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Facile synthesis of biocompatible cysteine-coated CuS nanoparticles with high photothermal conversion efficiency for cancer therapy† Xijian Liu,a,b Bo Li,a Fanfan Fu,c Kaibing Xu,a Rujia Zou,*a,d Qian Wang,a,e Bingjie Zhang,a Zhigang Chena and Junqing Hu*a The semiconductor compounds have been proven to be promising candidates as a new type of photothermal therapy agent, but unsatisfactory photothermal conversion efficiencies limit their widespread application in photothermal therapy (PTT). Herein, we synthesized cysteine-coated CuS nanoparticles (Cys-CuS NPs) as highly efficient PTT agents by a simple aqueous solution method. The Cys-CuS NPs have a good biocompatibility owing to their biocompatible cysteine coating and exhibit a strong absorption in the near-infrared region due to the localized surface plasma resonances of valence-band free carriers. The photothermal conversion efficiency of Cys-CuS NPs reaches 38.0%, which is much higher
Received 10th February 2014, Accepted 23rd May 2014
than that of the recently reported Cu9S5 and Cu2−xSe nanocrystals. More importantly, tumor growth can be efficiently inhibited in vivo by the fatal heat arising from the excellent photothermal effect of Cys-CuS
DOI: 10.1039/c4dt00424h
NPs at a low concentration under the irradiation of a 980 nm laser with a safe power density of 0.72 W cm−2.
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Therefore, the Cys-CuS NPs have great potential as ideal photothermal agents for cancer therapy.
1.
Introduction
Photothermal therapy (PTT) has attracted extensive research interest in recent years as a promising minimally invasive alternative to target cancerous cells without causing systemic effects.1–3 PTT is induced by a near-infrared (NIR, λ = 700–1100 nm) laser and is dependent on the heat converted by optical energy of the laser to ablate tumor cells.4,5 Thus, the photothermal conversion efficiency of PTT agents determines the therapy result of PTT. Currently, four types of nanomaterials have shown the encouraging PTT effects, including noble metal nanostructures,6–9 carbon-based materials,10,11 organic compounds,3,12,13 and semiconductor compounds.4,14–16 Among them, semiconductor compounds as a new type of PTT
a State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. E-mail: [email protected], [email protected] b College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China c College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China d Center of Super-Diamond and Advanced Films (COSDAF), Department of Physics and Materials Science, City University of Hong Kong, Hong Kong e Department of Orthopaedics, Shanghai First People’s Hospital, Shanghai Jiaotong University, 100 Haining Road, Hongkou District, Shanghai 200080, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4dt00424h
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agent including W18O49 nanowires,14 copper sulfide,4,15,16 and copper selenide17 nanoparticles have been proven to be promising alternatives due to their low cost and high stability.16 Although semiconductor compounds have shown effective PTT effects for cancer treatment, there is still considerable room for performance improvement of semiconductor compounds. The absorbance peaks of semiconductor compounds as PTT agents are far away from the illumination laser wavelength;5,17 thus the PTT agent cannot reach the highest temperature due to less absorption of light energy,18 and the photothermal conversion efficiencies are not high enough (such as 22% for Cu2−xSe nanocrystals with 800 nm light17 and 25.7% for Cu9S5 nanocrystals with 980 nm light5). In order to obtain sufficient heating to kill cancer cells, a relatively high optical power was required. For example, PEG-CuS NPs were irradiated with an 808 nm laser at 12 W cm−2 and an 800 nm laser at 30 W cm−2 to kill cancer cells,15,17 and the power densities were much higher than the conservative limit of intensity setting for human skin exposure (∼0.33 W cm−2 for 808 nm laser, ∼0.32 W cm−2 for 800 nm laser).19 The high power intensity probably harms the skin and normal tissues and thus limits the semiconductor compounds used as PTT agents for clinical cancer treatment. In order to meet the severe requirements of future photothermal therapy, the photothermal conversion efficiency of semiconductor compounds should be further promoted and it is still necessary to optimize semiconductor compounds as PTT agents. Mercapto-compounds as the capping ligands
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have been successfully used to prepare CdS,20 CdSe21 and CdTe22 NPs, which stimulated us to use biocompatible cysteine as capping ligands to synthesize CuS NPs. Herein, we report cysteine coated CuS nanoparticles (Cys-CuS NPs) as highly efficient PTT agents which were synthesized by a simple aqueous solution method. The Cys-CuS NPs have a strong NIR region absorption peak approximately at an illumination laser wavelength of 980 nm. The photothermal conversion efficiency of Cys-CuS NPs with the 980 nm laser irradiation reaches 38.0%, which is much higher than that of recently reported Cu9S5 nanocrystals and Cu2−xSe nanocrystals. The Cys-CuS NPs have a good biocompatibility due to cysteine coating; more importantly, tumor growth can be efficiently inhibited in vivo by the fatal heat arising from the excellent photothermal effect of Cys-CuS NPs at a low concentration (50 ppm) under the irradiation of the 980 nm laser with a safe power density of 0.72 W cm−2.19
2. Experimental section 2.1
Chemicals and reagents
All reagents were used without further purification. Copper nitrate, L-cysteine (Cys), sodium thiosulfate, hydrogen nitrate and anhydrous ethanol are analytically pure and were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). 2.2
Characterization
The sizes, morphologies, and microstructures of the CuS nanoparticles were measured using a transmission electron microscope (TEM; JEM-2100F). Powder X-ray diffraction (XRD) was determined using a D/max-2550 PC X-ray diffractometer (Rigaku, Japan). Fourier transform infrared (FTIR) spectra were measured using an IRPRESTIGE-21 spectrometer (Shimadzu) using KBr pressed pellets. UV-visible absorption spectra were determined using a UV-Vis 1901 spectrophotometer (Phoenix) using quartz cuvettes with an optical path length of 1 cm. The content of copper ions in the solution was determined by Leeman Laboratories Prodigy high-dispersion inductively coupled plasma atomic emission spectroscopy (ICP-AES). 2.3
Synthesis of Cys-CuS NPs
In a typical procedure, 0.12 mmol of L-cysteine was dissolved in 150 mL of deionized water, and 50 mL of 1.68 mmol Cu(NO3)2 was added to the above solution under constant stirring. After stirring for 30 min, the pH was adjusted to 3.0 by drop-wise addition of a hydrogen nitrate solution, and then 0.84 mL of sodium thiosulfate solution (0.2 M) was added. The solution was slowly heated to 90 °C under magnetic stirring and the temperature was maintained for 90 min. The solution turned from baby blue to brown and finally dark green. The synthesized product was centrifuged (10 000 rpm, 10 min) and then washed with deionized water three times. Finally, the Cys-CuS NPs were acquired and dispersed in deionized water for later use.
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2.4
Measurement of photothermal performance
Measurement of photothermal performance was carried out by our previous method.5,14 The solutions (0.3 mL) of Cys-CuS NPs with different concentrations (5, 10, 25, 50, and 100 ppm Cu2+) and gold nanorods (50 ppm) were irradiated with a laser (980 nm). The light source was a 980 nm semiconductor laser device (Xi’an Tours Radium Hirsh Laser Technology Co., Ltd, China) with an external adjustable power (0–0.3 W). The output power density was independently calibrated using a handy optical power meter (Newport model 1918-C, CA, USA) and was measured to be ∼0.72 W cm−2. The temperature of the solutions was measured using a digital thermometer (with an accuracy of 0.1 °C) using a thermocouple probe every 5 s. In order to investigate the photostability of the Cys-CuS NPs, a Cys-CuS NP solution (50 ppm Cu2+) was irradiated with a 980 nm NIR laser for 10 min (LASER ON), followed by naturally cooling to room temperature without NIR laser irradiation (LASER OFF). This cycle was repeated five times. 2.5
In vitro cytotoxicity of the Cys-CuS NPs
The in vitro cytotoxicity was measured using the MTT assay in the human cervical carcinoma cell line HeLa.5 HeLa cells were plated into a 96-well plate (1 × 104 cells per well) in complete medium at 37 °C and 5% CO2 for 24 h before the experiments. The culture medium was replaced and cells were incubated with complete medium containing the Cys-CuS NPs at a series of concentrations at 37 °C with 5% CO2 for further 24 h. Relative cell viabilities were determined by the standard MTT assay. Four replicates were prepared for each treatment group. 2.6
In vivo photothermal therapy
The photothermal therapy of cancer cells in vivo was carried out by our previously reported method.14 Osteosarcoma bearing mice were obtained by inoculating subcutaneously with K7M2 cells. When the tumors inside the mice had grown to 5–10 mm in diameter, the mice were randomly allocated into the control group and the treatment group. The mice were first anaesthetized by trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg kg−1 body weight. The mouse of the treatment group was intratumorally injected with 0.10 mL of phosphatebuffered saline (PBS) solution containing Cys-CuS NPs (50 ppm Cu2+), while the mouse of the control group was intratumorally injected with 0.1 mL of PBS solution. After 1 h, the injected areas of mice from both treatment and control groups were perpendicularly irradiated with similar 980 nm wavelength laser devices at a power density of 0.72 W cm−2 for 5 min. The temperatures of the tumor surface were measured using an infrared thermometer (GX-A300, Shanghai Guixin Corporation). The mice were killed after photothermal therapy, and tumors were removed, embedded in paraffin, and cryo-sectioned into 4 μm slices. The slides were stained with hematoxylin/eosin (H&E). The slices were examined under a Zeiss Axiovert 40 CFL inverted fluorescence microscope, and images were captured with a Zeiss AxioCam MRc5 digital camera.
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2.7
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In vivo antitumor effect
Osteosarcoma bearing mice were obtained by inoculating subcutaneously with K7M2 cells. When the tumor volume inside the mice reached ∼140 mm3, eight mice were randomly allocated into two groups and intratumorally injected with 150 μL of PBS and Cys-CuS solutions (50 ppm Cu2+). After injection, tumors were irradiated with NIR light (980 nm, 0.72 W cm−2) for 10 min. After the experiments were completed, mice were killed and the tumors were collected and weighed. All animal experiments were carried out according to protocols approved by the institutional committee for animal care and also in accordance with the policy of the National Ministry of Health.
3. Results and discussion Cys-CuS NPs were readily synthesized in aqueous solution by reacting Cu(NO3)2 and Na2S2O3 in the presence of L-cysteine at 90 °C for 90 min. As-prepared Cys-CuS NPs have a mean size of ∼18 nm (Fig. 1a,b, S1 in ESI†), and the Cys-CuS NPs tend to form small clusters probably due to the acting force of thiol and copper atoms. The size of the clusters was measured by dynamic light scattering (Zetasizer Nano Z) to be ∼152.9 nm (Fig. S2 in ESI†). Further microstructure information was obtained from the high resolution transmission electron microscope (HRTEM) image (Fig. 1b, inset), and it shows an interplanar spacing of ∼0.305 nm, which is in agreement with the lattice spacing of the (1,0,2) planes of hexagonal CuS nanostructures.23 We also synthesized several Cys-CuS NP samples with a different particle size. The absorbance spectra of these Cys-CuS NP samples did not show an obvious difference in absorbance peak position (Fig. S3 in ESI†). In fact, the absorption of the CuS NPs in the NIR region originated from the localized surface plasma resonances of valence-band free carriers ( positive holes),15 and thus shows little variation due to the
Fig. 1 (a) Low-magnification TEM and (b) TEM images of the synthesized Cys-CuS NPs (inset: HRTEM image of a Cys-CuS NP). (c) XRD patterns of the as-prepared product (upper) and the standard CuS (lower). (d) The FTIR spectrum of as-prepared Cys-CuS NPs.
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Fig. 2 (a) UV-vis-NIR absorbance spectra of the aqueous solution containing the Cys-CuS NPs with various concentrations of Cu2+ (i.e., 5, 10, 25, 50 and 100 ppm). An inset photograph shows Cys-CuS NPs in water after 1 week. (b) Temperature change of the aqueous solution containing the Cys-CuS NPs with different concentrations under irradiation of 980 nm laser with a power density of 0.72 W cm−2 as a function of irradiation time. (c) Plot of temperature elevation over a period of 300 s versus the concentration of Cys-CuS NPs. (d) Temperature change of Cys-CuS NPs over five ON/OFF cycles of 980 nm laser irradiation.
size change. The phase structure of the as-obtained Cys-CuS NPs was examined using XRD patterns, as shown in Fig. 2c. Several well-defined characteristic peaks, such as (1,0,2), (1,0,3) and (1,1,0), exhibit the hexagonal phase, referenced to the standard CuS phase (JCPDS card no. 6-0464). Corresponding to the standard hexagonal CuS phase, no obvious impure peaks were found, demonstrating that high quality covellite CuS was acquired. The Fourier transform infrared (FTIR) spectrum of the Cys-CuS NPs is shown in Fig. 1d. The adsorption peak at 3426 cm−1 corresponds to the stretching vibrations of NH2 or OH in cysteine.19 The adsorption peaks at 2927 cm−1 are assigned to the symmetrical stretching vibration of methylene (CH2) in the cysteine.24,25 Peaks at 1630 cm−1 and 1406 cm−1 are due to asymmetric stretching vibration and symmetric stretching vibration of the carboxyl group (COO−), respectively.26 These results suggest that cysteine was successfully anchored onto the surface of CuS NPs. Because cysteine is a needed amino acid for human beings, it has good biocompatibility in blood and tissues. So, the biocompatibility of CuS NPs was enhanced by coating with cysteine. The optical properties of the aqueous dispersion containing the Cys-CuS NPs with various concentrations were examined by UV-vis-NIR spectroscopy, as shown in Fig. 2a. The spectra exhibit a strong absorption in the NIR region due to the localized surface plasma resonances (SPR) of valence-band free carriers ( positive holes) of covellite CuS.15 The absorbance peak is located at 1010 nm, and is very close to the wavelength (980 nm) of the excitation laser, which is advantageous for full usage of the SPR effect of materials, enhancing the photothermal efficiency.18 Owing to coating with cysteine, the aqueous dispersion of Cys-CuS NPs has high stability and can even
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remain unchanged after being dispersed in water for one week (the inset of Fig. 2a). The strong SPR absorption of as-prepared Cys-CuS NPs in the NIR region motivates us to investigate the photothermal effect. A simple home-designed setup (Fig. S4 in ESI†) was used for the photothermal performance measurement. The photothermal conversion performance of the solutions containing various concentrations of Cys-CuS NPs was measured under laser irradiation (980 nm, 0.72 W cm−2). As shown in Fig. 2b, the temperature of the 50 ppm Cys-CuS NP solution was raised from 18.9 °C to 37.4 °C under NIR irradiation of 300 s, while the temperature of pure water was only increased by 3.8 °C under the same conditions. With the increase of the concentration of the Cys-CuS NPs (i.e., 5, 10, 25, 50, and 100 ppm), the temperatures of the solutions containing CysCuS NPs can increase by 8.4 to 21.5 °C in 300 s, as shown in Fig. 2c. These data confirm that the Cys-CuS NPs can effectively absorb the 980 nm laser energy and effectively convert laser energy into heat. The ideal PTT agents should not only have high photothermal conversion efficiency, but also possess good NIR photostability. To investigate the NIR photostability, the solution of Cys-CuS NPs was irradiated with the 980 nm laser (0.72 W cm−2) for 5 min, followed by naturally cooling to room temperature without laser irradiation. This cycle was repeated five times. As shown in Fig. 2d, at the first cycle, the temperature of the Cys-CuS NP solution increased from 16.9 °C to 35.5 °C, compared with the fifth cycle that increased from 17 °C to 34.9 °C. The temperature elevation after five cycles of irradiation only decreased by less than 4.0%, indicating that as-synthesized Cys-CuS NPs have good NIR photostability, which is beneficial to repeated photothermal therapy in long term clinical treatments. Cys-CuS NPs have good photothermal effects and NIR photostability that inspire us to examine the photothermal conversion efficiency of the Cys-CuS NPs. Following a previous method,5,17 the photothermal conversion efficiency (η) of the Cys-CuS NPs was measured. The aqueous solution of the CysCuS NPs (50 ppm) was under continuous irradiation of the laser (980 nm, 0.72 W cm−2) until a steady state temperature was reached. Subsequently, the laser was shut off, and the temperature decrease of the aqueous solution was recorded to measure the rate of heat transfer from the Cys-CuS NP solution system to the environment (Fig. 3a). The η value was calculated (see ESI† for details) as follows:5,17 η¼
hSðT Max T Surr Þ QDis Ið1 10A980 Þ
ð1Þ
where h is the heat transfer coefficient, S is the surface area of the container, and the value of hS was gained from Fig. 3b. Tmax − TSurr is the temperature change of the Cys-CuS NP solution at the maximum steady-environmental temperature, I is the power of the laser, A980 is the absorbance of Cys-CuS NPs at 980 nm, and QDis expresses heat dissipated from light absorbed by the solvent and the container.
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Fig. 3 (a) Photothermal effect of the irradiation of the aqueous solution of the Cys-CuS NPs (50 ppm) with the NIR laser (980 nm, 0.72 W cm−2), in which the irradiation lasted for 660 s, and then the laser was shut off. (b) Linear time data versus −ln(θ) obtained from the cooling period of (a).
According to eqn (1), the 980 nm laser heat conversion efficiency (η) of the Cys-CuS NPs was determined to be 38.0%, which is relatively high compared with those of previously reported materials, such as the widely used Au rods (∼21.0%, 800 nm laser), Cu2−xSe nanocrystals (∼22%, 800 nm laser), and Cu9S5 nanocrystals (∼25.7%, 980 nm laser).5 In order to further compare the Cys-CuS NPs with other materials, we have measured the photothermal conversion efficiency of the Au nanorods and Cys-CuS NPs under the same experimental conditions. As shown in Fig. S5 (in ESI†), the photothermal conversion efficiency (η) of the Au nanorods was 21.3%, which is much lower than that of the CuS NPs. The high photothermal conversion efficiency of the Cys-CuS NPs should be attributed to the strong NIR absorption related to the plasmon resonance. Like the Au nanorods, the highest temperature can be reached when the plasmon resonance wavelength is equal to the illumination laser wavelength, and the lower temperature elevation was obtained when the plasmon resonance peak wavelength was either shorter or longer than the illumination laser wavelength.25 It can be inferred that more laser energy conversion to heat will occur when the plasmon resonance peak wavelength is near the illumination laser wavelength. Our previous work also demonstrated that the photothermal efficiency of Au nanorods (absorbance peaks at ∼808 nm) illuminated with an 808 nm laser was much higher than that of Au nanorods illuminated with a 980 nm laser.27 Thus, it can be inferred that higher photothermal conversion efficiency could be acquired when the absorbance peak of the photothermal agents is close to the illumination laser wavelength. The absorbance peak (∼1010 nm) of the Cys-CuS NPs is approximately at the wavelength (980 nm) of the illumination laser; thus, the Cys-CuS NP solution converts the absorbed light into heat more effectively. The high η of the Cys-CuS NPs makes them highly superior as promising PTT agents. The ideal PTT should be nontoxic or low toxic for biological applications. The high photothermal conversion efficiency of the Cys-CuS NPs prompted us to further evaluate their feasibility as PTT agents for cancer therapy. So we first incubated HeLa cells with the Cys-CuS NPs at different concentrations for 24 h and then the MTT assay was carried out to test the cell viability. It was found that the as-prepared Cys-CuS NPs demonstrate a good biocompatibility (Fig. 4). Even at the
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Fig. 4 Viabilities of HeLa cells incubated for 24 h with different concentrations of the Cys-CuS NPs.
highest tested dose of the Cys-CuS NPs (75 ppm Cu2+), cell viability still remained approximately 93.7%. These results can be attributed to that CuS NPs were coated with biocompatible cysteine. The Cys-CuS NPs have high photothermal conversion efficiency and good biocompatibility in vitro. Because of the complexity of the in vivo environment, the therapy efficacy should be examined in vivo to confirm that the Cys-CuS NPs are good PTT agents. When the tumors inside the mice had grown to 5–10 mm in diameter, the mice were randomly allocated into treatment and control groups. The treatment mouse was intratumorally injected with 0.10 mL of the Cys-CuS NP solution, while the control mouse was intratumorally injected with 0.1 mL PBS solution. After 1 h, the injected areas of the mice from two groups were perpendicularly irradiated by the 980 nm laser device (0.72 W cm−2) for 5 min. For the mouse treated with PBS solution, the surface temperature of the tumor showed no significant change during the entire irradiation process (Fig. 5). In contrast, for the Cys-CuS NPs injected mouse, the tumor surface temperature increased rapidly from 31.8 °C, and reached 51.0 °C after 300 s. As is well known, the hyperthermic therapy is the use of heat between 40 and 45 °C to damage cancer cells.14 The tumor surface temperature of the treatment mouse can easily reach over 45 °C after 2 min of laser irradiation, so the tumor tissue can be efficiently destroyed after 5 min of laser irradiation for hyperthermia. These results reveal that the Cys-CuS NPs within the tumor can also effectively adsorb and convert the NIR light to fatal heat due to the deep penetration depth of the 980 nm laser in biological tissues.4,14,28 The mice were killed after the photothermal therapy, and tumors were removed, embedded in paraffin, and cryo-sectioned into 4 μm slices. The slides were stained with hematoxylin/eosin (H&E). The histopathological images of the mouse treated with PBS (Fig. 6a,c) show that the cells remained normal after irradiation. Because the heat of PBS solution converted from the 980 nm laser irradiation was very little, the
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Fig. 5 Plots of the temperature within the irradiated tumor area in the mice injected with PBS solution and Cys-CuS NP solution as a function of irradiation time, respectively. ( p < 0.001 was considered to be statistically significant difference.)
Fig. 6 H&E-stained histological images of mice (a, c) injected with PBS solution and (b, d) injected with the Cys-CuS NP solution after 5 min of laser irradiation, respectively.
temperature of the tumor surface is only 35.7 °C, which is not high enough to kill the cancer cells. The histopathological images of the Cys-CuS NP treatment are shown in Fig. 6b,d. Almost all of the tumor tissue was necrotized on areas of the examined tumor slide, exhibiting cell shrinkage, loss of contact, coagulation, cytoplasmic acidophilia, and corruption of the tumor extracellular matrix. These facts suggest that the Cys-CuS NPs can still effectively convert NIR light into fatal heat in vivo and tumor cells can be efficiently destroyed by hyperthermia arising from the excellent photothermal effect of the Cys-CuS NPs. To further determine the antitumor effect of Cys-CuS mediated photothermal therapy in vivo, osteosarcoma bearing mice were randomly allocated into two groups and intratumorally injected with 150 μL of PBS and Cys-CuS solutions. After injection, tumors were irradiated with NIR light (980 nm,
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weight of mice in two groups every two days. No obvious weight loss was observed in both the PBS group and the Cys-CuS group (Fig. S6 in ESI†), indicating that the toxicity of treatments was low. Therefore, the Cys-CuS NPs have great potential as ideal photothermal agents for the photothermal therapy in vivo of tumor tissues.
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4.
Fig. 7 (a) Representative photos of mice bearing osteosarcoma tumors before and after treatments of PBS, Cys-CuS. (b) Photos of the tumors collected from PBS and Cys-CuS groups of mice at the end of treatments (day 12). (c) Mean weights of tumors collected from mice at the end of PBS and Cys-CuS treatments. ( p < 0.05 was considered to be statistically significant difference.)
0.72 W cm−2) for 10 min. 12 days after treatment, mice were sacrificed and tumors were excised and weighed. As shown in Fig. 7a–c, the tumors treated with PBS plus laser irradiation grew rapidly, suggesting that the osteosarcoma tumor growth was not affected by laser irradiation. While the tumors treated with Cys-CuS solutions plus laser irradiation were greatly inhibited, half tumors were even completely eradicated. As a result, tumor of the Cys-CuS group was not completely inhibited but showed much smaller tumor sizes than the PBS group after treatment, probably due to a small part of the larger size of tumor area free of irradiation (Fig. 7b). The mean tumor weight of the Cys-CuS group is only 0.088 g, while that of the PBS group is 0.627 g, which is more than seven times that of the Cys-CuS group (Fig. 7c). There are significant differences between the group under treatment with CuS NPs and the group under treatment with PBS ( p = 0.018). The CuS photothermal materials had a negligible impact on tumor growth without illumination,29,30 and only 980 nm laser illumination (below 1.5 W cm−2) has little influence on the tumor growth,29 so the inhibition of tumor growth could be attributed to the excellent photothermal effect of Cys-CuS NPs under 980 nm illumination. These results indicate that the Cys-CuS NPs still can effectively convert NIR light into fatal heat in vivo and tumor growth can be efficiently inhibited by the photothermal effect of Cys-CuS NP solutions. The potential in vivo toxicity is always a primary concern for nanomaterials applied in biomedicine.31 High toxicity usually leads to a significant weight loss, so we measured the body
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Conclusion
In summary, we have successfully synthesized hydrophilic PTT agents of the Cys-CuS NPs by a facile aqueous solution method. The Cys-CuS NPs were coated with cysteine, and have good photostability and biocompatibility. The solution of the Cys-CuS NPs exhibits an intense absorbance in the NIR region, and the temperature of the 50 ppm solution can be increased by 18.5 °C in 300 s under the irradiation of the 980 nm laser with a safe power density of 0.72 W cm−2. The photothermal conversion efficiency of the Cys-CuS NPs can be as high as 38.0%, which is higher than that of the reported Au nanorods, Cu2−xSe nanocrystals and Cu9S5 nanocrystals. More importantly, tumor growth can be efficiently inhibited by hyperthermia arising from the excellent photothermal effect of the Cys-CuS NPs at a low concentration (50 ppm) under the irradiation of the 980 nm laser. Owing to their high photothermal conversion efficiency, photostability, and good biocompatibility, these Cys-CuS NPs could be used as promising PTT agents for cancer therapy.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 51302035 and 21171035), the Key Grant Project of Chinese Ministry of Education (Grant No. 313015), Ph.D. Programs Foundation of Ministry of Education of China (Grant No. 20110075110008), the National 863 Program of China (Grant No. 2013AA031903), the Fundamental Research Funds for the Central Universities, the Science and Technology Commission of Shanghai Municipality (Grant No. 13ZR1451200), the Hong Kong Scholars Program, the Project funded by China Postdoctoral Science Foundation, the Program for Changjiang Scholars and Innovative Research Team in University (IRT1221), 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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17 C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell and B. A. Korgel, Nano Lett., 2011, 11, 2560–2566. 18 H. Chen, L. Shao, T. Ming, Z. Sun, C. Zhao, B. Yang and J. Wang, Small, 2010, 6, 2272–2280. 19 American National Standard Institute, ANSI Z136. 1-2000, American National Standard for Safe Use of Lasers, Orlando, Fl, 2000. 20 X. Xue, Y. Huang, Z. Zhuang, F. Huang and Z. Lin, CrystEngComm, 2013, 15, 4963–4969. 21 W. Mi, J. Tian, J. Jia, W. Tian, J. Dai and X. Wang, J. Phys. D: Appl. Phys., 2012, 45, 435303. 22 D. Zhou, M. Lin, X. Liu, J. Li, Z. Chen, D. Yao, H. Sun, H. Zhang and B. Yang, ACS Nano, 2013, 7, 2273–2283. 23 C. Wu, S. H. Yu, S. Chen, G. Liu and B. Liu, J. Mater. Chem., 2006, 16, 3326–3331. 24 M. L. Cui, J. M. Liu, X. X. Wang, L. P. Lin, L. Jiao, L. H. Zhang, Z. Y. Zheng and S.-Q. Lin, Analyst, 2012, 137, 5346–5351. 25 H. Kumar, R. Srivastava and P. K. Dutta, Carbohydr. Polym., 2013, 97, 327–334. 26 M. Balaz, P. Balaz, G. Tjuliev, A. Zubrik, M. Jesus Sayagues, A. Zorkovska and N. Kostova, J. Mater. Sci., 2013, 48, 2424–2432. 27 B. Li, Q. Wang, R. Zou, X. Liu, K. Xu, W. Li and J. Hu, Nanoscale, 2014, 6, 3274–3282. 28 Z. Chen, L. Zhang, Y. Sun, J. Hu and D. Wang, Adv. Funct. Mater., 2009, 19, 3815–3820. 29 Q. Xiao, X. Zheng, W. Bu, W. Ge, S. Zhang, F. Chen, H. Xing, Q. Ren, W. Fan, K. Zhao, Y. Hua and J. Shi, J. Am. Chem. Soc., 2013, 135, 13041–13048. 30 K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452–4458. 31 K. Yang, S. Zhang, G. Zhang, X. Sun, S. T. Lee and Z. Liu, Nano Lett., 2010, 10, 3318–3323.
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Facile synthesis of biocompatible cysteine-coated CuS nanoparticles with high photothermal conversion efficiency for cancer therapy† Xijian Liu,a,b Bo Li,a Fanfan Fu,c Kaibing Xu,a Rujia Zou,*a,d Qian Wang,a,e Bingjie Zhang,a Zhigang Chena and Junqing Hu*a The semiconductor compounds have been proven to be promising candidates as a new type of photothermal therapy agent, but unsatisfactory photothermal conversion efficiencies limit their widespread application in photothermal therapy (PTT). Herein, we synthesized cysteine-coated CuS nanoparticles (Cys-CuS NPs) as highly efficient PTT agents by a simple aqueous solution method. The Cys-CuS NPs have a good biocompatibility owing to their biocompatible cysteine coating and exhibit a strong absorption in the near-infrared region due to the localized surface plasma resonances of valence-band free carriers. The photothermal conversion efficiency of Cys-CuS NPs reaches 38.0%, which is much higher
Received 10th February 2014, Accepted 23rd May 2014
than that of the recently reported Cu9S5 and Cu2−xSe nanocrystals. More importantly, tumor growth can be efficiently inhibited in vivo by the fatal heat arising from the excellent photothermal effect of Cys-CuS
DOI: 10.1039/c4dt00424h
NPs at a low concentration under the irradiation of a 980 nm laser with a safe power density of 0.72 W cm−2.
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Therefore, the Cys-CuS NPs have great potential as ideal photothermal agents for cancer therapy.
1.
Introduction
Photothermal therapy (PTT) has attracted extensive research interest in recent years as a promising minimally invasive alternative to target cancerous cells without causing systemic effects.1–3 PTT is induced by a near-infrared (NIR, λ = 700–1100 nm) laser and is dependent on the heat converted by optical energy of the laser to ablate tumor cells.4,5 Thus, the photothermal conversion efficiency of PTT agents determines the therapy result of PTT. Currently, four types of nanomaterials have shown the encouraging PTT effects, including noble metal nanostructures,6–9 carbon-based materials,10,11 organic compounds,3,12,13 and semiconductor compounds.4,14–16 Among them, semiconductor compounds as a new type of PTT
a State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China. E-mail: [email protected], [email protected] b College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China c College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China d Center of Super-Diamond and Advanced Films (COSDAF), Department of Physics and Materials Science, City University of Hong Kong, Hong Kong e Department of Orthopaedics, Shanghai First People’s Hospital, Shanghai Jiaotong University, 100 Haining Road, Hongkou District, Shanghai 200080, China † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4dt00424h
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agent including W18O49 nanowires,14 copper sulfide,4,15,16 and copper selenide17 nanoparticles have been proven to be promising alternatives due to their low cost and high stability.16 Although semiconductor compounds have shown effective PTT effects for cancer treatment, there is still considerable room for performance improvement of semiconductor compounds. The absorbance peaks of semiconductor compounds as PTT agents are far away from the illumination laser wavelength;5,17 thus the PTT agent cannot reach the highest temperature due to less absorption of light energy,18 and the photothermal conversion efficiencies are not high enough (such as 22% for Cu2−xSe nanocrystals with 800 nm light17 and 25.7% for Cu9S5 nanocrystals with 980 nm light5). In order to obtain sufficient heating to kill cancer cells, a relatively high optical power was required. For example, PEG-CuS NPs were irradiated with an 808 nm laser at 12 W cm−2 and an 800 nm laser at 30 W cm−2 to kill cancer cells,15,17 and the power densities were much higher than the conservative limit of intensity setting for human skin exposure (∼0.33 W cm−2 for 808 nm laser, ∼0.32 W cm−2 for 800 nm laser).19 The high power intensity probably harms the skin and normal tissues and thus limits the semiconductor compounds used as PTT agents for clinical cancer treatment. In order to meet the severe requirements of future photothermal therapy, the photothermal conversion efficiency of semiconductor compounds should be further promoted and it is still necessary to optimize semiconductor compounds as PTT agents. Mercapto-compounds as the capping ligands
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have been successfully used to prepare CdS,20 CdSe21 and CdTe22 NPs, which stimulated us to use biocompatible cysteine as capping ligands to synthesize CuS NPs. Herein, we report cysteine coated CuS nanoparticles (Cys-CuS NPs) as highly efficient PTT agents which were synthesized by a simple aqueous solution method. The Cys-CuS NPs have a strong NIR region absorption peak approximately at an illumination laser wavelength of 980 nm. The photothermal conversion efficiency of Cys-CuS NPs with the 980 nm laser irradiation reaches 38.0%, which is much higher than that of recently reported Cu9S5 nanocrystals and Cu2−xSe nanocrystals. The Cys-CuS NPs have a good biocompatibility due to cysteine coating; more importantly, tumor growth can be efficiently inhibited in vivo by the fatal heat arising from the excellent photothermal effect of Cys-CuS NPs at a low concentration (50 ppm) under the irradiation of the 980 nm laser with a safe power density of 0.72 W cm−2.19
2. Experimental section 2.1
Chemicals and reagents
All reagents were used without further purification. Copper nitrate, L-cysteine (Cys), sodium thiosulfate, hydrogen nitrate and anhydrous ethanol are analytically pure and were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). 2.2
Characterization
The sizes, morphologies, and microstructures of the CuS nanoparticles were measured using a transmission electron microscope (TEM; JEM-2100F). Powder X-ray diffraction (XRD) was determined using a D/max-2550 PC X-ray diffractometer (Rigaku, Japan). Fourier transform infrared (FTIR) spectra were measured using an IRPRESTIGE-21 spectrometer (Shimadzu) using KBr pressed pellets. UV-visible absorption spectra were determined using a UV-Vis 1901 spectrophotometer (Phoenix) using quartz cuvettes with an optical path length of 1 cm. The content of copper ions in the solution was determined by Leeman Laboratories Prodigy high-dispersion inductively coupled plasma atomic emission spectroscopy (ICP-AES). 2.3
Synthesis of Cys-CuS NPs
In a typical procedure, 0.12 mmol of L-cysteine was dissolved in 150 mL of deionized water, and 50 mL of 1.68 mmol Cu(NO3)2 was added to the above solution under constant stirring. After stirring for 30 min, the pH was adjusted to 3.0 by drop-wise addition of a hydrogen nitrate solution, and then 0.84 mL of sodium thiosulfate solution (0.2 M) was added. The solution was slowly heated to 90 °C under magnetic stirring and the temperature was maintained for 90 min. The solution turned from baby blue to brown and finally dark green. The synthesized product was centrifuged (10 000 rpm, 10 min) and then washed with deionized water three times. Finally, the Cys-CuS NPs were acquired and dispersed in deionized water for later use.
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2.4
Measurement of photothermal performance
Measurement of photothermal performance was carried out by our previous method.5,14 The solutions (0.3 mL) of Cys-CuS NPs with different concentrations (5, 10, 25, 50, and 100 ppm Cu2+) and gold nanorods (50 ppm) were irradiated with a laser (980 nm). The light source was a 980 nm semiconductor laser device (Xi’an Tours Radium Hirsh Laser Technology Co., Ltd, China) with an external adjustable power (0–0.3 W). The output power density was independently calibrated using a handy optical power meter (Newport model 1918-C, CA, USA) and was measured to be ∼0.72 W cm−2. The temperature of the solutions was measured using a digital thermometer (with an accuracy of 0.1 °C) using a thermocouple probe every 5 s. In order to investigate the photostability of the Cys-CuS NPs, a Cys-CuS NP solution (50 ppm Cu2+) was irradiated with a 980 nm NIR laser for 10 min (LASER ON), followed by naturally cooling to room temperature without NIR laser irradiation (LASER OFF). This cycle was repeated five times. 2.5
In vitro cytotoxicity of the Cys-CuS NPs
The in vitro cytotoxicity was measured using the MTT assay in the human cervical carcinoma cell line HeLa.5 HeLa cells were plated into a 96-well plate (1 × 104 cells per well) in complete medium at 37 °C and 5% CO2 for 24 h before the experiments. The culture medium was replaced and cells were incubated with complete medium containing the Cys-CuS NPs at a series of concentrations at 37 °C with 5% CO2 for further 24 h. Relative cell viabilities were determined by the standard MTT assay. Four replicates were prepared for each treatment group. 2.6
In vivo photothermal therapy
The photothermal therapy of cancer cells in vivo was carried out by our previously reported method.14 Osteosarcoma bearing mice were obtained by inoculating subcutaneously with K7M2 cells. When the tumors inside the mice had grown to 5–10 mm in diameter, the mice were randomly allocated into the control group and the treatment group. The mice were first anaesthetized by trichloroacetaldehyde hydrate (10%) at a dosage of 40 mg kg−1 body weight. The mouse of the treatment group was intratumorally injected with 0.10 mL of phosphatebuffered saline (PBS) solution containing Cys-CuS NPs (50 ppm Cu2+), while the mouse of the control group was intratumorally injected with 0.1 mL of PBS solution. After 1 h, the injected areas of mice from both treatment and control groups were perpendicularly irradiated with similar 980 nm wavelength laser devices at a power density of 0.72 W cm−2 for 5 min. The temperatures of the tumor surface were measured using an infrared thermometer (GX-A300, Shanghai Guixin Corporation). The mice were killed after photothermal therapy, and tumors were removed, embedded in paraffin, and cryo-sectioned into 4 μm slices. The slides were stained with hematoxylin/eosin (H&E). The slices were examined under a Zeiss Axiovert 40 CFL inverted fluorescence microscope, and images were captured with a Zeiss AxioCam MRc5 digital camera.
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2.7
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In vivo antitumor effect
Osteosarcoma bearing mice were obtained by inoculating subcutaneously with K7M2 cells. When the tumor volume inside the mice reached ∼140 mm3, eight mice were randomly allocated into two groups and intratumorally injected with 150 μL of PBS and Cys-CuS solutions (50 ppm Cu2+). After injection, tumors were irradiated with NIR light (980 nm, 0.72 W cm−2) for 10 min. After the experiments were completed, mice were killed and the tumors were collected and weighed. All animal experiments were carried out according to protocols approved by the institutional committee for animal care and also in accordance with the policy of the National Ministry of Health.
3. Results and discussion Cys-CuS NPs were readily synthesized in aqueous solution by reacting Cu(NO3)2 and Na2S2O3 in the presence of L-cysteine at 90 °C for 90 min. As-prepared Cys-CuS NPs have a mean size of ∼18 nm (Fig. 1a,b, S1 in ESI†), and the Cys-CuS NPs tend to form small clusters probably due to the acting force of thiol and copper atoms. The size of the clusters was measured by dynamic light scattering (Zetasizer Nano Z) to be ∼152.9 nm (Fig. S2 in ESI†). Further microstructure information was obtained from the high resolution transmission electron microscope (HRTEM) image (Fig. 1b, inset), and it shows an interplanar spacing of ∼0.305 nm, which is in agreement with the lattice spacing of the (1,0,2) planes of hexagonal CuS nanostructures.23 We also synthesized several Cys-CuS NP samples with a different particle size. The absorbance spectra of these Cys-CuS NP samples did not show an obvious difference in absorbance peak position (Fig. S3 in ESI†). In fact, the absorption of the CuS NPs in the NIR region originated from the localized surface plasma resonances of valence-band free carriers ( positive holes),15 and thus shows little variation due to the
Fig. 1 (a) Low-magnification TEM and (b) TEM images of the synthesized Cys-CuS NPs (inset: HRTEM image of a Cys-CuS NP). (c) XRD patterns of the as-prepared product (upper) and the standard CuS (lower). (d) The FTIR spectrum of as-prepared Cys-CuS NPs.
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Fig. 2 (a) UV-vis-NIR absorbance spectra of the aqueous solution containing the Cys-CuS NPs with various concentrations of Cu2+ (i.e., 5, 10, 25, 50 and 100 ppm). An inset photograph shows Cys-CuS NPs in water after 1 week. (b) Temperature change of the aqueous solution containing the Cys-CuS NPs with different concentrations under irradiation of 980 nm laser with a power density of 0.72 W cm−2 as a function of irradiation time. (c) Plot of temperature elevation over a period of 300 s versus the concentration of Cys-CuS NPs. (d) Temperature change of Cys-CuS NPs over five ON/OFF cycles of 980 nm laser irradiation.
size change. The phase structure of the as-obtained Cys-CuS NPs was examined using XRD patterns, as shown in Fig. 2c. Several well-defined characteristic peaks, such as (1,0,2), (1,0,3) and (1,1,0), exhibit the hexagonal phase, referenced to the standard CuS phase (JCPDS card no. 6-0464). Corresponding to the standard hexagonal CuS phase, no obvious impure peaks were found, demonstrating that high quality covellite CuS was acquired. The Fourier transform infrared (FTIR) spectrum of the Cys-CuS NPs is shown in Fig. 1d. The adsorption peak at 3426 cm−1 corresponds to the stretching vibrations of NH2 or OH in cysteine.19 The adsorption peaks at 2927 cm−1 are assigned to the symmetrical stretching vibration of methylene (CH2) in the cysteine.24,25 Peaks at 1630 cm−1 and 1406 cm−1 are due to asymmetric stretching vibration and symmetric stretching vibration of the carboxyl group (COO−), respectively.26 These results suggest that cysteine was successfully anchored onto the surface of CuS NPs. Because cysteine is a needed amino acid for human beings, it has good biocompatibility in blood and tissues. So, the biocompatibility of CuS NPs was enhanced by coating with cysteine. The optical properties of the aqueous dispersion containing the Cys-CuS NPs with various concentrations were examined by UV-vis-NIR spectroscopy, as shown in Fig. 2a. The spectra exhibit a strong absorption in the NIR region due to the localized surface plasma resonances (SPR) of valence-band free carriers ( positive holes) of covellite CuS.15 The absorbance peak is located at 1010 nm, and is very close to the wavelength (980 nm) of the excitation laser, which is advantageous for full usage of the SPR effect of materials, enhancing the photothermal efficiency.18 Owing to coating with cysteine, the aqueous dispersion of Cys-CuS NPs has high stability and can even
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remain unchanged after being dispersed in water for one week (the inset of Fig. 2a). The strong SPR absorption of as-prepared Cys-CuS NPs in the NIR region motivates us to investigate the photothermal effect. A simple home-designed setup (Fig. S4 in ESI†) was used for the photothermal performance measurement. The photothermal conversion performance of the solutions containing various concentrations of Cys-CuS NPs was measured under laser irradiation (980 nm, 0.72 W cm−2). As shown in Fig. 2b, the temperature of the 50 ppm Cys-CuS NP solution was raised from 18.9 °C to 37.4 °C under NIR irradiation of 300 s, while the temperature of pure water was only increased by 3.8 °C under the same conditions. With the increase of the concentration of the Cys-CuS NPs (i.e., 5, 10, 25, 50, and 100 ppm), the temperatures of the solutions containing CysCuS NPs can increase by 8.4 to 21.5 °C in 300 s, as shown in Fig. 2c. These data confirm that the Cys-CuS NPs can effectively absorb the 980 nm laser energy and effectively convert laser energy into heat. The ideal PTT agents should not only have high photothermal conversion efficiency, but also possess good NIR photostability. To investigate the NIR photostability, the solution of Cys-CuS NPs was irradiated with the 980 nm laser (0.72 W cm−2) for 5 min, followed by naturally cooling to room temperature without laser irradiation. This cycle was repeated five times. As shown in Fig. 2d, at the first cycle, the temperature of the Cys-CuS NP solution increased from 16.9 °C to 35.5 °C, compared with the fifth cycle that increased from 17 °C to 34.9 °C. The temperature elevation after five cycles of irradiation only decreased by less than 4.0%, indicating that as-synthesized Cys-CuS NPs have good NIR photostability, which is beneficial to repeated photothermal therapy in long term clinical treatments. Cys-CuS NPs have good photothermal effects and NIR photostability that inspire us to examine the photothermal conversion efficiency of the Cys-CuS NPs. Following a previous method,5,17 the photothermal conversion efficiency (η) of the Cys-CuS NPs was measured. The aqueous solution of the CysCuS NPs (50 ppm) was under continuous irradiation of the laser (980 nm, 0.72 W cm−2) until a steady state temperature was reached. Subsequently, the laser was shut off, and the temperature decrease of the aqueous solution was recorded to measure the rate of heat transfer from the Cys-CuS NP solution system to the environment (Fig. 3a). The η value was calculated (see ESI† for details) as follows:5,17 η¼
hSðT Max T Surr Þ QDis Ið1 10A980 Þ
ð1Þ
where h is the heat transfer coefficient, S is the surface area of the container, and the value of hS was gained from Fig. 3b. Tmax − TSurr is the temperature change of the Cys-CuS NP solution at the maximum steady-environmental temperature, I is the power of the laser, A980 is the absorbance of Cys-CuS NPs at 980 nm, and QDis expresses heat dissipated from light absorbed by the solvent and the container.
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Fig. 3 (a) Photothermal effect of the irradiation of the aqueous solution of the Cys-CuS NPs (50 ppm) with the NIR laser (980 nm, 0.72 W cm−2), in which the irradiation lasted for 660 s, and then the laser was shut off. (b) Linear time data versus −ln(θ) obtained from the cooling period of (a).
According to eqn (1), the 980 nm laser heat conversion efficiency (η) of the Cys-CuS NPs was determined to be 38.0%, which is relatively high compared with those of previously reported materials, such as the widely used Au rods (∼21.0%, 800 nm laser), Cu2−xSe nanocrystals (∼22%, 800 nm laser), and Cu9S5 nanocrystals (∼25.7%, 980 nm laser).5 In order to further compare the Cys-CuS NPs with other materials, we have measured the photothermal conversion efficiency of the Au nanorods and Cys-CuS NPs under the same experimental conditions. As shown in Fig. S5 (in ESI†), the photothermal conversion efficiency (η) of the Au nanorods was 21.3%, which is much lower than that of the CuS NPs. The high photothermal conversion efficiency of the Cys-CuS NPs should be attributed to the strong NIR absorption related to the plasmon resonance. Like the Au nanorods, the highest temperature can be reached when the plasmon resonance wavelength is equal to the illumination laser wavelength, and the lower temperature elevation was obtained when the plasmon resonance peak wavelength was either shorter or longer than the illumination laser wavelength.25 It can be inferred that more laser energy conversion to heat will occur when the plasmon resonance peak wavelength is near the illumination laser wavelength. Our previous work also demonstrated that the photothermal efficiency of Au nanorods (absorbance peaks at ∼808 nm) illuminated with an 808 nm laser was much higher than that of Au nanorods illuminated with a 980 nm laser.27 Thus, it can be inferred that higher photothermal conversion efficiency could be acquired when the absorbance peak of the photothermal agents is close to the illumination laser wavelength. The absorbance peak (∼1010 nm) of the Cys-CuS NPs is approximately at the wavelength (980 nm) of the illumination laser; thus, the Cys-CuS NP solution converts the absorbed light into heat more effectively. The high η of the Cys-CuS NPs makes them highly superior as promising PTT agents. The ideal PTT should be nontoxic or low toxic for biological applications. The high photothermal conversion efficiency of the Cys-CuS NPs prompted us to further evaluate their feasibility as PTT agents for cancer therapy. So we first incubated HeLa cells with the Cys-CuS NPs at different concentrations for 24 h and then the MTT assay was carried out to test the cell viability. It was found that the as-prepared Cys-CuS NPs demonstrate a good biocompatibility (Fig. 4). Even at the
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Fig. 4 Viabilities of HeLa cells incubated for 24 h with different concentrations of the Cys-CuS NPs.
highest tested dose of the Cys-CuS NPs (75 ppm Cu2+), cell viability still remained approximately 93.7%. These results can be attributed to that CuS NPs were coated with biocompatible cysteine. The Cys-CuS NPs have high photothermal conversion efficiency and good biocompatibility in vitro. Because of the complexity of the in vivo environment, the therapy efficacy should be examined in vivo to confirm that the Cys-CuS NPs are good PTT agents. When the tumors inside the mice had grown to 5–10 mm in diameter, the mice were randomly allocated into treatment and control groups. The treatment mouse was intratumorally injected with 0.10 mL of the Cys-CuS NP solution, while the control mouse was intratumorally injected with 0.1 mL PBS solution. After 1 h, the injected areas of the mice from two groups were perpendicularly irradiated by the 980 nm laser device (0.72 W cm−2) for 5 min. For the mouse treated with PBS solution, the surface temperature of the tumor showed no significant change during the entire irradiation process (Fig. 5). In contrast, for the Cys-CuS NPs injected mouse, the tumor surface temperature increased rapidly from 31.8 °C, and reached 51.0 °C after 300 s. As is well known, the hyperthermic therapy is the use of heat between 40 and 45 °C to damage cancer cells.14 The tumor surface temperature of the treatment mouse can easily reach over 45 °C after 2 min of laser irradiation, so the tumor tissue can be efficiently destroyed after 5 min of laser irradiation for hyperthermia. These results reveal that the Cys-CuS NPs within the tumor can also effectively adsorb and convert the NIR light to fatal heat due to the deep penetration depth of the 980 nm laser in biological tissues.4,14,28 The mice were killed after the photothermal therapy, and tumors were removed, embedded in paraffin, and cryo-sectioned into 4 μm slices. The slides were stained with hematoxylin/eosin (H&E). The histopathological images of the mouse treated with PBS (Fig. 6a,c) show that the cells remained normal after irradiation. Because the heat of PBS solution converted from the 980 nm laser irradiation was very little, the
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Fig. 5 Plots of the temperature within the irradiated tumor area in the mice injected with PBS solution and Cys-CuS NP solution as a function of irradiation time, respectively. ( p < 0.001 was considered to be statistically significant difference.)
Fig. 6 H&E-stained histological images of mice (a, c) injected with PBS solution and (b, d) injected with the Cys-CuS NP solution after 5 min of laser irradiation, respectively.
temperature of the tumor surface is only 35.7 °C, which is not high enough to kill the cancer cells. The histopathological images of the Cys-CuS NP treatment are shown in Fig. 6b,d. Almost all of the tumor tissue was necrotized on areas of the examined tumor slide, exhibiting cell shrinkage, loss of contact, coagulation, cytoplasmic acidophilia, and corruption of the tumor extracellular matrix. These facts suggest that the Cys-CuS NPs can still effectively convert NIR light into fatal heat in vivo and tumor cells can be efficiently destroyed by hyperthermia arising from the excellent photothermal effect of the Cys-CuS NPs. To further determine the antitumor effect of Cys-CuS mediated photothermal therapy in vivo, osteosarcoma bearing mice were randomly allocated into two groups and intratumorally injected with 150 μL of PBS and Cys-CuS solutions. After injection, tumors were irradiated with NIR light (980 nm,
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weight of mice in two groups every two days. No obvious weight loss was observed in both the PBS group and the Cys-CuS group (Fig. S6 in ESI†), indicating that the toxicity of treatments was low. Therefore, the Cys-CuS NPs have great potential as ideal photothermal agents for the photothermal therapy in vivo of tumor tissues.
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4.
Fig. 7 (a) Representative photos of mice bearing osteosarcoma tumors before and after treatments of PBS, Cys-CuS. (b) Photos of the tumors collected from PBS and Cys-CuS groups of mice at the end of treatments (day 12). (c) Mean weights of tumors collected from mice at the end of PBS and Cys-CuS treatments. ( p < 0.05 was considered to be statistically significant difference.)
0.72 W cm−2) for 10 min. 12 days after treatment, mice were sacrificed and tumors were excised and weighed. As shown in Fig. 7a–c, the tumors treated with PBS plus laser irradiation grew rapidly, suggesting that the osteosarcoma tumor growth was not affected by laser irradiation. While the tumors treated with Cys-CuS solutions plus laser irradiation were greatly inhibited, half tumors were even completely eradicated. As a result, tumor of the Cys-CuS group was not completely inhibited but showed much smaller tumor sizes than the PBS group after treatment, probably due to a small part of the larger size of tumor area free of irradiation (Fig. 7b). The mean tumor weight of the Cys-CuS group is only 0.088 g, while that of the PBS group is 0.627 g, which is more than seven times that of the Cys-CuS group (Fig. 7c). There are significant differences between the group under treatment with CuS NPs and the group under treatment with PBS ( p = 0.018). The CuS photothermal materials had a negligible impact on tumor growth without illumination,29,30 and only 980 nm laser illumination (below 1.5 W cm−2) has little influence on the tumor growth,29 so the inhibition of tumor growth could be attributed to the excellent photothermal effect of Cys-CuS NPs under 980 nm illumination. These results indicate that the Cys-CuS NPs still can effectively convert NIR light into fatal heat in vivo and tumor growth can be efficiently inhibited by the photothermal effect of Cys-CuS NP solutions. The potential in vivo toxicity is always a primary concern for nanomaterials applied in biomedicine.31 High toxicity usually leads to a significant weight loss, so we measured the body
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Conclusion
In summary, we have successfully synthesized hydrophilic PTT agents of the Cys-CuS NPs by a facile aqueous solution method. The Cys-CuS NPs were coated with cysteine, and have good photostability and biocompatibility. The solution of the Cys-CuS NPs exhibits an intense absorbance in the NIR region, and the temperature of the 50 ppm solution can be increased by 18.5 °C in 300 s under the irradiation of the 980 nm laser with a safe power density of 0.72 W cm−2. The photothermal conversion efficiency of the Cys-CuS NPs can be as high as 38.0%, which is higher than that of the reported Au nanorods, Cu2−xSe nanocrystals and Cu9S5 nanocrystals. More importantly, tumor growth can be efficiently inhibited by hyperthermia arising from the excellent photothermal effect of the Cys-CuS NPs at a low concentration (50 ppm) under the irradiation of the 980 nm laser. Owing to their high photothermal conversion efficiency, photostability, and good biocompatibility, these Cys-CuS NPs could be used as promising PTT agents for cancer therapy.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 51302035 and 21171035), the Key Grant Project of Chinese Ministry of Education (Grant No. 313015), Ph.D. Programs Foundation of Ministry of Education of China (Grant No. 20110075110008), the National 863 Program of China (Grant No. 2013AA031903), the Fundamental Research Funds for the Central Universities, the Science and Technology Commission of Shanghai Municipality (Grant No. 13ZR1451200), the Hong Kong Scholars Program, the Project funded by China Postdoctoral Science Foundation, the Program for Changjiang Scholars and Innovative Research Team in University (IRT1221), the Shanghai Leading Academic Discipline Project (Grant No. B603), and the Program of Introducing Talents of Discipline to Universities (Grant No. 111-2-04).
Notes and references 1 Z. Zha, S. Wang, S. Zhang, E. Qu, H. Ke, J. Wang and Z. Dai, Nanoscale, 2013, 5, 3216–3219. 2 Z. Zha, X. Yue, Q. Ren and Z. Dai, Adv. Mater., 2013, 25, 777–782. 3 K. Yang, H. Xu, L. Cheng, C. Sun, J. Wang and Z. Liu, Adv. Mater., 2012, 24, 5586–5592.
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