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Cu7.2S4 nanocrystals: a novel photothermal agent with a 56.7% photothermal conversion efficiency for 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,
Received 25th November 2013 Accepted 2nd January 2014
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.
DOI: 10.1039/c3nr06242b
Importantly, these NCs can be very compatibly used as a 980 nm laser-driven PTT agent for the efficient
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PTT of cancer cells in vitro and in vivo.
1. Introduction Photothermal therapy (PTT), employing hyperthermia generated by photoabsorbers from near-infrared (NIR) laser energy to “cook” cancer cells, has gained increasing attention in recent years as a potentially effective way to target cancerous cell death without damaging surrounding healthy tissue.1–13 Currently, nanoparticles with unique optical properties which are extensively used as photothermal agents mainly include four classes, i.e., noble metal nanostructures (such as Au nanorods,1 Au nanoshells,2 Au nanocages,3 Pd-based nanosheets,4 and Ge nanoparticles5), organic compounds (indocyanine green (ICG) dye,6 polyaniline,7 and polypyrrole8), carbon-based materials (e.g., carbon nanotubes (CNTs)9 and graphene10), and semiconductor nanostructures (Cu2xS nanocrystals (NCs),11 Cu2xSe NCs,12 and W18O49 nanowires13). Among these photothermal agents, Au nanostructures are the most studied photothermal agent, which have an excellent photothermal conversion effect, but poor photostability aer a long period of laser irradiation.14,15 To improve the photostability, several new photothermal agents have been developed, however, there remains a key issue about the promotion of their photothermal conversion efficiency, which is essential in practically realizing
a
State Key Laboratory for Modication of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. E-mail: [email protected]; Fax: +86-21-6779-2947; Tel: +8621-6779-2947
b
Department of Orthopaedics, Shanghai First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China † Electronic supplementary information (ESI) available: Figures. See DOI: 10.1039/c3nr06242b
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the application of PTT.16 For instance, the photothermal conversion efficiency of polypyrrole,17 Cu9S5 NCs,11 and Cu2xSe NCs12 is 44.7% from an 808 nm laser, 25.7% from a 980 nm laser and 22% from an 808 nm laser, respectively, and thus is still low for PTT. Generally, with a higher photothermal conversion efficiency, photothermal agents could cause equally cancerous cell death with a lower concentration of nanoparticles, a shorter irradiation 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 higher concentration of nanoparticles or a higher power density of the NIR laser is needed. Due to the lower photothermal conversion efficiency, for example, the power density of the NIR laser for Cu2xSe NCs reaches 30 W cm2 under an 808 nm laser,12 which is greatly beyond the safe power density limit according to the American National Standard for the Safe Use of Lasers (0.33 W cm2 for the 808 nm laser9 and 0.726 W cm2 for the 980 nm laser18). Moreover, the diameter of some photothermal agents deviates the right size range, as the optimum intravenously administered nanoparticles with a diameter between 10 and 50 nm could increase bloodstream circulation time.19,20 Au nanoshells,2 polyaniline,7 CuS superstructures,18 W18O49 nanowires,13 and hollow CuS nanoparticles21 can be considerably large with a size of more than 100 nm, while Ge nanoparticles5 and Fe3O4@Cu2xS15 can be considerably small with a size of less than 10 nm, which limits their biological applications as larger nanoparticles could be removed by the reticuloendothelial system, primarily by the liver and spleen, and smaller particles by the renal system.22,23 To meet the severe demands of PTT in future, it is still necessary to develop new photothermal agents with high photothermal conversion efficiency, good photostability, small size (between 10 and
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50 nm), lack of toxicity and immunogenicity for the efficient PTT of cancer cells. It has been revealed that highly self-doped semiconductor copper sulphide NCs show strong NIR localized surface plasmon resonances (LSPRs) of free holes in the valence band.24 Additionally, the relatively high photothermal conversion efficiency, good photostability, synthetic simplicity, low toxicity and low cost make copper sulphide NCs promising platforms as photothermal agents.11,15,18,21,25,26 Also, the photothermal conversion efficiency could be promoted when the plasmon resonance wavelength of as-synthesized NCs is tuned by selfdoping to be equal to the illumination laser wavelength.16 These features trigger our interest in developing novel copper sulphide NCs with a suitable size, highly self-doping, and maximum absorption wavelength close to the illumination laser wavelength. To the best of our knowledge, this work is the rst one that reports the preparation of Cu7.2S4 NCs by a thermal decomposition reaction with a mean size of 20 nm, exhibiting strong NIR absorption, a 56.7% photothermal conversion efficiency, and good photostability when excited by NIR light at 980 nm. Importantly, these NCs can be very compatibly used as a 980 nm laser-driven PTT agent at a safe power density (0.72 W cm2) for the efficient PTT of cancer cells in vitro and in vivo.
2.
Experimental section
2.1 Synthesis of copper diethyldithiocarbamate [Cu(DEDTC)2] precursor Diethyldithiocarbamate (DEDTC) and CuCl2$2H2O were rst dissolved 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 dark brown precipitate was ltered, washed several times with deionized 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 to 130 C in a 100 mL 3-neck ask under magnetic stirring for 15 min to remove residual water and oxygen under a dry nitrogen gas ow. The ask was then slowly heated to 280 C under nitrogen 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 injected into the above hot solution; the solution boiled vigorously following the injection. The solution became dark brown upon injection and was held at 240–260 C for 4–10 minutes. The ask was removed from the heating mantle and allowed to cool to 60 C. The solution became dark green during the cooling procedure and was allowed to cool to 40 C by the addition of ethanol. Green precipitates were collected by centrifugation and washed with ethanol twice at 10 000 rpm for 10 min with a Allegra 64R Centrifuge purchased from Shanghai Sincereland Science-Instrument Co., Ltd., P. R. China. The precipitates were then dispersed in 10 mL of chloroform and the dispersions were
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centrifuged at 4000 rpm for 2 min to remove bigger and aggregated poorly capped nanocrystals. The green supernatant was 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 an amphiphilic hydrolyzed polymaleic anhydride premodied with oleylamine according to a modied literature procedure.15,27,28 To a 100 mL round-bottom ask was added 0.15 mM of monomer units (dissolved in 5 mL of chloroform and 5 mL of ethanol) and 10 mL of the oleylamine and oleic acid passivated nanocrystals (6.0 mg mL1 in anhydrous CHCl3). The resulting mixture was stirred for 30 min. Subsequent rotary evaporation of the solvent resulted in a dark-green lm of polymer coated nanocrystals attached to the inner wall of the ask. 10 mL of aqueous sodium borate buffer (SBB, pH ¼ 12) was then added to the ask and subject to ultrasonication for 15 min. Aer phase transfer from chloroform to aqueous solution, the hydrophilic nanocrystals were puried by centrifugation at 10 000 rpm for 20 min. 2.4 Synthesis of cetyltrimethyl-ammoniumbromide (CTAB) stabilized gold nanorods CTAB stabilized gold nanorods were synthesized using the silver ion-assisted seed-mediated method, by referring to the previous literature.16 Briey, 0.25 mL of HAuCl4 solution (0.01 mM) was rst mixed with 10 mL of CTAB solution (0.1 M) with gentle mixing. Then 0.60 mL of a freshly prepared, ice-cold NaBH4 solution (0.01 M) was then injected into the mixture solution and vigorously stirred for 2 min. The seed solution was kept at room 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) were mixed with 40 mL of CTAB (0.1 M). 0.4 mL of HCl (1.0 M) was then 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 the growth solution. The solution was gently mixed for 10 s and le undisturbed at room temperature for at least 6 h before being centrifuged at 10 000 rpm for 10 min, three times. The collected CTAB gold nanorods were re-dispersed in deionized water. 2.5
Characterization
The size, morphology, and microstructure of the Cu7.2S4 NCs were determined by HRTEM (JEOL JEM-2010F). XRD measurements were performed on a Bruker D4 X-ray diffractometer using Cu Ka radiation (l ¼ 0.15418 nm). UV-vis absorption spectra were recorded on a Shimadzu UV-2550 UV-visible-NIR spectrophotometer using quartz cuvettes with an optical path of 1 cm. The content of copper ions released (from Cu7.2S4 NCs) and gold ions released (from Au nanorods) in the solution was determined using a Leeman Laboratories Prodigy high-dispersion inductively coupled plasma atomic emission spectrometer (ICP-AES). To measure the photothermal conversion performances of the Cu7.2S4 NCs and Au nanorods, radiation from a 980 nm laser was 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 laser device (Xi'an Tours Radium Hirsh Laser Technology Co., Ltd., P. R. of China) whose power could be adjusted externally (0–0.3 W). The output power was independently calibrated using a hand-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 into the aqueous dispersion at such a position that the direct irradiation of the laser on the probe was avoided. The temperature was recorded by an online type thermocouple thermometer (DT8891E Shenzhen Everbest Machinery Industry Co., Ltd., China) every 5 s. To measure the photothermal conversion performances of the Au nanorods under the irradiation from an 808 nm laser, an 808 nm laser was chosen instead of a 980 nm laser without changing other conditions. To compare the photostability between the Cu7.2S4 NCs and Au nanorods, 32 ppm Cu7.2S4 NC solution was chosen due to the same absorbance at 980 nm as 40 ppm CTAB capped Au nanorods. 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 for 10 min (LASER ON), followed by naturally cooling to room temperature without irradiation for 20 min (LASER OFF). The temperature was measured every 10 s. This cycle was repeated four times and then UV-vis absorption spectra and TEM images of the irradiated samples were obtained for characterizing the absorption and morphology properties, respectively. 2.6 In vitro photothermal therapy of cancer cells with Cu7.2S4 nanocrystals HeLa cells were seeded into a 24 well plate at a density of 10 000 cells mL1 in RPMI-1640 culture medium at 37 C in the presence of 5% CO2 for 24 h prior to treatment. Aer incubation, the cell medium was removed, and the cells were washed with PBS buffer solution three times. 100 mL of the polymer-modied Cu7.2S4 NCs dispersed in a PBS solution was then added into the wells at gradient concentrations (0, 20, and 40 ppm). Aer incubation for another 12 h, the cells were irradiated for 0 min and 7 min, respectively, using a 980 nm laser with an output power density of 0.72 W cm2 (0.29 W for a spot size of 0.40 cm2). The cell viability was measured using the MTT assay according to the procedures suggested by the manufacturer. All of the tests were independently performed twice. 2.7 In vitro quantitative analysis of Cu7.2S4 NC uptake by cancer cells The cellular uptake of Cu7.2S4 NCs by HeLa cells was evaluated by ICP-AES. In brief, HeLa cells were seeded in a 24-well plate at a density of 5 105 cells per well in RPMI-1640 culture medium at 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 PBS solution was then added into the wells at gradient concentrations (0, 20, and 40 ppm). Aer 12 hour incubation, the media were removed. The cells were carefully washed 5 times with PBS before being digested by aqua regia and diluted by ultrapure water for the ICP analysis.
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2.8 In vivo photothermal therapy of cancer cells with Cu7.2S4 nanocrystals Severe combined immunodeciency (SCID) mice were inoculated subcutaneously with 2 106 K7M2 cells for 20 days. When the tumors inside the mice had grown to 5–10 mm in diameter, the SCID nude mice were randomly allocated into treatment and control groups. The SCID nude mice in the treatment group were injected with 100 mL of phosphate-buffered saline (PBS) solution containing 40 ppm Cu7.2S4 NCs via the hypodermic injection to the central region of the tumor with a depth of 4 mm, while the SCID nude mice in the control group were injected with 100 mL of saline solution. Aer 1 h, mice from both the control and treatment groups were simultaneously irradiated with a 980 nm laser at 0.72 W cm2 power density (0.29 W for a spot size of 0.40 cm2) for 7 min. During the laser treatment, full-body infrared thermal images were captured in real time using a photothermal medical device (GX-300; Shanghai Infratest Electronics Co., Ltd, P. R. China) with an infrared camera. Aer the laser treatment, the SCID mice were killed, and tumors were removed, embedded in paraffin, and cryosectioned into 4 mm slices. The slides were stained with H&E. The slices were examined under a Zeiss Axiovert 40 CFL inverted uorescence microscope, and images were captured with a Zeiss AxioCam MRc5 digital camera.
3.
Results and discussion
Hydrophobic Cu7.2S4 NCs capped with oleylamine and oleic acid ligands were prepared by a simple thermal decomposition route in the presence of a mixture of oleylamine and oleic acid at 280 C. As-synthesized Cu7.2S4 NCs that were dispersed in chloroform and kept at room temperature for two months retained their strong NIR absorption, indicating that the particles are not undergoing aggregation (Fig. S1, see ESI†). The transmission electron microscopy (TEM) image of Cu7.2S4 NCs aer two months gives further evidence towards lack of aggregation (Fig. S2, see ESI†). The TEM image shows good monodispersity of the resulting Cu7.2S4 NCs with a mean size of 20 nm (Fig. 1a, and S3, see ESI†). Further microstructure information of the as-synthesized Cu7.2S4 NCs is obtained from the high-resolution transmission electron microscope (HRTEM) image and electron diffraction (ED) pattern. The HRTEM image (Fig. 1b) shows a single crystal with an interplanar spacing of 0.196 nm, which corresponds to the d-spacing for (220) planes of the cubic structured Cu7.2S4 crystal. The diffraction pattern (Fig. 1c) of the fast Fourier transform (FFT) from the HRTEM image in Fig. 1b can be indexed to the [220] zone axis of the cubic 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 to cubic Cu7.2S4 with lattice parameters similar to those on JCPDS le (card no.: 24-0061), indicating the formation of a pure cubic phase of Cu7.2S4 with high crystallinity. The cell constants of ˚ which agree well Cu7.2S4 are calculated to be a ¼ b ¼ c ¼ 5.57 A, with the data obtained from JCPDS card. In addition, the injection temperature plays an important role in the formation
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of pure cubic Cu7.2S4 NCs. A mixed crystal structure of cubic phase and hexagonal phase was obtained when the injection temperature was below 240 C (Fig. S4, see ESI†). The most striking feature of the as-obtained cubic Cu7.2S4 NCs is that they exhibit broad and very strong peaks in the NIR region. To make them well-suited for PTT applications, the NCs were coated with an amphiphilic polymer (Fig. S5, see ESI†).15,27,28 Fig. 2a shows the room temperature UV-vis absorbance spectra for Cu7.2S4 NCs dispersed in water with various solution concentrations of Cu2+. It exhibits a short-wavelength absorption edge at approximately 560 nm and reaches a minimum at around 570 nm, conrming the effect of quantum size connement,25 which can be further demonstrated by Fig. S6 (see ESI†). There is a blue-shi from 580 nm to 560 nm with the decrease of the size of the NCs from 25 nm to 20 nm. Obviously, Cu7.2S4 NCs show an increased and strong absorption in the NIR region, which originated from a localized
Fig. 2 (a) UV-vis absorbance spectra for the aqueous dispersion of hydrophilic 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 980 nm versus concentration for the aqueous dispersion of hydrophilic Cu7.2S4 NCs.
(a) Low-magnification and (b) HRTEM images of as-synthesized Cu7.2S4 NCs via 4 min. (c) Corresponding FFT diffraction pattern from the 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. 240061). Fig. 1
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surface plasmon resonance of the free holes in the p-type Cu7.2S4 NCs.24 The strong absorption intensity should be mainly attributed to many Cu deciencies and high monodispersity as well as the lack of inter-particle aggregation.24 The spectral position of Cu7.2S4 NCs is different from that of Cu9S5 NCs possibly because the spectral position depends on the crystal phase.29 Luther’s study can give further evidence that LSPR spectroscopy can be tuned by the degree of crystal phase.24 Moreover, the absorbance increases linearly at 980 nm as the concentration of Cu7.2S4 NCs in water is elevated, indicating the good dispersity of Cu7.2S4 NCs in aqueous solution. Owing to their strong NIR absorption features and the location (968 nm) of maximum absorption wavelength, these Cu7.2S4 NCs are of interest for investigating their potential in photothermal ablation therapy of cancer using a 980 nm wavelength laser. Under continuous irradiation of a 980 nm laser with a power of 0.29 W, the temperature elevation of aqueous dispersions containing Cu7.2S4 NCs at different concentrations (0–40 ppm) was measured, as shown in Fig. 3 (a and b). The concentration of Cu2+ was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES).
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(a) Temperature elevation of pure water and the aqueous dispersion 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 with a power of 0.29 W as a function of irradiation time (0–420 s). (b) Plot of temperature change (DT) over a period of 420 s versus the aqueous dispersion of Cu7.2S4 NCs (with different concentrations of Cu2+). Fig. 3
The control experiment demonstrates that the temperature of pure water (without Cu7.2S4 NCs) is only increased by less than 3.0 C from room temperature (17.7 C) in 7 min. With the addition of the Cu7.2S4 NCs (i.e., 2.5, 5.0, 10, 20 and 40 ppm), the temperature of the aqueous dispersion increased by 6.0–19.5 C aer 7 min irradiation, indicating that Cu7.2S4 NCs can rapidly and efficiently convert the 980 nm wavelength laser energy into heat energy resulting from strong photoabsorption at 980 nm. For further study of the photothermal performance of the Cu7.2S4 NCs, we then measured the photothermal transduction efficiencies of the NCs (40 ppm) by a modied method similar to the report by Roper et al.30 and our previous report on Cu9S5 NCs.11 Nanoparticle dispersions were continuously illuminated by a 980 nm laser with a power of 0.29 W until reaching a steadystate temperature increase. The irradiation source was then shut off and the temperature decrease was monitored to determine the rate of heat transfer from the system. Fig. 4a shows the typical thermal prole of the Cu7.2S4 NCs dispersed in water. Following Roper's report30 the photothermal conversion efficiency, hT, was calculated using eqn (1).
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Fig. 4 (a) Photothermal effect of 40 ppm Cu7.2S4 NCs upon being irradiated 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 ss ¼ 162.9 s by applying the linear time data from the cooling period of (a) versus negative natural logarithm of driving force temperature.
hT ¼
hAðTmax Tamb Þ Q0 Ið1 10Al Þ
(1)
where h is the heat transfer coefficient, A is the surface area of the container, and the value of hA can be obtained from Fig. 4b. Tmax is the maximum system temperature, Tamb is the ambient surrounding temperature, and (Tmax Tamb) is 20.6 C according to Fig. 3c. I is the laser power (in units of mW, 290 mW) and Al is the absorbance (0.8517) at an excitation wavelength of 980 nm. Q0 is the rate of heat input (in units of mW) due to light absorption by the solvent. The lumped quantity hA was determined by measuring the rate of temperature drop aer removing the light source. The value of hA is derived according to eqn (2): ss ¼
mD CD hA
(2)
where ss is the sample system time constant, mD and CD are the mass (0.3 g) and heat capacity (4.2 J g1) of deionized water used as solvent, respectively. The Q0 was measured independently using a quartz cuvette cell containing pure water without the NCs and found to be 17.9 mW. Thus, the 980 nm laser heat
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For comparison, we subsequently investigated NIR photostability of the Cu7.2S4 NCs as well as that of the well-known photothermal agent of Au nanorods (65 17 nm, Fig. S7, see ESI†) by using four cycles of LASER ON/OFF with the NIR light according to the method described in the literature.14 The aqueous dispersion of the Cu7.2S4 NCs and Au nanorods was irradiated 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 ppm Cu7.2S4 NC solution has been chosen due to the same absorbance at 980 nm as 40 ppm cetyltrimethylammonium bromide (CTAB) capped Au nanorods. The concentration was determined by ICP-AES. The temperature elevation of 42.0–37.5 C and 34.3–24.4 C was achieved over the four LASER ON for 32 ppm Cu7.2S4 NC solution and 40 ppm Au nanorod solution, respectively, which indicated that the Cu7.2S4 NCs also show higher photothermal conversion efficiency than Au nanorods (24.6%, Fig. S8, see ESI†) under the irradiation from a 980 nm laser. As the 808 nm laser is more suitable for Au nanorods, the photothermal conversion efficiency of Au nanorods irradiated by 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
Fig. 5 (a) UV-vis spectra of Cu7.2S4 NCs and Au nanorods before and after four LASER ON/OFF cycles of NIR light (980 nm, 2 W) irradiation (LASER ON time: 10 min; LASER OFF time: 30 min). (b) Temperature elevation of Cu7.2S4 NCs and Au nanorods over four LASER ON/OFF cycles of NIR laser irradiation.
conversion efficiency (hT) of the Cu7.2S4 NCs can be calculated to be 56.7%. With a higher photothermal conversion efficiency, photothermal agents could cause equally cancerous cell death with a lower concentration of nanoparticles, a shorter irradiation time, or a lower power density of the NIR laser, which is safer for healthy tissues of the body. The value (56.7%) is noticeably higher than that of Cu9S5 NCs (25.7%)11 whose degree of copper deciency is the same as that of Cu7.2S4 NCs. As demonstrated, when the plasmon resonance wavelength is close to the wavelength of the NIR laser, Au nanorods can convert the absorbed light into heat more effectively.16 In our experiment, the plasmon resonance peak of the synthesized Cu7.2S4 NCs is centered at 968 nm which is close to the illumination laser wavelength (980 nm), and the Cu7.2S4 NCs exhibit higher NIR absorption (0.8517) at 980 nm than that (0.6581) of the Cu9S5 NCs. Furthermore, this heat conversion efficiency of the Cu7.2S4 NCs is also higher than those of previously reported materials on polypyrrole nanoparticles (44.7%)17 due to the size-dependence of the relative amounts of light scattering and absorption,31–33 and Cu2xSe NCs (22%)12 due to effective nonradiative electron relaxation dynamics.11
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(a) Cell viability after treatment with different concentrations of the Cu7.2S4 NCs and different NIR laser irradiation times. (b) Cellular uptake of the Cu7.2S4 NC in vitro cells treated with the Cu7.2S4 NCs with different concentrations. Fig. 6
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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 the maximal temperature elevation for Au nanorods, while there is less loss of the maximum temperature elevation of 10.7% for 32 ppm 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, the absorption spectrum of Au nanorods showed a signicant decrease in optical absorbance (black dot line), while the Cu7.2S4 NCs displayed no notable changes in their absorption (red dot line). The TEM test was also conducted aer 4 cycles of LASER ON/OFF cycles. The morphologies of the Cu7.2S4 NCs were retained well (Fig. S10, see ESI†) while the morphology of Au nanorods almost disappeared. These results indicate that the Cu7.2S4 NCs showed higher photothermal conversion efficiency and NIR photostability than Au nanorods. Due to such high photothermal conversion efficiency and good NIR photostability of the as-synthesized Cu7.2S4 NCs, we thus believe that these NCs can be used as excellent PTT agents. To verify our hypothesis, we rst evaluated the photothermal cytotoxicity of the Cu7.2S4 NCs with and without laser irradiation on 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
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980 nm laser (an output power density of 0.72 W cm2) in the presence of NCs (40 ppm), thus indicating a signicant photothermal therapeutic effect for HeLa cells. To better understand the efficient PTT of cancer cells in vitro, it is essential to investigate the cellular uptake of the NCs by cancer cells. ICP-AES was used to quantify the NC uptake aer treatment with NCs at different 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), the uptake of the NCs increased by 0.10–2.75 pg per cell aer incubation for 12 h, indicating that Cu7.2S4 NCs could be a promising candidate for efficient photothermal killing of cancer cells owing to the high uptake. To shed more light on the photothermal effect of the Cu7.2S4 NCs, the in vivo therapeutic efficacy of the Cu7.2S4 (40 ppm)induced photothermal therapy cancer treatment using a 980 nm laser (0.72 W cm2) for 7 min was studied (Fig. S11, see ESI†). During the laser treatment, full-body infrared thermal images were captured using an IR camera. Inspiringly, infrared thermal images with high contrast could be achieved (Fig. 7a). One can clearly see that the region 11 framed area injected with the Cu7.2S4 NCs generates more signicant temperature increases under irradiation, while, as a control, very little temperature
Fig. 7 (a) Infrared thermal images of two mice injected with the Cu7.2S4 NCs (the left mouse, indicated region 12) or saline (the right mouse, indicated region 11) via the hypodermic injection, respectively, irradiated with a 980 nm laser (0.72 W cm2) at a time point of 0 and 240 s. (b) The temperature profiles in regions 11 and 12 as a function of the irradiation time. (c and d) The representative hematoxylin and eosin stained histological images of the corresponding ex vivo tumor sections, after irradiation for 7 min. The irradiation source is a 980 nm laser with the safe power density of 0.72 W cm2.
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change was detected on the region 12 framed area. The temperature of the irradiated area was also recorded as a function of the irradiation time (Fig. 7b). For the mice injected with saline solution (region 12), the surface temperature of the tumor increased by less than 2 C, and remained below 35 C in the whole irradiation process. However, in the case of Cu7.2S4 NC injected mice (region 11), the tumor surface temperature increased rapidly and reached up to 43.4 C at 30 s and 46.0 C at 60 s, and then change to plateau at about 46.0 0.7 C aer 80 s, as demonstrated vividly in Fig. S12, see ESI.† These results reveal a rapid elevation of temperature of the in vivo tumor, which suggests that the Cu7.2S4 NCs in vivo still have an excellent photothermal effect. To further evaluate photothermal ablation of cancer cells in vivo, the histological examination of tumors was performed by means of microscopic imaging (Fig. 6c, d and S9, see ESI†). As expected, signicant cancer cell damage was noticed only in the tumor with the Cu7.2S4 NC injection, but not in the control group. The treatment injected with the Cu7.2S4 NCs shows severe cellular damage (pyknosis, karyorrhexis, and karyolysis) as well as a decrease in the number of cells in comparison with the control injected with saline. These facts suggest that in vivo cancer cells can be efficiently destroyed by the high temperature (46 C) arising from the excellent photothermal effect of the Cu7.2S4 NCs. Taken together, these results unambiguously prove that the photothermal effects of the synthesized Cu7.2S4 NCs have great potential to be used as a novel and excellent photothermal agent for PTT of cancer cells.
4. Conclusions In conclusion, hydrophilic Cu7.2S4 NCs with a mean size of 20 nm as a novel photothermal agent have been prepared by a simple thermal decomposition route in the presence of a mixture of oleylamine and oleic acid and a subsequent hydrophilic modication process with an amphiphilic polymer. The amphiphilic polymer-coated Cu7.2S4 NCs exhibit good photostability and signicant photothermal conversion efficiency up to 56.7% due to strong NIR absorption, higher than that of the as-synthesized Au nanorods, Cu9S5 NCs, Cu2xSe NCs, and the recently reported polypyrrole nanoparticles. Importantly, cancer cells in vitro and in vivo can be efficiently killed by the photothermal effects, which are realized by a very low concentration (40 ppm) of Cu7.2S4 NCs in PBS solution under the irradiation of a 980 nm laser with a safe power density of 0.72 W cm2. Therefore, these Cu7.2S4 NCs have a great superiority as a novel PTT agent of cancer cells, owing to their right size (20 nm), high photothermal conversion efficiency, and good photostability. Further studies of targeted cancer therapy and in vivo distribution are underway.
Acknowledgements B.L. and Q.W. contributed equally to this work. This work was nancially supported by the National Natural Science Foundation of China (Grant Nos. 21171035 and 51302035), the Key Grant Project of Chinese Ministry of Education (Grant No. 313015), the PhD Programs Foundation of the Ministry of
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Education of China (Grant Nos. 20110075110008 and 20130075120001), the National 863 Program of China (Grant No. 2013AA031903), the Science and Technology Commission of Shanghai Municipality (Grant No. 13ZR1451200), the Fundamental Research Funds for the Central Universities, the Shanghai Leading Academic Discipline Project (Grant no. B603), and the Program of Introducing Talents of Discipline to Universities (Grant no. 111-2-04).
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Cu7.2S4 nanocrystals: a novel photothermal agent with a 56.7% photothermal conversion efficiency for 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,
Received 25th November 2013 Accepted 2nd January 2014
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.
DOI: 10.1039/c3nr06242b
Importantly, these NCs can be very compatibly used as a 980 nm laser-driven PTT agent for the efficient
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PTT of cancer cells in vitro and in vivo.
1. Introduction Photothermal therapy (PTT), employing hyperthermia generated by photoabsorbers from near-infrared (NIR) laser energy to “cook” cancer cells, has gained increasing attention in recent years as a potentially effective way to target cancerous cell death without damaging surrounding healthy tissue.1–13 Currently, nanoparticles with unique optical properties which are extensively used as photothermal agents mainly include four classes, i.e., noble metal nanostructures (such as Au nanorods,1 Au nanoshells,2 Au nanocages,3 Pd-based nanosheets,4 and Ge nanoparticles5), organic compounds (indocyanine green (ICG) dye,6 polyaniline,7 and polypyrrole8), carbon-based materials (e.g., carbon nanotubes (CNTs)9 and graphene10), and semiconductor nanostructures (Cu2xS nanocrystals (NCs),11 Cu2xSe NCs,12 and W18O49 nanowires13). Among these photothermal agents, Au nanostructures are the most studied photothermal agent, which have an excellent photothermal conversion effect, but poor photostability aer a long period of laser irradiation.14,15 To improve the photostability, several new photothermal agents have been developed, however, there remains a key issue about the promotion of their photothermal conversion efficiency, which is essential in practically realizing
a
State Key Laboratory for Modication of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. E-mail: [email protected]; Fax: +86-21-6779-2947; Tel: +8621-6779-2947
b
Department of Orthopaedics, Shanghai First People's Hospital, Shanghai Jiaotong University, Shanghai 200080, China † Electronic supplementary information (ESI) available: Figures. See DOI: 10.1039/c3nr06242b
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the application of PTT.16 For instance, the photothermal conversion efficiency of polypyrrole,17 Cu9S5 NCs,11 and Cu2xSe NCs12 is 44.7% from an 808 nm laser, 25.7% from a 980 nm laser and 22% from an 808 nm laser, respectively, and thus is still low for PTT. Generally, with a higher photothermal conversion efficiency, photothermal agents could cause equally cancerous cell death with a lower concentration of nanoparticles, a shorter irradiation 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 higher concentration of nanoparticles or a higher power density of the NIR laser is needed. Due to the lower photothermal conversion efficiency, for example, the power density of the NIR laser for Cu2xSe NCs reaches 30 W cm2 under an 808 nm laser,12 which is greatly beyond the safe power density limit according to the American National Standard for the Safe Use of Lasers (0.33 W cm2 for the 808 nm laser9 and 0.726 W cm2 for the 980 nm laser18). Moreover, the diameter of some photothermal agents deviates the right size range, as the optimum intravenously administered nanoparticles with a diameter between 10 and 50 nm could increase bloodstream circulation time.19,20 Au nanoshells,2 polyaniline,7 CuS superstructures,18 W18O49 nanowires,13 and hollow CuS nanoparticles21 can be considerably large with a size of more than 100 nm, while Ge nanoparticles5 and Fe3O4@Cu2xS15 can be considerably small with a size of less than 10 nm, which limits their biological applications as larger nanoparticles could be removed by the reticuloendothelial system, primarily by the liver and spleen, and smaller particles by the renal system.22,23 To meet the severe demands of PTT in future, it is still necessary to develop new photothermal agents with high photothermal conversion efficiency, good photostability, small size (between 10 and
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50 nm), lack of toxicity and immunogenicity for the efficient PTT of cancer cells. It has been revealed that highly self-doped semiconductor copper sulphide NCs show strong NIR localized surface plasmon resonances (LSPRs) of free holes in the valence band.24 Additionally, the relatively high photothermal conversion efficiency, good photostability, synthetic simplicity, low toxicity and low cost make copper sulphide NCs promising platforms as photothermal agents.11,15,18,21,25,26 Also, the photothermal conversion efficiency could be promoted when the plasmon resonance wavelength of as-synthesized NCs is tuned by selfdoping to be equal to the illumination laser wavelength.16 These features trigger our interest in developing novel copper sulphide NCs with a suitable size, highly self-doping, and maximum absorption wavelength close to the illumination laser wavelength. To the best of our knowledge, this work is the rst one that reports the preparation of Cu7.2S4 NCs by a thermal decomposition reaction with a mean size of 20 nm, exhibiting strong NIR absorption, a 56.7% photothermal conversion efficiency, and good photostability when excited by NIR light at 980 nm. Importantly, these NCs can be very compatibly used as a 980 nm laser-driven PTT agent at a safe power density (0.72 W cm2) for the efficient PTT of cancer cells in vitro and in vivo.
2.
Experimental section
2.1 Synthesis of copper diethyldithiocarbamate [Cu(DEDTC)2] precursor Diethyldithiocarbamate (DEDTC) and CuCl2$2H2O were rst dissolved 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 dark brown precipitate was ltered, washed several times with deionized 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 to 130 C in a 100 mL 3-neck ask under magnetic stirring for 15 min to remove residual water and oxygen under a dry nitrogen gas ow. The ask was then slowly heated to 280 C under nitrogen 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 injected into the above hot solution; the solution boiled vigorously following the injection. The solution became dark brown upon injection and was held at 240–260 C for 4–10 minutes. The ask was removed from the heating mantle and allowed to cool to 60 C. The solution became dark green during the cooling procedure and was allowed to cool to 40 C by the addition of ethanol. Green precipitates were collected by centrifugation and washed with ethanol twice at 10 000 rpm for 10 min with a Allegra 64R Centrifuge purchased from Shanghai Sincereland Science-Instrument Co., Ltd., P. R. China. The precipitates were then dispersed in 10 mL of chloroform and the dispersions were
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centrifuged at 4000 rpm for 2 min to remove bigger and aggregated poorly capped nanocrystals. The green supernatant was 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 an amphiphilic hydrolyzed polymaleic anhydride premodied with oleylamine according to a modied literature procedure.15,27,28 To a 100 mL round-bottom ask was added 0.15 mM of monomer units (dissolved in 5 mL of chloroform and 5 mL of ethanol) and 10 mL of the oleylamine and oleic acid passivated nanocrystals (6.0 mg mL1 in anhydrous CHCl3). The resulting mixture was stirred for 30 min. Subsequent rotary evaporation of the solvent resulted in a dark-green lm of polymer coated nanocrystals attached to the inner wall of the ask. 10 mL of aqueous sodium borate buffer (SBB, pH ¼ 12) was then added to the ask and subject to ultrasonication for 15 min. Aer phase transfer from chloroform to aqueous solution, the hydrophilic nanocrystals were puried by centrifugation at 10 000 rpm for 20 min. 2.4 Synthesis of cetyltrimethyl-ammoniumbromide (CTAB) stabilized gold nanorods CTAB stabilized gold nanorods were synthesized using the silver ion-assisted seed-mediated method, by referring to the previous literature.16 Briey, 0.25 mL of HAuCl4 solution (0.01 mM) was rst mixed with 10 mL of CTAB solution (0.1 M) with gentle mixing. Then 0.60 mL of a freshly prepared, ice-cold NaBH4 solution (0.01 M) was then injected into the mixture solution and vigorously stirred for 2 min. The seed solution was kept at room 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) were mixed with 40 mL of CTAB (0.1 M). 0.4 mL of HCl (1.0 M) was then 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 the growth solution. The solution was gently mixed for 10 s and le undisturbed at room temperature for at least 6 h before being centrifuged at 10 000 rpm for 10 min, three times. The collected CTAB gold nanorods were re-dispersed in deionized water. 2.5
Characterization
The size, morphology, and microstructure of the Cu7.2S4 NCs were determined by HRTEM (JEOL JEM-2010F). XRD measurements were performed on a Bruker D4 X-ray diffractometer using Cu Ka radiation (l ¼ 0.15418 nm). UV-vis absorption spectra were recorded on a Shimadzu UV-2550 UV-visible-NIR spectrophotometer using quartz cuvettes with an optical path of 1 cm. The content of copper ions released (from Cu7.2S4 NCs) and gold ions released (from Au nanorods) in the solution was determined using a Leeman Laboratories Prodigy high-dispersion inductively coupled plasma atomic emission spectrometer (ICP-AES). To measure the photothermal conversion performances of the Cu7.2S4 NCs and Au nanorods, radiation from a 980 nm laser was 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 laser device (Xi'an Tours Radium Hirsh Laser Technology Co., Ltd., P. R. of China) whose power could be adjusted externally (0–0.3 W). The output power was independently calibrated using a hand-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 into the aqueous dispersion at such a position that the direct irradiation of the laser on the probe was avoided. The temperature was recorded by an online type thermocouple thermometer (DT8891E Shenzhen Everbest Machinery Industry Co., Ltd., China) every 5 s. To measure the photothermal conversion performances of the Au nanorods under the irradiation from an 808 nm laser, an 808 nm laser was chosen instead of a 980 nm laser without changing other conditions. To compare the photostability between the Cu7.2S4 NCs and Au nanorods, 32 ppm Cu7.2S4 NC solution was chosen due to the same absorbance at 980 nm as 40 ppm CTAB capped Au nanorods. 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 for 10 min (LASER ON), followed by naturally cooling to room temperature without irradiation for 20 min (LASER OFF). The temperature was measured every 10 s. This cycle was repeated four times and then UV-vis absorption spectra and TEM images of the irradiated samples were obtained for characterizing the absorption and morphology properties, respectively. 2.6 In vitro photothermal therapy of cancer cells with Cu7.2S4 nanocrystals HeLa cells were seeded into a 24 well plate at a density of 10 000 cells mL1 in RPMI-1640 culture medium at 37 C in the presence of 5% CO2 for 24 h prior to treatment. Aer incubation, the cell medium was removed, and the cells were washed with PBS buffer solution three times. 100 mL of the polymer-modied Cu7.2S4 NCs dispersed in a PBS solution was then added into the wells at gradient concentrations (0, 20, and 40 ppm). Aer incubation for another 12 h, the cells were irradiated for 0 min and 7 min, respectively, using a 980 nm laser with an output power density of 0.72 W cm2 (0.29 W for a spot size of 0.40 cm2). The cell viability was measured using the MTT assay according to the procedures suggested by the manufacturer. All of the tests were independently performed twice. 2.7 In vitro quantitative analysis of Cu7.2S4 NC uptake by cancer cells The cellular uptake of Cu7.2S4 NCs by HeLa cells was evaluated by ICP-AES. In brief, HeLa cells were seeded in a 24-well plate at a density of 5 105 cells per well in RPMI-1640 culture medium at 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 PBS solution was then added into the wells at gradient concentrations (0, 20, and 40 ppm). Aer 12 hour incubation, the media were removed. The cells were carefully washed 5 times with PBS before being digested by aqua regia and diluted by ultrapure water for the ICP analysis.
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2.8 In vivo photothermal therapy of cancer cells with Cu7.2S4 nanocrystals Severe combined immunodeciency (SCID) mice were inoculated subcutaneously with 2 106 K7M2 cells for 20 days. When the tumors inside the mice had grown to 5–10 mm in diameter, the SCID nude mice were randomly allocated into treatment and control groups. The SCID nude mice in the treatment group were injected with 100 mL of phosphate-buffered saline (PBS) solution containing 40 ppm Cu7.2S4 NCs via the hypodermic injection to the central region of the tumor with a depth of 4 mm, while the SCID nude mice in the control group were injected with 100 mL of saline solution. Aer 1 h, mice from both the control and treatment groups were simultaneously irradiated with a 980 nm laser at 0.72 W cm2 power density (0.29 W for a spot size of 0.40 cm2) for 7 min. During the laser treatment, full-body infrared thermal images were captured in real time using a photothermal medical device (GX-300; Shanghai Infratest Electronics Co., Ltd, P. R. China) with an infrared camera. Aer the laser treatment, the SCID mice were killed, and tumors were removed, embedded in paraffin, and cryosectioned into 4 mm slices. The slides were stained with H&E. The slices were examined under a Zeiss Axiovert 40 CFL inverted uorescence microscope, and images were captured with a Zeiss AxioCam MRc5 digital camera.
3.
Results and discussion
Hydrophobic Cu7.2S4 NCs capped with oleylamine and oleic acid ligands were prepared by a simple thermal decomposition route in the presence of a mixture of oleylamine and oleic acid at 280 C. As-synthesized Cu7.2S4 NCs that were dispersed in chloroform and kept at room temperature for two months retained their strong NIR absorption, indicating that the particles are not undergoing aggregation (Fig. S1, see ESI†). The transmission electron microscopy (TEM) image of Cu7.2S4 NCs aer two months gives further evidence towards lack of aggregation (Fig. S2, see ESI†). The TEM image shows good monodispersity of the resulting Cu7.2S4 NCs with a mean size of 20 nm (Fig. 1a, and S3, see ESI†). Further microstructure information of the as-synthesized Cu7.2S4 NCs is obtained from the high-resolution transmission electron microscope (HRTEM) image and electron diffraction (ED) pattern. The HRTEM image (Fig. 1b) shows a single crystal with an interplanar spacing of 0.196 nm, which corresponds to the d-spacing for (220) planes of the cubic structured Cu7.2S4 crystal. The diffraction pattern (Fig. 1c) of the fast Fourier transform (FFT) from the HRTEM image in Fig. 1b can be indexed to the [220] zone axis of the cubic 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 to cubic Cu7.2S4 with lattice parameters similar to those on JCPDS le (card no.: 24-0061), indicating the formation of a pure cubic phase of Cu7.2S4 with high crystallinity. The cell constants of ˚ which agree well Cu7.2S4 are calculated to be a ¼ b ¼ c ¼ 5.57 A, with the data obtained from JCPDS card. In addition, the injection temperature plays an important role in the formation
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of pure cubic Cu7.2S4 NCs. A mixed crystal structure of cubic phase and hexagonal phase was obtained when the injection temperature was below 240 C (Fig. S4, see ESI†). The most striking feature of the as-obtained cubic Cu7.2S4 NCs is that they exhibit broad and very strong peaks in the NIR region. To make them well-suited for PTT applications, the NCs were coated with an amphiphilic polymer (Fig. S5, see ESI†).15,27,28 Fig. 2a shows the room temperature UV-vis absorbance spectra for Cu7.2S4 NCs dispersed in water with various solution concentrations of Cu2+. It exhibits a short-wavelength absorption edge at approximately 560 nm and reaches a minimum at around 570 nm, conrming the effect of quantum size connement,25 which can be further demonstrated by Fig. S6 (see ESI†). There is a blue-shi from 580 nm to 560 nm with the decrease of the size of the NCs from 25 nm to 20 nm. Obviously, Cu7.2S4 NCs show an increased and strong absorption in the NIR region, which originated from a localized
Fig. 2 (a) UV-vis absorbance spectra for the aqueous dispersion of hydrophilic 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 980 nm versus concentration for the aqueous dispersion of hydrophilic Cu7.2S4 NCs.
(a) Low-magnification and (b) HRTEM images of as-synthesized Cu7.2S4 NCs via 4 min. (c) Corresponding FFT diffraction pattern from the 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. 240061). Fig. 1
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surface plasmon resonance of the free holes in the p-type Cu7.2S4 NCs.24 The strong absorption intensity should be mainly attributed to many Cu deciencies and high monodispersity as well as the lack of inter-particle aggregation.24 The spectral position of Cu7.2S4 NCs is different from that of Cu9S5 NCs possibly because the spectral position depends on the crystal phase.29 Luther’s study can give further evidence that LSPR spectroscopy can be tuned by the degree of crystal phase.24 Moreover, the absorbance increases linearly at 980 nm as the concentration of Cu7.2S4 NCs in water is elevated, indicating the good dispersity of Cu7.2S4 NCs in aqueous solution. Owing to their strong NIR absorption features and the location (968 nm) of maximum absorption wavelength, these Cu7.2S4 NCs are of interest for investigating their potential in photothermal ablation therapy of cancer using a 980 nm wavelength laser. Under continuous irradiation of a 980 nm laser with a power of 0.29 W, the temperature elevation of aqueous dispersions containing Cu7.2S4 NCs at different concentrations (0–40 ppm) was measured, as shown in Fig. 3 (a and b). The concentration of Cu2+ was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES).
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(a) Temperature elevation of pure water and the aqueous dispersion 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 with a power of 0.29 W as a function of irradiation time (0–420 s). (b) Plot of temperature change (DT) over a period of 420 s versus the aqueous dispersion of Cu7.2S4 NCs (with different concentrations of Cu2+). Fig. 3
The control experiment demonstrates that the temperature of pure water (without Cu7.2S4 NCs) is only increased by less than 3.0 C from room temperature (17.7 C) in 7 min. With the addition of the Cu7.2S4 NCs (i.e., 2.5, 5.0, 10, 20 and 40 ppm), the temperature of the aqueous dispersion increased by 6.0–19.5 C aer 7 min irradiation, indicating that Cu7.2S4 NCs can rapidly and efficiently convert the 980 nm wavelength laser energy into heat energy resulting from strong photoabsorption at 980 nm. For further study of the photothermal performance of the Cu7.2S4 NCs, we then measured the photothermal transduction efficiencies of the NCs (40 ppm) by a modied method similar to the report by Roper et al.30 and our previous report on Cu9S5 NCs.11 Nanoparticle dispersions were continuously illuminated by a 980 nm laser with a power of 0.29 W until reaching a steadystate temperature increase. The irradiation source was then shut off and the temperature decrease was monitored to determine the rate of heat transfer from the system. Fig. 4a shows the typical thermal prole of the Cu7.2S4 NCs dispersed in water. Following Roper's report30 the photothermal conversion efficiency, hT, was calculated using eqn (1).
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Fig. 4 (a) Photothermal effect of 40 ppm Cu7.2S4 NCs upon being irradiated 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 ss ¼ 162.9 s by applying the linear time data from the cooling period of (a) versus negative natural logarithm of driving force temperature.
hT ¼
hAðTmax Tamb Þ Q0 Ið1 10Al Þ
(1)
where h is the heat transfer coefficient, A is the surface area of the container, and the value of hA can be obtained from Fig. 4b. Tmax is the maximum system temperature, Tamb is the ambient surrounding temperature, and (Tmax Tamb) is 20.6 C according to Fig. 3c. I is the laser power (in units of mW, 290 mW) and Al is the absorbance (0.8517) at an excitation wavelength of 980 nm. Q0 is the rate of heat input (in units of mW) due to light absorption by the solvent. The lumped quantity hA was determined by measuring the rate of temperature drop aer removing the light source. The value of hA is derived according to eqn (2): ss ¼
mD CD hA
(2)
where ss is the sample system time constant, mD and CD are the mass (0.3 g) and heat capacity (4.2 J g1) of deionized water used as solvent, respectively. The Q0 was measured independently using a quartz cuvette cell containing pure water without the NCs and found to be 17.9 mW. Thus, the 980 nm laser heat
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For comparison, we subsequently investigated NIR photostability of the Cu7.2S4 NCs as well as that of the well-known photothermal agent of Au nanorods (65 17 nm, Fig. S7, see ESI†) by using four cycles of LASER ON/OFF with the NIR light according to the method described in the literature.14 The aqueous dispersion of the Cu7.2S4 NCs and Au nanorods was irradiated 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 ppm Cu7.2S4 NC solution has been chosen due to the same absorbance at 980 nm as 40 ppm cetyltrimethylammonium bromide (CTAB) capped Au nanorods. The concentration was determined by ICP-AES. The temperature elevation of 42.0–37.5 C and 34.3–24.4 C was achieved over the four LASER ON for 32 ppm Cu7.2S4 NC solution and 40 ppm Au nanorod solution, respectively, which indicated that the Cu7.2S4 NCs also show higher photothermal conversion efficiency than Au nanorods (24.6%, Fig. S8, see ESI†) under the irradiation from a 980 nm laser. As the 808 nm laser is more suitable for Au nanorods, the photothermal conversion efficiency of Au nanorods irradiated by 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
Fig. 5 (a) UV-vis spectra of Cu7.2S4 NCs and Au nanorods before and after four LASER ON/OFF cycles of NIR light (980 nm, 2 W) irradiation (LASER ON time: 10 min; LASER OFF time: 30 min). (b) Temperature elevation of Cu7.2S4 NCs and Au nanorods over four LASER ON/OFF cycles of NIR laser irradiation.
conversion efficiency (hT) of the Cu7.2S4 NCs can be calculated to be 56.7%. With a higher photothermal conversion efficiency, photothermal agents could cause equally cancerous cell death with a lower concentration of nanoparticles, a shorter irradiation time, or a lower power density of the NIR laser, which is safer for healthy tissues of the body. The value (56.7%) is noticeably higher than that of Cu9S5 NCs (25.7%)11 whose degree of copper deciency is the same as that of Cu7.2S4 NCs. As demonstrated, when the plasmon resonance wavelength is close to the wavelength of the NIR laser, Au nanorods can convert the absorbed light into heat more effectively.16 In our experiment, the plasmon resonance peak of the synthesized Cu7.2S4 NCs is centered at 968 nm which is close to the illumination laser wavelength (980 nm), and the Cu7.2S4 NCs exhibit higher NIR absorption (0.8517) at 980 nm than that (0.6581) of the Cu9S5 NCs. Furthermore, this heat conversion efficiency of the Cu7.2S4 NCs is also higher than those of previously reported materials on polypyrrole nanoparticles (44.7%)17 due to the size-dependence of the relative amounts of light scattering and absorption,31–33 and Cu2xSe NCs (22%)12 due to effective nonradiative electron relaxation dynamics.11
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(a) Cell viability after treatment with different concentrations of the Cu7.2S4 NCs and different NIR laser irradiation times. (b) Cellular uptake of the Cu7.2S4 NC in vitro cells treated with the Cu7.2S4 NCs with different concentrations. Fig. 6
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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 the maximal temperature elevation for Au nanorods, while there is less loss of the maximum temperature elevation of 10.7% for 32 ppm 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, the absorption spectrum of Au nanorods showed a signicant decrease in optical absorbance (black dot line), while the Cu7.2S4 NCs displayed no notable changes in their absorption (red dot line). The TEM test was also conducted aer 4 cycles of LASER ON/OFF cycles. The morphologies of the Cu7.2S4 NCs were retained well (Fig. S10, see ESI†) while the morphology of Au nanorods almost disappeared. These results indicate that the Cu7.2S4 NCs showed higher photothermal conversion efficiency and NIR photostability than Au nanorods. Due to such high photothermal conversion efficiency and good NIR photostability of the as-synthesized Cu7.2S4 NCs, we thus believe that these NCs can be used as excellent PTT agents. To verify our hypothesis, we rst evaluated the photothermal cytotoxicity of the Cu7.2S4 NCs with and without laser irradiation on 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
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980 nm laser (an output power density of 0.72 W cm2) in the presence of NCs (40 ppm), thus indicating a signicant photothermal therapeutic effect for HeLa cells. To better understand the efficient PTT of cancer cells in vitro, it is essential to investigate the cellular uptake of the NCs by cancer cells. ICP-AES was used to quantify the NC uptake aer treatment with NCs at different 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), the uptake of the NCs increased by 0.10–2.75 pg per cell aer incubation for 12 h, indicating that Cu7.2S4 NCs could be a promising candidate for efficient photothermal killing of cancer cells owing to the high uptake. To shed more light on the photothermal effect of the Cu7.2S4 NCs, the in vivo therapeutic efficacy of the Cu7.2S4 (40 ppm)induced photothermal therapy cancer treatment using a 980 nm laser (0.72 W cm2) for 7 min was studied (Fig. S11, see ESI†). During the laser treatment, full-body infrared thermal images were captured using an IR camera. Inspiringly, infrared thermal images with high contrast could be achieved (Fig. 7a). One can clearly see that the region 11 framed area injected with the Cu7.2S4 NCs generates more signicant temperature increases under irradiation, while, as a control, very little temperature
Fig. 7 (a) Infrared thermal images of two mice injected with the Cu7.2S4 NCs (the left mouse, indicated region 12) or saline (the right mouse, indicated region 11) via the hypodermic injection, respectively, irradiated with a 980 nm laser (0.72 W cm2) at a time point of 0 and 240 s. (b) The temperature profiles in regions 11 and 12 as a function of the irradiation time. (c and d) The representative hematoxylin and eosin stained histological images of the corresponding ex vivo tumor sections, after irradiation for 7 min. The irradiation source is a 980 nm laser with the safe power density of 0.72 W cm2.
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change was detected on the region 12 framed area. The temperature of the irradiated area was also recorded as a function of the irradiation time (Fig. 7b). For the mice injected with saline solution (region 12), the surface temperature of the tumor increased by less than 2 C, and remained below 35 C in the whole irradiation process. However, in the case of Cu7.2S4 NC injected mice (region 11), the tumor surface temperature increased rapidly and reached up to 43.4 C at 30 s and 46.0 C at 60 s, and then change to plateau at about 46.0 0.7 C aer 80 s, as demonstrated vividly in Fig. S12, see ESI.† These results reveal a rapid elevation of temperature of the in vivo tumor, which suggests that the Cu7.2S4 NCs in vivo still have an excellent photothermal effect. To further evaluate photothermal ablation of cancer cells in vivo, the histological examination of tumors was performed by means of microscopic imaging (Fig. 6c, d and S9, see ESI†). As expected, signicant cancer cell damage was noticed only in the tumor with the Cu7.2S4 NC injection, but not in the control group. The treatment injected with the Cu7.2S4 NCs shows severe cellular damage (pyknosis, karyorrhexis, and karyolysis) as well as a decrease in the number of cells in comparison with the control injected with saline. These facts suggest that in vivo cancer cells can be efficiently destroyed by the high temperature (46 C) arising from the excellent photothermal effect of the Cu7.2S4 NCs. Taken together, these results unambiguously prove that the photothermal effects of the synthesized Cu7.2S4 NCs have great potential to be used as a novel and excellent photothermal agent for PTT of cancer cells.
4. Conclusions In conclusion, hydrophilic Cu7.2S4 NCs with a mean size of 20 nm as a novel photothermal agent have been prepared by a simple thermal decomposition route in the presence of a mixture of oleylamine and oleic acid and a subsequent hydrophilic modication process with an amphiphilic polymer. The amphiphilic polymer-coated Cu7.2S4 NCs exhibit good photostability and signicant photothermal conversion efficiency up to 56.7% due to strong NIR absorption, higher than that of the as-synthesized Au nanorods, Cu9S5 NCs, Cu2xSe NCs, and the recently reported polypyrrole nanoparticles. Importantly, cancer cells in vitro and in vivo can be efficiently killed by the photothermal effects, which are realized by a very low concentration (40 ppm) of Cu7.2S4 NCs in PBS solution under the irradiation of a 980 nm laser with a safe power density of 0.72 W cm2. Therefore, these Cu7.2S4 NCs have a great superiority as a novel PTT agent of cancer cells, owing to their right size (20 nm), high photothermal conversion efficiency, and good photostability. Further studies of targeted cancer therapy and in vivo distribution are underway.
Acknowledgements B.L. and Q.W. contributed equally to this work. This work was nancially supported by the National Natural Science Foundation of China (Grant Nos. 21171035 and 51302035), the Key Grant Project of Chinese Ministry of Education (Grant No. 313015), the PhD Programs Foundation of the Ministry of
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Education of China (Grant Nos. 20110075110008 and 20130075120001), the National 863 Program of China (Grant No. 2013AA031903), the Science and Technology Commission of Shanghai Municipality (Grant No. 13ZR1451200), the Fundamental Research Funds for the Central Universities, the Shanghai Leading Academic Discipline Project (Grant no. B603), and the Program of Introducing Talents of Discipline to Universities (Grant no. 111-2-04).
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