 nm as theconcentration of Cu7.2S4 NCs in water is elevated, indicating thegood dispersity of Cu7.2S4 NCs in aqueous solution.

Owing to their strong NIR absorption features and thelocation (968 nm) of maximum absorption wavelength, theseCu7.2S4 NCs are of interest for investigating their potential inphotothermal ablation therapy of cancer using a 980 nmwavelength laser. Under continuous irradiation of a 980 nmlaser with a power of 0.29 W, the temperature elevation ofaqueous dispersions containing Cu7.2S4 NCs at differentconcentrations (0–40 ppm) was measured, as shown in Fig. 3 (aand b). The concentration of Cu2+ was determined by induc-tively coupled plasma atomic emission spectroscopy (ICP-AES).

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Fig. 3 (a) Temperature elevation of pure water and the aqueousdispersion of Cu7.2S4 NCs with different concentrations of Cu2+ (i.e.,2.5, 5, 10, 20, and 40 ppm) under the irradiation of a 980 nm laser witha power of 0.29W as a function of irradiation time (0–420 s). (b) Plot oftemperature change (DT) over a period of 420 s versus the aqueousdispersion of Cu7.2S4 NCs (with different concentrations of Cu2+).

Fig. 4 (a) Photothermal effect of 40 ppm Cu7.2S4 NCs upon beingirradiated for 10 min (980 nm, 0.29 W) and shutting off the laser. (b)Time constant for heat transfer from the system is determined to be ss¼ 162.9 s by applying the linear time data from the cooling period of (a)versus negative natural logarithm of driving force temperature.

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The control experiment demonstrates that the temperature ofpure water (without Cu7.2S4 NCs) is only increased by less than3.0 �C from room temperature (17.7 �C) in 7 min. With theaddition of the Cu7.2S4 NCs (i.e., 2.5, 5.0, 10, 20 and 40 ppm), thetemperature of the aqueous dispersion increased by 6.0–19.5 �Caer 7 min irradiation, indicating that Cu7.2S4 NCs can rapidlyand efficiently convert the 980 nm wavelength laser energy intoheat energy resulting from strong photoabsorption at 980 nm.

For further study of the photothermal performance of theCu7.2S4 NCs, we then measured the photothermal transductionefficiencies of the NCs (40 ppm) by a modied method similarto the report by Roper et al.30 and our previous report on Cu9S5NCs.11 Nanoparticle dispersions were continuously illuminatedby a 980 nm laser with a power of 0.29W until reaching a steady-state temperature increase. The irradiation source was thenshut off and the temperature decrease was monitored todetermine the rate of heat transfer from the system. Fig. 4ashows the typical thermal prole of the Cu7.2S4 NCs dispersed inwater. Following Roper's report30 the photothermal conversionefficiency, hT, was calculated using eqn (1).

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hT ¼ hAðTmax � TambÞ �Q0

Ið1� 10�AlÞ (1)

where h is the heat transfer coefficient, A is the surface area ofthe container, and the value of hA can be obtained from Fig. 4b.Tmax is the maximum system temperature, Tamb is the ambientsurrounding temperature, and (Tmax � Tamb) is 20.6 �Caccording to Fig. 3c. I is the laser power (in units of mW,290 mW) and Al is the absorbance (0.8517) at an excitationwavelength of 980 nm. Q0 is the rate of heat input (in units ofmW) due to light absorption by the solvent. The lumpedquantity hA was determined by measuring the rate of temper-ature drop aer removing the light source. The value of hA isderived according to eqn (2):

ss ¼ mDCD

hA(2)

where ss is the sample system time constant, mD and CD are themass (0.3 g) and heat capacity (4.2 J g�1) of deionized water usedas solvent, respectively. The Q0 was measured independentlyusing a quartz cuvette cell containing pure water without theNCs and found to be 17.9 mW. Thus, the 980 nm laser heat

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Fig. 5 (a) UV-vis spectra of Cu7.2S4 NCs and Au nanorods before andafter four LASER ON/OFF cycles of NIR light (980 nm, 2 W) irradiation(LASER ON time: 10 min; LASER OFF time: 30 min). (b) Temperatureelevation of Cu7.2S4 NCs and Au nanorods over four LASER ON/OFFcycles of NIR laser irradiation.

Fig. 6 (a) Cell viability after treatment with different concentrations ofthe Cu7.2S4 NCs and different NIR laser irradiation times. (b) Cellularuptake of the Cu7.2S4 NC in vitro cells treatedwith the Cu7.2S4 NCswithdifferent concentrations.

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conversion efficiency (hT) of the Cu7.2S4 NCs can be calculated tobe 56.7%. With a higher photothermal conversion efficiency,photothermal agents could cause equally cancerous cell deathwith a lower concentration of nanoparticles, a shorter irradia-tion time, or a lower power density of the NIR laser, which issafer for healthy tissues of the body. The value (56.7%) isnoticeably higher than that of Cu9S5 NCs (25.7%)11 whosedegree of copper deciency is the same as that of Cu7.2S4 NCs.As demonstrated, when the plasmon resonance wavelength isclose to the wavelength of the NIR laser, Au nanorods canconvert the absorbed light into heat more effectively.16 In ourexperiment, the plasmon resonance peak of the synthesizedCu7.2S4 NCs is centered at 968 nm which is close to the illu-mination laser wavelength (980 nm), and the Cu7.2S4 NCsexhibit higher NIR absorption (0.8517) at 980 nm than that(0.6581) of the Cu9S5 NCs. Furthermore, this heat conversionefficiency of the Cu7.2S4 NCs is also higher than those ofpreviously reported materials on polypyrrole nanoparticles(44.7%)17 due to the size-dependence of the relative amounts oflight scattering and absorption,31–33 and Cu2�xSe NCs (22%)12

due to effective nonradiative electron relaxation dynamics.11

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For comparison, we subsequently investigated NIR photo-stability of the Cu7.2S4 NCs as well as that of the well-knownphotothermal agent of Au nanorods (65 � 17 nm, Fig. S7, seeESI†) by using four cycles of LASER ON/OFF with the NIR lightaccording to the method described in the literature.14 Theaqueous dispersion of the Cu7.2S4 NCs and Au nanorods wasirradiated with a 980 nm laser for 10 min (LASER ON, Fig. 5b),followed by naturally cooling to room temperature for 30 min(without irradiation, LASER OFF). As shown in Fig. 5a, 32 ppmCu7.2S4 NC solution has been chosen due to the same absor-bance at 980 nm as 40 ppm cetyltrimethylammonium bromide(CTAB) capped Au nanorods. The concentration was deter-mined by ICP-AES. The temperature elevation of 42.0–37.5 �Cand 34.3–24.4 �C was achieved over the four LASER ON for 32ppm Cu7.2S4 NC solution and 40 ppm Au nanorod solution,respectively, which indicated that the Cu7.2S4 NCs also showhigher photothermal conversion efficiency than Au nanorods(24.6%, Fig. S8, see ESI†) under the irradiation from a 980 nmlaser. As the 808 nm laser is more suitable for Au nanorods, thephotothermal conversion efficiency of Au nanorods irradiatedby a 808 nm laser was also investigated and found to be 35.8%,which is still lower than that of Cu7.2S4 NCs under the

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irradiation of a 980 nm laser (Fig. S9, see ESI†). What is more,the decrease of temperature elevation is about 28.8% of themaximal temperature elevation for Au nanorods, while there isless loss of the maximum temperature elevation of 10.7% for 32ppm Cu7.2S4 NCs aer 4 cycles of LASER ON/OFF. In addition,aer four cycles of LASER ON/OFF by a 2 W, 980 nm laser, theabsorption spectrum of Au nanorods showed a signicantdecrease in optical absorbance (black dot line), while theCu7.2S4 NCs displayed no notable changes in their absorption(red dot line). The TEM test was also conducted aer 4 cycles ofLASER ON/OFF cycles. The morphologies of the Cu7.2S4 NCswere retained well (Fig. S10, see ESI†) while the morphology ofAu nanorods almost disappeared. These results indicate thatthe Cu7.2S4 NCs showed higher photothermal conversion effi-ciency and NIR photostability than Au nanorods.

Due to such high photothermal conversion efficiency andgood NIR photostability of the as-synthesized Cu7.2S4 NCs, wethus believe that these NCs can be used as excellent PTT agents.To verify our hypothesis, we rst evaluated the photothermalcytotoxicity of the Cu7.2S4 NCs with and without laser irradiationon HeLa cells using a standard MTT assay. As seen from Fig. 6a,�95% of the cells was killed only aer 7 min irradiation of a

Fig. 7 (a) Infrared thermal images of two mice injected with the Cu7.2Sindicated region 11) via the hypodermic injection, respectively, irradiated wtemperature profiles in regions 11 and 12 as a function of the irradiatihistological images of the corresponding ex vivo tumor sections, after irrapower density of 0.72 W cm�2.

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980 nm laser (an output power density of 0.72 W cm�2) in thepresence of NCs (40 ppm), thus indicating a signicant photo-thermal therapeutic effect for HeLa cells. To better understandthe efficient PTT of cancer cells in vitro, it is essential to inves-tigate the cellular uptake of the NCs by cancer cells. ICP-AES wasused to quantify the NC uptake aer treatment with NCs atdifferent Cu2+ concentrations for 12 h. As illustrated in Fig. 6b,with the addition of the Cu7.2S4 NCs (i.e., 0, 20 and 40 ppm), theuptake of the NCs increased by 0.10–2.75 pg per cell aerincubation for 12 h, indicating that Cu7.2S4 NCs could be apromising candidate for efficient photothermal killing of cancercells owing to the high uptake.

To shed more light on the photothermal effect of the Cu7.2S4NCs, the in vivo therapeutic efficacy of the Cu7.2S4 (40 ppm)-induced photothermal therapy cancer treatment using a 980 nmlaser (0.72 W cm�2) for 7 min was studied (Fig. S11, see ESI†).During the laser treatment, full-body infrared thermal imageswere captured using an IR camera. Inspiringly, infrared thermalimages with high contrast could be achieved (Fig. 7a). One canclearly see that the region 11 framed area injected with theCu7.2S4 NCs generates more signicant temperature increasesunder irradiation, while, as a control, very little temperature

4 NCs (the left mouse, indicated region 12) or saline (the right mouse,ith a 980 nm laser (0.72W cm�2) at a time point of 0 and 240 s. (b) The

on time. (c and d) The representative hematoxylin and eosin staineddiation for 7 min. The irradiation source is a 980 nm laser with the safe

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change was detected on the region 12 framed area. Thetemperature of the irradiated area was also recorded as afunction of the irradiation time (Fig. 7b). For the mice injectedwith saline solution (region 12), the surface temperature of thetumor increased by less than 2 �C, and remained below 35 �C inthe whole irradiation process. However, in the case of Cu7.2S4NC injected mice (region 11), the tumor surface temperatureincreased rapidly and reached up to 43.4 �C at 30 s and 46.0 �Cat 60 s, and then change to plateau at about 46.0 � 0.7 �C aer80 s, as demonstrated vividly in Fig. S12, see ESI.† These resultsreveal a rapid elevation of temperature of the in vivo tumor,which suggests that the Cu7.2S4 NCs in vivo still have an excellentphotothermal effect. To further evaluate photothermal ablationof cancer cells in vivo, the histological examination of tumors wasperformed by means of microscopic imaging (Fig. 6c, d and S9,see ESI†). As expected, signicant cancer cell damage was noticedonly in the tumor with the Cu7.2S4 NC injection, but not in thecontrol group. The treatment injected with the Cu7.2S4 NCs showssevere cellular damage (pyknosis, karyorrhexis, and karyolysis) aswell as a decrease in the number of cells in comparison with thecontrol injected with saline. These facts suggest that in vivocancer cells can be efficiently destroyed by the high temperature(�46 �C) arising from the excellent photothermal effect of theCu7.2S4 NCs. Taken together, these results unambiguously provethat the photothermal effects of the synthesized Cu7.2S4 NCs havegreat potential to be used as a novel and excellent photothermalagent for PTT of cancer cells.

4. Conclusions

In conclusion, hydrophilic Cu7.2S4 NCs with a mean size of �20nm as a novel photothermal agent have been prepared by asimple thermal decomposition route in the presence of amixture of oleylamine and oleic acid and a subsequent hydro-philic modication process with an amphiphilic polymer. Theamphiphilic polymer-coated Cu7.2S4 NCs exhibit good photo-stability and signicant photothermal conversion efficiency upto 56.7% due to strong NIR absorption, higher than that ofthe as-synthesized Au nanorods, Cu9S5 NCs, Cu2�xSe NCs, andthe recently reported polypyrrole nanoparticles. Importantly,cancer cells in vitro and in vivo can be efficiently killed by thephotothermal effects, which are realized by a very low concen-tration (40 ppm) of Cu7.2S4 NCs in PBS solution under theirradiation of a 980 nm laser with a safe power density of0.72 W cm�2. Therefore, these Cu7.2S4 NCs have a great supe-riority as a novel PTT agent of cancer cells, owing to their rightsize (�20 nm), high photothermal conversion efficiency, andgood photostability. Further studies of targeted cancer therapyand in vivo distribution are underway.

Acknowledgements

B.L. and Q.W. contributed equally to this work. This work wasnancially supported by the National Natural Science Founda-tion of China (Grant Nos. 21171035 and 51302035), the KeyGrant Project of Chinese Ministry of Education (Grant No.313015), the PhD Programs Foundation of the Ministry of

This journal is © The Royal Society of Chemistry 2014

Education of China (Grant Nos. 20110075110008 and20130075120001), the National 863 Program of China (GrantNo. 2013AA031903), the Science and Technology Commissionof Shanghai Municipality (Grant No. 13ZR1451200), theFundamental Research Funds for the Central Universities, theShanghai Leading Academic Discipline Project (Grant no.B603), and the Program of Introducing Talents of Discipline toUniversities (Grant no. 111-2-04).

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