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PEGylated nickel carbide nanocrystals as efficient near-infrared laser induced photothermal therapy for treatment of cancer cells in vivo† Zhiguo Zhou,a Jun Wang,a Wei Liu,a Chao Yu,a Bin Kong,a Yanan Sun,a Hong Yang,a Shiping Yang*a and Wei Wang*a,b Photothermal therapy has attracted significant attention as a minimally invasive therapy methodology. In this work, we report PEGylated nickel carbide nanocrystals (Ni3C NCs) as an efficient photothermal agent for the first time. The nanoparticles exhibit a broad absorption from the visible to the near-infrared regions and a rapid rise in temperature when irradiated by an 808 nm laser even at a concentration of 100 μg mL−1. In vitro and in vivo cytotoxicity assays demonstrate they have good biocompatibility, which lays an important foundation for their biological application. In vitro studies reveal the efficient damage of cancer cells by the exposure of 808 nm laser with a power density of 0.50 W cm−2. Furthermore, hema-
Received 4th July 2014, Accepted 13th August 2014
toxylin and eosin (H & E) and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) staining of tumor slices confirmed the obvious destruction of cancer cells in vivo by an 808 nm
DOI: 10.1039/c4nr03727h
laser (0.50 W cm−2) after only a 5 min application. Our work may open up a new application domain for
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transition metal carbides for biomedicine.
1 Introduction Near-infrared (NIR) laser-induced photothermal therapy (PTT) has emerged as an attractive strategy for cancer treatment.1–3 The PTT employing NIR light as a highly orthogonal external stimulus enables spatial and temporal exposure in the tumor region to maximize the treatment efficacy while minimizing the side effects. Several PTT agents have been the subject of extensive studies and their drawbacks have also been realized. Organic dyes of indocyanine green (ICG) dye4 and polyaniline nanoparticles5 and copper chalcogenide semiconductors6–10 have the disadvantage of being unstable against NIR irradiation. Carbon-based materials such as carbon nanotubes (CNTs)11 and graphenes,12 have relatively low absorption ability in the NIR region. The most studied photothermal nanomaterials, including Ge nanoparticles,13 Pd-based nanosheets,14 and Au nanostructures15–21 in PTT, exhibit intense NIR photoabsorption, but the cost of these noble
a The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, China b Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131-000. E-mail: [email protected], [email protected]; Fax: +86-21-64322346; Tel: +86-21-64322346 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4nr03727h
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metals limits their wide use in applications. Therefore, there is a significant demand on the development of a new class of PTT agents,22–27 to ultimately become clinically useful tools for cancer treatment. Such materials should be of low toxicity and be easily functionalized with bioactive molecules as well as have the property of the high NIR-induced photothermal conversion efficiency. Transition metal carbide nanocrystals have received considerable attention recently owing to their unique chemical and physical properties, such as excellent mechanical properties, high melting points, high chemical stability, superconductivity and catalytic performance.28–31 Among the metal carbides, nickel carbide nanocrystals (Ni3C NCs), which are formed with an interstitial carbon in a hexagonal close packed nickel (hcp-Ni), have been much less investigated mainly because of their complicated synthetic procedures.32 The methods for their preparation include mechanical alloying,33 carbon-ion implantation into nickel,34 magnetron sputtering35,36 and high energy ball milling.37 These protocols rely on the expensive, specialized apparatus that are difficult for most researchers to access. Recently, a protocol utilizing thermal decomposition of a nickel precursor in the presence of organic surfactants has been developed for a convenient synthesis of high quality Ni3C NCs,38–40 which opens a door to explore their applications, especially in the field of biological science. We envisioned that the Ni3C NCs could serve as a new type of PTT agents, which may possess unique properties. Herein,
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we wish to disclose the results of the investigation. As demonstrated experimentally, the cost effective, conveniently prepared PEGylated Ni3C nanocrystals (NCs) with a uniform and small size exhibit a high photostability, relatively low toxicity, and high photothermal efficiency. Furthermore, the PEGylated Ni3C NCs have good photothermal conversion efficiency at a desirable NIR irradiation wavelength. The efficiency has been transformed to efficiently destroy cancer cells in vitro under the 808 nm laser irradiation with a power density of 0.50 W cm−2. Moreover, under the same operating conditions, the effective destruction of cancer cells in vivo was achieved within only 5 min with a low dose PEGylated Ni3C NCs (10 mg kg−1 body weight). The high photothermal therapeutic efficiency in vitro and in vivo has also been evaluated quantitatively by the MTT assay and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) staining, respectively. To the best of our knowledge, this study represents the first example of using transition metal carbides in PTT.
2
Experimental
2.1
Materials and characterization
Octadecylene and oleylamine were purchased from Beijing InnoChem Technology Co., Ltd. Poly(ethylene glycol) carboxylic acid (PEG-COOH, Mw: ∼2000) was purchased from Shanghai Seebio Biotech, Inc. Nickel acetylacetonate hydrate [Ni(acac)2] was purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were used without further purification. Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 MΩ cm. XRD was performed using a Rigaku DMAX 2000 diffractometer equipped with Cu/Kα radiation at a scanning rate of 4° min−1 in the 2θ range from 30 to 80° (λ = 0.15405 nm) (40 kV, 40 mA). TEM was carried out using a JEOL JEM-2010 transmission electron microscope operating at 200 kV. TEM samples were prepared by depositing a diluted NCs suspension (200 μg mL−1, 5 μL) onto a carbon-coated copper grid and air-dried before the measurements. FT-IR spectra were collected using a Nicolet Avatar 370 spectrometer. The samples were pelletized with KBr before measurements. Hysteresis loop was measured with a Quantum Design SQUID MPMS XL-7 magnetometer. X-ray photoelectron spectroscopy (XPS) was performed on a Kratos AXIS-165 surface analysis system. The hydrodynamic size was measured using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, UK) equipped with a standard 633 nm laser. The absorption spectra were collected on a DU 730 UV-visible spectrophotometer (Nucleic acid, protein analyzer) at room temperature. 2.2
Synthesis of Ni3C nanocrystals (Ni3C NCs)
In a typical procedure, Ni(acac)2 (0.1 g) was dissolved in a cosolvent mixture of octadecylene (20 mL) and oleylamine (8 mL) in a 100 mL three-neck flask. The solution was deaerated under a nitrogen flow at 110 °C for 15 min, heated to 285 °C
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under vigorous magnetic stirring, and kept for 30 min at this temperature before it was cooled down to room temperature. After centrifugation at 12 000 rpm for 8 min, the supernatant was removed. The resulting black precipitation was washed with ethanol three times to acquire pure Ni3C NCs. Finally, the obtained NCs were stored in 20 mL of chloroform before use. 2.3
Synthesis of PEGylated Ni3C NCs
In a typical process, methoxypoly (ethylene glycol) carboxyl acid (50 mg) was reacted with NaOH (2 mg) in mixed co-solvents ethanol (5 mL) and chloroform (10 mL) for 4 h, followed by addition of the chloroform solution of Ni3C NCs (5 mL, 2000 μg mL−1). The solution was stirred overnight. The formed PEGylated NCs were purified by washing with chloroform and ethanol each three times before further characterization. 2.4
Photothermal effect of PEGylated Ni3C NCs
An aqueous suspension (2 mL) containing PEGylated Ni3C NCs with different concentrations (100, 200, 500 and 1000 μg mL−1, respectively) was put in a quartz cuvette with an optical path length of 0.5 cm. The cuvette was illuminated by an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co., Ltd) with a power density of 0.50 W cm−2 for 300 seconds. The diameter of the laser spot was 3.5 cm. The increase in temperature was monitored by a digital thermocouple device with an accuracy of ±0.1 °C. The temperature was recorded every 10 s. 2.5
Cell culture
A human cervical carcinoma cell line (HeLa cells) was provided by the Shanghai Institutes of Biological Sciences (SIBS), Chinese Academy of Sciences (CAS, China). The cells were cultured in RPMI-1640 (Thermo, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin–streptomycin (Thermo) at 37 °C and 5% CO2. Cells were plated in a cell culture flask (Corning, USA) under the 100% humidity and allowed to adhere for 24 h, then harvested by treatment with 0.25% trypsin-EDTA solution (Gibco, USA). 2.6
MTT assay
In vitro cytotoxicity of PEGylated Ni3C NCs was evaluated by performing a methyl thiazolyl tetrazolium (MTT) assay of HeLa cells. The cells were seeded into a 96-well cell culture plate at a density of 5000 per well and cultured in RPMI-1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The next day, the cells were incubated with PEGylated Ni3C NCs with different concentrations (diluted in RPMI-1640 at 0, 12.5, 25, 50, 100, 200, 350, and 700 μg mL−1) for another 12 h and 24 h at 37 °C under 5% CO2. Thereafter, MTT (20 μL, 5 mg mL−1) was added to each well and the plate was incubated for an additional 4 h at 37 °C. After the removal of the medium, the purple formazan product was dissolved with DMSO for 15 min. Finally, the optical absorption of formazan at 490 nm was measured by an enzyme-linked immunosorbent
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assay reader, with background subtraction at 690 nm was measured by a microplate reader (Multiskan MK3, USA).
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2.7
Photothermal therapy of HeLa cells in vitro
HeLa cells were seeded into a 96-well cell culture plate at a density of 1 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The culture medium was replaced without or with PEGylated Ni3C NCs (50 μg mL−1) dispersed in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh medium (200 μL) was added to the wells, followed by the 808 nm laser irradiation for 10 min with the different power density of 0.25 and 0.50 W cm−2, respectively. After the laser irradiation, HeLa cells were cultured for additional 1 h for MTT assay. All measurements were conducted in triplicate.
2.8
Trypan blue staining
HeLa cells were seeded into a 12-well cell culture plate at a density of 5 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The culture medium was then replaced without or with PEGylated Ni3C NCs (50 μg mL−1) dispersed in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh complete medium (800 μL) was added to the wells, followed by the 808 nm laser irradiation for 10 min with a power density of 0.50 W cm−2. HeLa cells were stained with 0.4% Trypan blue solution for 15 min. Then the suspension was removed and the cells were washed with PBS twice. Cell morphology of the adherent cells in PBS (500 μL) was observed by an inverted optical microscope (Olympus, IX71, Japan) with a magnification of 20×. Cells stained by Trypan blue were counted as dead cells.
2.9
Laser scanning confocal microscopy
Confocal microscopic imaging was performed with a Leica TCS SP5 II inverted microscopy (Leica DMI 6000B, Solms, Germany). HeLa cells were seeded into a 12-well cell culture plate at a density of 5 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. Then, the culture medium was replaced with PEGylated Ni3C NCs (50 μg mL−1) in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh complete medium (800 μL) was added to the wells, followed by the 808 nm laser irradiation for 10 min with a power density of 0.50 W cm−2. After laser irradiation, the cells were co-stained by a mixture solution containing Calcein-AM (2 μmol L−1) and propidium iodide (PI) (4 μmol L−1) to differentiate live (green) and dead (red) cells for 15 min. Calcein-AM and PI was excited by 488 nm and 543 nm lasers, respectively. Labeled HeLa cells were observed on a laser scanning confocal microscope.
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2.10 Flow cytometry HeLa cells were seeded into a 12-well cell culture plate at a density of 5 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The culture medium was then replaced without or with PEGylated Ni3C NCs (50 μg mL−1) dispersed in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh complete medium (800 μL) was added to the wells followed by the 808 nm laser irradiation for 10 min with the power density of 0.25 and 0.50 W cm−2, respectively. After the laser irradiation, HeLa cells were washed twice with PBS and harvested by treatment with 0.25% trypsin-EDTA solution. After centrifugation, the obtained HeLa cells were suspended in PBS solution and analyzed with a flow cytometer (Beckman Coulter, Quanta SC, USA). The collected data were analyzed using Flow Jo software 7.6.5. 2.11 Animal experiments ICR rats (∼40 g body weight) and HeLa tumor-bearing nude mice (∼20 g body weight) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Animal care and handling procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments. The nude mice were used for further experiments when the tumor volume reached about 360 mm3. 2.12 In vivo cytotoxicity assay Three healthy ICR rats (∼40 g body weight) were intravenously injected with a dose of 10 mg kg−1 PEGylated Ni3C NCs in the saline solution. For a control group, three other ICR rats were treated with saline (200 μL). Over a one month period, ICR rats were sacrificed to collect the blood (1 mL) and the tissues, including the heart, liver, lung, kidney, and spleen. For the short term in vivo cytotoxicity, another six rats were used (three rats for each group) as previously described. The only difference was that the rats were sacrificed 24 h after the injection instead of a month. The blood analysis data were measured in Shanghai SLAC Laboratory Animal Co., Ltd. All the tissues were stained with hematoxylin and eosin (H & E) to assess tissue and cellular morphology. 2.13 Photothermal imaging in vivo HeLa tumor-bearing nude mice were intratumorally or intravenously injected with a dose of 10 mg kg−1 of PEGylated Ni3C NCs in the saline solution or saline (200 μL). After 1 h, the tumors in the mice were irradiated with an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co., Ltd) for 5 min with a power density of 0.25 and 0.50 W cm−2, respectively, then imaged and recorded by a FLIR A300 (USA, spectral band: 7.5–13 μm). 2.14 Photothermal therapy of HeLa cells in vivo HeLa tumor-bearing nude mice were intratumorally or intravenously injected with a dose of 10 mg kg−1 of PEGylated Ni3C
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NCs or saline (200 μL). After 1 h, the tumors in the mice were irradiated with an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co., Ltd) for 5 min with a power density of 0.25 and 0.50 W cm−2, respectively, then imaged and recorded by a FLIR A300 (USA). The mice were euthanized after the laser treatment. The tumors were removed, fixed in 4% paraformaldehyde at 4 °C overnight, and embedded in paraffin for H & E staining and Tunel staining. 2.15 H & E and TUNEL staining Serial 5 μm thick tumor sections were prepared and stained with haematoxylin/eosin (HE, Beyotime, China). The histology and morphology of the tumors were observed under the Eclipse E800 microscope (Nikon, Japan). The DeadEnd Colorimetric TUNEL System from Promega (Mannheim, Germany) was used. Biotinylated nucleotides were incorporated at the 3′-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase (TdT). Horseradish peroxidase-labeled streptavidin was then bound to these biotinylated nucleotides. Peroxidase activity was visualized using the liquid DAB substrate chromogen system (Dako, Hamburg, Germany). In case of antibodyTUNEL double staining, Cy2-labeled streptavidin (Dianova, Hamburg, Germany) was used. The number and percentage of
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TUNEL-positive cells were counted and determined by counting 1 × 103 cells from five random selected fields.
3
Results and discussion
3.1
Synthesis and characterization of Ni3C NCs
The prerequisite Ni3C NCs were synthesized by the high-temperature pyrolysis method from the precursor of nickel acetylacetonate in the mixture of octadecylene and oleylamine at 285 °C. The resulting organic ligand-capped Ni3C NCs show excellent dispersibility and stability in chloroform (Fig. 1b inset), with an average diameter of 42.7 ± 5.0 nm without obvious aggregation as depicted in the field of view from a representative transmission electron microscope (TEM) image (Fig. 1b and S1a†). A high-resolution TEM image (Fig. S1b†) of the nanoparticles reveals the high crystalline nature of Ni3C NCs. The lattice spacing between two adjacent planes is 0.203 nm, which corresponds very well to the d-spacing for the (113) lattice plane. The crystallography of NCs was verified by powder X-ray diffraction (XRD) (Fig. 1c). All the diffraction peaks of the as-prepared Ni3C NCs are well indexed to the hexagonal Ni3C phase (JCPDS card no. 06-0697). The mean
Fig. 1 (a) Illustration of the synthesis and in vivo near-infrared laser driven photothermal therapy of PEGylated Ni3C NCs. (b) TEM image of Ni3C NCs and a photograph in chloroform solution (2 mg mL−1, inset). (c) XRD pattern of Ni3C NCs. (d) XPS spectra of Ni 2p3/2 region. (e) Absorption spectra of PEGylated Ni3C aqueous solution at different concentrations in water (Inset: the absorbance of PEGylated Ni3C aqueous solution at 808 nm as a function of concentration related to (e).) (f ) Temperature changes of the aqueous solution of PEGylated Ni3C NCs at different concentration under the 808 nm laser irradiation with a power density 0.50 W cm−2 for 5 min. (g) Temperature changes of the aqueous solution of PEGylated Ni3C NCs (100 μg mL−1) over four Laser on/off cycles (0.50 W cm−2).
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crystalline size is estimated to be ∼45 nm from the halfmaximum full width of (113) peak according to the Scherrer equation, which matches the size observed from TEM analysis. Considering that the crystal structure of Ni3C phase is almost identical to that of hcp Ni phase, X-ray photoelectron spectroscopy (XPS) was performed (Fig. 1d). In the Ni 2p region, the Ni 2p3/2 peak is observed at 853.1 eV, which is consistent with the binding energy of nickel carbide reported in the literature.41 In addition, the very weak magnetization properties of Ni3C NCs from the hysteresis loops further confirms the Ni3C phase, but not the hcp-Ni phase (Fig. S1e†).42 To make the Ni3C NCs water soluble and biocompatible, PEG chains were anchored on the surface of Ni3C NCs via coordination between carboxylate ions and the Ni3C surface. The immobilization is confirmed by the absorption band at 1105 cm−1 in the Fourier transform infrared spectroscopy (FT-IR) spectra (Fig. S1f†). Moreover, such modification does not change the morphology of NCs with a diameter of 44.4 ± 4.8 nm verified by TEM (Fig. S1c and S1d†). The hydrodynamic diameter measured by dynamic light scattering (DLS) is 107 ± 4.5 nm, which is larger than that observed by TEM owing to the slight aggregation. The PEGylated Ni3C NCs display the high dispersity in water, PBS and RPMI with 10% fetal bovine calf serum (FBS) as simulated in vivo plasma (Fig. S1c† inset). The property is essential for the biological studies. 3.2
Photothermal effect of PEGylated Ni3C NCs
The absorption spectra of PEGylated Ni3C NCs (Fig. 1e) exhibit a broad absorption ranging from visible to NIR region, and there is a good linear correlation between the absorbance at 808 nm and the concentration of PEGylated Ni3C NCs (Fig. 1e inset). The absorption co-efficient of PEGylated Ni3C NCs was ∼4.0 L g−1 cm−1. The absorption of PEGylated Ni3C NCs in the NIR region is probably attributed to the charge transfer transition between Ni atom and C atom in the Ni3C phase, which is confirmed by the increased Ni 2p3/2 peak compared to that of the metallic Ni (852.7 eV).41 The absorption in the NIR region of PEGylated Ni3C NCs encouraged us to demonstrate the possibility as a photothermal agent for cancer therapy. Different concentrations of PEGylated Ni3C NCs (100, 200, 500 and 1000 μg mL−1) were irradiated by an 808 nm NIR laser with a power density of 0.50 W cm−2 for 5 min (Fig. 1f ). Notably, the PEGylated Ni3C NCs solution showed a rapid rise in temperature during laser irradiation while no obvious temperature change was observed for pure water in the absence of the material under the same experimental conditions. Even at the concentration of 100 μg mL−1, the temperature was raised by 16.3 °C. Furthermore, higher concentration of NCs leads to a more prominent temperature increase. For example, an increase of 30 °C was obtained at the concentration of 1000 μg mL−1 in 5 min. According to a literature reported method,43 the photothermal conversion efficiency was calculated to be ∼16.9% (Fig. S1g and S1h†). To evaluate the NIR photostability of PEGylated Ni3C NCs, four on/off laser cycles were performed by irradiating the aqueous solution of PEGylated Ni3C NCs via an 808 nm
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laser for 5 min (laser on, Fig. 1g), followed by cooling down to room temperature without NIR laser irradiation for 20 min (laser off, Fig. 1g). The temperature increase of 16.3 °C was achieved after the first laser on at the NCs concentration of 100 μg mL−1. No significant change in the temperature increase was observed after four cycles. These findings indicate that PEGylated Ni3C NCs have the good NIR photostability. 3.3
In vitro and in vivo cytotoxicity assays
As discussed above, in addition to high photothermal conversion efficiency and NIR photostability, an ideal photothermal agent should be of low toxicity for biological applications.44,45 To evaluate the cytotoxicity of PEGylated Ni3C NCs, a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with HeLa cells was used. The concentrationdependent effect of PEGylated Ni3C NCs on the cell viability incubated for 12 and 24 h was determined (Fig. S2†). These studies clearly showed that the high cell viability (>80%) can still be attained within the concentration of 100 μg mL−1 incubated for 12 h, which provides the basis for in vivo photothermal therapy. Meanwhile, in order to further investigate the potential in vivo toxicity, healthy ICR rats were intravenously injected with a dose of PEGylated Ni3C NCs (10 mg kg−1 of body weight). The rats were sacrificed for blood analysis and histological examination after 24 h and 30 d, respectively. The liver function index (Fig. 2a1, 2a2, S3a1, and S3a2†) including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), kidney function index (Fig. 2b1, 2b2, S3b1, and S3b2†) including urea nitrogen (BUN), creatinine (CREA) and a complete blood panel were tested (Fig. 2c1–2c8 and Fig. S3c1– S3c8†). According to the analysis of blood indexes with the control groups, except for the white blood cells value that was likely caused by inflammation from the PEGylated Ni3C NCs, there was no significant difference and all measured parameters showed only slight variations within normal ranges, suggesting no obvious hepatic and kidney damage. In addition, organs were excised, including the heart, liver, lung, kidney, and spleen, and stained with hematoxylin and eosin (H&E) for a histology analysis (Fig. 2d and Fig. S3d†). Compared with the control group, few differences were observed for mice injected with PEGylated Ni3C NCs. Therefore, PEGylated Ni3C NCs showed no noticeable toxicology in vivo experiments under our experimental conditions and dosage. 3.4
Photothermal therapy of HeLa cells in vitro
We then evaluated the in vitro photothermal therapeutic capability of the PEGylated Ni3C NCs to cancer cells under the laser irradiation by optical microscopy. HeLa cells were incubated with or without PEGylated Ni3C NCs at a concentration of 50 μg mL−1 for 4 h and then were irradiated by an 808 nm laser of 0.50 W cm−2 for 10 min. After NIR laser exposure, the cells were stained blue by treating with 0.4% Trypan blue for 5 min. As shown in the optical microscope images in Fig. 3a1–3a3, there were no obvious changes for the control
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Fig. 2 Blood analysis data of ICR rats intravenously injected with PEGylated Ni3C NCs (10 mg kg−1 body weight). Untreated rats were used as the control group. Liver function index includes alanine aminotransferase (ALT, a1) and aspartate aminotransferase (AST, a2). Kidney function index includes urea nitrogen (BUN, b1) and creatinine (CREA, b2). The complete blood analysis includes red blood cells (RBC, c1), white blood cells (WBC, c2), haemoglobin (HGB, c3), hematocrit (HCT, c4), mean corpuscular volume (MCV, c5) mean corpuscular hemoglobin concentration (MCHC, c6), mean corpuscular hemoglobin (MCH, c7) and platelets (PLT, c8). Statistics were based on three mice per data point. (d) Histological changes in the heart, liver, spleen, lung and kidney of ICR rats without or with intravenous injection of PEGylated Ni3C NCs (10 mg kg−1 body weight) after 30 days. These organs were stained with H & E and observed under an optical microscope at a 40× magnification.
groups of HeLa cells cultured without or with PEGylated Ni3C NCs (50 μg mL−1, Fig. 3a1 and 3a2). Moreover, negligible cell destruction was also observed for HeLa cells with 808 nm laser irradiation alone (Fig. 3a3). In contrast, nearly all HeLa cells incubated with PEGylated Ni3C NCs after the 808 nm laser irradiation were stained blue (Fig. 3a4), indicating that cancer cells were damaged by the irradiation. The photothermal therapeutic effect of PEGylated Ni3C NCs on cancer cells was also confirmed by laser confocal fluorescence microscopy. Following laser irradiation, HeLa cells were co-stained by CalceinAM and propidium iodide (PI) to differentiate live (green) and dead (red) cells, respectively. These studies indicate that the PEGylated Ni3C NCs are able to kill HeLa cells via photothermal destruction (Fig. 3b4). In contrast, HeLa cells without treatment (Fig. 3b1), incubated with PEGylated Ni3C NCs alone (Fig. 3b2) and irradiated by an 808 nm laser only (Fig. 3b3) were not affected under similar experimental conditions. These results also corroborated well with the above optical microscopic imaging data. We further quantitatively investigated the photothermal effect of PEGylated Ni3C NCs to HeLa cells using a MTT assay and a fluorescence activating cell sorter (FACS) at a concen-
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tration of 50 μg mL−1 PEGylated Ni3C NCs. It was determined that the viability of HeLa cells significantly dropped as an 808 nm laser power density of PEGylated Ni3C NCs increased. As shown in Fig. 3d, while the cell viability remained 74.5 ± 1.7% by the MTT assay (∼74.5% by FACS analysis, Fig. S4b†) with a power density of 0.25 W cm−2, the viability decreased rapidly to 13.7 ± 0.7% by the MTT assay (∼7.2% by FACS analysis, Fig. 3c3) with a power density of 0.50 W cm−2. As expected, neither PEGylated Ni3C NCs (50 μg mL−1) nor an 808 nm laser treatment showed obvious destructive effect on HeLa cells in the control experiments (Fig. 3c1, 3c2 and Fig. S4a†). These experimental findings demonstrated that the combination of PEGylated Ni3C NCs and an 808 nm laser illumination of 0.50 W cm−2 could kill localized tumor cells efficiently. To clarify the cell death mode upon photothermal treatment with a power density of 0.50 W cm−2, annexin V-FITC/propidium iodide (PI) double staining analysis was used. The quantity of apoptotic and necrotic cells was determined by the percentage of AV+/PI−, while the quantity of necrotic cells was determined by the percentages of AV+/PI+. Statistical analysis of the data showed that the laser irradiation plus PEGylated Ni3C NCs treatment induced ∼6.1% cell apoptosis and ∼84.6% cell
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Fig. 3 Trypan blue staining (a1–a4) and confocal fluorescence images (b1–b4) of HeLa cells. Control HeLa cells (a1, b1), HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1, a2, b2), HeLa cells treated with the 808 nm laser irradiation for 10 min (0.5 W cm−2, a3, b3), HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1) and irradiated by an 808 nm laser for 10 min (0.50 W cm−2, a4, b4). (c) Representative FACS plots. (c1) HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1), (c2) HeLa cells after 808 nm laser irradiation for 10 min with a power density of 0.50 W cm−2, (c3) HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1) and then irradiated by an 808 nm laser for 10 min with a power density of 0.50 W cm−2. (d) Cell viability of HeLa cells with different treatments.
necrosis (Fig. 3c3). Therefore, these results show that the main mechanism of in vitro photothermal therapy induced cell death is necrosis rather than apoptosis. 3.5
Photothermal experiments in vivo
Encouraged by the effective photothermal therapeutic capability of the newly developed PEGylated Ni3C NCs to cancer cells, we then turned our attention on the in vivo studies of cancer PTT using HeLa tumor mouse model. HeLa tumorbearing nude mice were intratumorally injected with PEGylated Ni3C NCs (10 mg kg−1 of body weight) and then exposed to an 808 nm laser for 5 min. The temperature change was monitored by an infrared thermal camera (Fig. 4a). To our delight, we found that the temperature injected with PEGylated Ni3C NCs under an 808 nm laser irradiation with a power density of 0.25 and 0.50 W cm−2 rapidly increased from ∼30 to
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∼36.5 and ∼44 °C within 5 min, respectively (Fig. 4b). It is established that photothermal therapy can destruct cancer cells when the temperature is greater than 43 °C.46,47 In contrast, the temperature at the tumor site without PEGylated Ni3C NCs treatment was marginally affected even after being exposed to the laser irradiation with a power density of 0.50 W cm−2, showing only a temperature increase of 3 °C after 5 min. More importantly, no obvious temperature increase was observed on other parts near the tumor, which effectively minimizes the damage of normal tissues. Although a significant accumulation of PEGylated Ni3C NCs in the tumor was observed due to the enhanced permeability and retention (EPR) effect (Fig. S7†), the low absolute amount of NCs in the tumor did not measurably increase the temperature of the tumor under the 808 nm laser irradiation after the intravenous injection with the same dose (Fig. S5†).
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Fig. 4 (a) Thermal infrared images of tumor area intratumorally injected with PEGylated Ni3C NCs (10 mg kg−1) recorded at different time intervals. Mice injected saline was taken as the control group. The tumor was exposed to an 808 nm laser with a power density of 0.25 and 0.5 W cm−2, respectively. (b) Plots of tumor temperature as a function of time that corresponds to (a).
Finally, to assess the in vivo therapeutic effect in a HeLa tumor mouse model, hematoxylin and eosin (H & E) staining, and terminal deoxynucleotidyl transferase biotin-dUTP nickend labelling (TUNEL) staining studies of tumor slices were carried out. H & E staining of tumours treated with PEGylated Ni3C NCs (10 mg kg−1 of body weight) under the 808 nm laser
irradiation with a power density of 0.50 W cm−2 showed significant thermal damage, including shrinkage and nuclear chromatin that disintegrated into small fragments. Nevertheless, tumor tissues of the control cells remained normal without changes (Fig. 5a and 5b). TUNEL staining is a method for probing of DNA fragmentation, which results from apoptotic
Fig. 5 H&E (a) and TUNEL (b) stained histological images of tumor sections. (c) The percentage of TUNEL-positive cells in tumor sections with different experiment groups.
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signalling cascades by labelling the terminal end of nucleic acids. Therefore, TUNEL enables to identify extensive thermonecrotic or apoptotic cells, in which dead cells are stained in brown.48,49 As expected, there were more brown cells than in the control groups (Fig. 5a and 5b). Moreover, as shown in Fig. 5c, 18.5% ± 1.7% and 21.7% ± 3.0% TUNEL positive cells were found only cultured with PEGylated Ni3C NCs and under the laser alone, respectively. However, after laser irradiation, the quantity of TUNEL positive cells increased to 44.1% ± 1.7% after treatment with the NCs. Under such a short irradiation time, a significant tumor killing effect is achieved. However, no obvious damage of the tumor tissue under the similar 808 nm laser irradiation was observed after the intravenous injection with the same dose, which was probably attributed to the accumulation with this low absolute amount in tumor (Fig. S6 and S7†).
4 Conclusions In summary, we have developed PEGylated transition-metal carbide Ni3C NCs as a new class of PTT agents. The Ni3C NCs can be conveniently prepared via a cost-effective thermal decomposition and ligand exchange method to give uniformly small-sized NCs. The PEGylated Ni3C NCs had a photothermal conversion efficiency of ∼16.9% at a desired NIR irradiation wavelength and NIR photostability in the aqueous solution. As demonstrated, cancer cells in vitro and in vivo photothermal treatment can be efficiently destroyed under the 808 nm laser irradiation with a power density of 0.50 W cm−2. Therefore, the features of their small size, high photothermal conversion efficiency, low cost and low cytotoxicity enable the PEGylated Ni3C NCs as a promising tool for cancer treatment. Furthermore, the work described here opens a new avenue for the application of transition metal carbides in the field of biomedical science.
Acknowledgements This work was partially supported by National Natural Science Foundation of China (No. 21271130 and 21371122), program for Changjiang Scholars and Innovative Research Team in University (no. IRT1269), Shanghai Natural Science Fund Project (no. 12ZR1421800 and 13520502800), Shanghai Pujiang Program (13PJ1406600), Shanghai Municipal Education Commission (no. 13ZZ110) and Shanghai Normal University (no. DXL122 and SK201339).
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PEGylated nickel carbide nanocrystals as efficient near-infrared laser induced photothermal therapy for treatment of cancer cells in vivo† Zhiguo Zhou,a Jun Wang,a Wei Liu,a Chao Yu,a Bin Kong,a Yanan Sun,a Hong Yang,a Shiping Yang*a and Wei Wang*a,b Photothermal therapy has attracted significant attention as a minimally invasive therapy methodology. In this work, we report PEGylated nickel carbide nanocrystals (Ni3C NCs) as an efficient photothermal agent for the first time. The nanoparticles exhibit a broad absorption from the visible to the near-infrared regions and a rapid rise in temperature when irradiated by an 808 nm laser even at a concentration of 100 μg mL−1. In vitro and in vivo cytotoxicity assays demonstrate they have good biocompatibility, which lays an important foundation for their biological application. In vitro studies reveal the efficient damage of cancer cells by the exposure of 808 nm laser with a power density of 0.50 W cm−2. Furthermore, hema-
Received 4th July 2014, Accepted 13th August 2014
toxylin and eosin (H & E) and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) staining of tumor slices confirmed the obvious destruction of cancer cells in vivo by an 808 nm
DOI: 10.1039/c4nr03727h
laser (0.50 W cm−2) after only a 5 min application. Our work may open up a new application domain for
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transition metal carbides for biomedicine.
1 Introduction Near-infrared (NIR) laser-induced photothermal therapy (PTT) has emerged as an attractive strategy for cancer treatment.1–3 The PTT employing NIR light as a highly orthogonal external stimulus enables spatial and temporal exposure in the tumor region to maximize the treatment efficacy while minimizing the side effects. Several PTT agents have been the subject of extensive studies and their drawbacks have also been realized. Organic dyes of indocyanine green (ICG) dye4 and polyaniline nanoparticles5 and copper chalcogenide semiconductors6–10 have the disadvantage of being unstable against NIR irradiation. Carbon-based materials such as carbon nanotubes (CNTs)11 and graphenes,12 have relatively low absorption ability in the NIR region. The most studied photothermal nanomaterials, including Ge nanoparticles,13 Pd-based nanosheets,14 and Au nanostructures15–21 in PTT, exhibit intense NIR photoabsorption, but the cost of these noble
a The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Shanghai Normal University, Shanghai 200234, China b Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131-000. E-mail: [email protected], [email protected]; Fax: +86-21-64322346; Tel: +86-21-64322346 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4nr03727h
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metals limits their wide use in applications. Therefore, there is a significant demand on the development of a new class of PTT agents,22–27 to ultimately become clinically useful tools for cancer treatment. Such materials should be of low toxicity and be easily functionalized with bioactive molecules as well as have the property of the high NIR-induced photothermal conversion efficiency. Transition metal carbide nanocrystals have received considerable attention recently owing to their unique chemical and physical properties, such as excellent mechanical properties, high melting points, high chemical stability, superconductivity and catalytic performance.28–31 Among the metal carbides, nickel carbide nanocrystals (Ni3C NCs), which are formed with an interstitial carbon in a hexagonal close packed nickel (hcp-Ni), have been much less investigated mainly because of their complicated synthetic procedures.32 The methods for their preparation include mechanical alloying,33 carbon-ion implantation into nickel,34 magnetron sputtering35,36 and high energy ball milling.37 These protocols rely on the expensive, specialized apparatus that are difficult for most researchers to access. Recently, a protocol utilizing thermal decomposition of a nickel precursor in the presence of organic surfactants has been developed for a convenient synthesis of high quality Ni3C NCs,38–40 which opens a door to explore their applications, especially in the field of biological science. We envisioned that the Ni3C NCs could serve as a new type of PTT agents, which may possess unique properties. Herein,
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we wish to disclose the results of the investigation. As demonstrated experimentally, the cost effective, conveniently prepared PEGylated Ni3C nanocrystals (NCs) with a uniform and small size exhibit a high photostability, relatively low toxicity, and high photothermal efficiency. Furthermore, the PEGylated Ni3C NCs have good photothermal conversion efficiency at a desirable NIR irradiation wavelength. The efficiency has been transformed to efficiently destroy cancer cells in vitro under the 808 nm laser irradiation with a power density of 0.50 W cm−2. Moreover, under the same operating conditions, the effective destruction of cancer cells in vivo was achieved within only 5 min with a low dose PEGylated Ni3C NCs (10 mg kg−1 body weight). The high photothermal therapeutic efficiency in vitro and in vivo has also been evaluated quantitatively by the MTT assay and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) staining, respectively. To the best of our knowledge, this study represents the first example of using transition metal carbides in PTT.
2
Experimental
2.1
Materials and characterization
Octadecylene and oleylamine were purchased from Beijing InnoChem Technology Co., Ltd. Poly(ethylene glycol) carboxylic acid (PEG-COOH, Mw: ∼2000) was purchased from Shanghai Seebio Biotech, Inc. Nickel acetylacetonate hydrate [Ni(acac)2] was purchased from Sinopharm Chemical Reagent Co., Ltd. All reagents were used without further purification. Water used in all experiments was purified using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) with resistivity higher than 18 MΩ cm. XRD was performed using a Rigaku DMAX 2000 diffractometer equipped with Cu/Kα radiation at a scanning rate of 4° min−1 in the 2θ range from 30 to 80° (λ = 0.15405 nm) (40 kV, 40 mA). TEM was carried out using a JEOL JEM-2010 transmission electron microscope operating at 200 kV. TEM samples were prepared by depositing a diluted NCs suspension (200 μg mL−1, 5 μL) onto a carbon-coated copper grid and air-dried before the measurements. FT-IR spectra were collected using a Nicolet Avatar 370 spectrometer. The samples were pelletized with KBr before measurements. Hysteresis loop was measured with a Quantum Design SQUID MPMS XL-7 magnetometer. X-ray photoelectron spectroscopy (XPS) was performed on a Kratos AXIS-165 surface analysis system. The hydrodynamic size was measured using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, UK) equipped with a standard 633 nm laser. The absorption spectra were collected on a DU 730 UV-visible spectrophotometer (Nucleic acid, protein analyzer) at room temperature. 2.2
Synthesis of Ni3C nanocrystals (Ni3C NCs)
In a typical procedure, Ni(acac)2 (0.1 g) was dissolved in a cosolvent mixture of octadecylene (20 mL) and oleylamine (8 mL) in a 100 mL three-neck flask. The solution was deaerated under a nitrogen flow at 110 °C for 15 min, heated to 285 °C
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under vigorous magnetic stirring, and kept for 30 min at this temperature before it was cooled down to room temperature. After centrifugation at 12 000 rpm for 8 min, the supernatant was removed. The resulting black precipitation was washed with ethanol three times to acquire pure Ni3C NCs. Finally, the obtained NCs were stored in 20 mL of chloroform before use. 2.3
Synthesis of PEGylated Ni3C NCs
In a typical process, methoxypoly (ethylene glycol) carboxyl acid (50 mg) was reacted with NaOH (2 mg) in mixed co-solvents ethanol (5 mL) and chloroform (10 mL) for 4 h, followed by addition of the chloroform solution of Ni3C NCs (5 mL, 2000 μg mL−1). The solution was stirred overnight. The formed PEGylated NCs were purified by washing with chloroform and ethanol each three times before further characterization. 2.4
Photothermal effect of PEGylated Ni3C NCs
An aqueous suspension (2 mL) containing PEGylated Ni3C NCs with different concentrations (100, 200, 500 and 1000 μg mL−1, respectively) was put in a quartz cuvette with an optical path length of 0.5 cm. The cuvette was illuminated by an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co., Ltd) with a power density of 0.50 W cm−2 for 300 seconds. The diameter of the laser spot was 3.5 cm. The increase in temperature was monitored by a digital thermocouple device with an accuracy of ±0.1 °C. The temperature was recorded every 10 s. 2.5
Cell culture
A human cervical carcinoma cell line (HeLa cells) was provided by the Shanghai Institutes of Biological Sciences (SIBS), Chinese Academy of Sciences (CAS, China). The cells were cultured in RPMI-1640 (Thermo, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin–streptomycin (Thermo) at 37 °C and 5% CO2. Cells were plated in a cell culture flask (Corning, USA) under the 100% humidity and allowed to adhere for 24 h, then harvested by treatment with 0.25% trypsin-EDTA solution (Gibco, USA). 2.6
MTT assay
In vitro cytotoxicity of PEGylated Ni3C NCs was evaluated by performing a methyl thiazolyl tetrazolium (MTT) assay of HeLa cells. The cells were seeded into a 96-well cell culture plate at a density of 5000 per well and cultured in RPMI-1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The next day, the cells were incubated with PEGylated Ni3C NCs with different concentrations (diluted in RPMI-1640 at 0, 12.5, 25, 50, 100, 200, 350, and 700 μg mL−1) for another 12 h and 24 h at 37 °C under 5% CO2. Thereafter, MTT (20 μL, 5 mg mL−1) was added to each well and the plate was incubated for an additional 4 h at 37 °C. After the removal of the medium, the purple formazan product was dissolved with DMSO for 15 min. Finally, the optical absorption of formazan at 490 nm was measured by an enzyme-linked immunosorbent
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assay reader, with background subtraction at 690 nm was measured by a microplate reader (Multiskan MK3, USA).
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2.7
Photothermal therapy of HeLa cells in vitro
HeLa cells were seeded into a 96-well cell culture plate at a density of 1 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The culture medium was replaced without or with PEGylated Ni3C NCs (50 μg mL−1) dispersed in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh medium (200 μL) was added to the wells, followed by the 808 nm laser irradiation for 10 min with the different power density of 0.25 and 0.50 W cm−2, respectively. After the laser irradiation, HeLa cells were cultured for additional 1 h for MTT assay. All measurements were conducted in triplicate.
2.8
Trypan blue staining
HeLa cells were seeded into a 12-well cell culture plate at a density of 5 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The culture medium was then replaced without or with PEGylated Ni3C NCs (50 μg mL−1) dispersed in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh complete medium (800 μL) was added to the wells, followed by the 808 nm laser irradiation for 10 min with a power density of 0.50 W cm−2. HeLa cells were stained with 0.4% Trypan blue solution for 15 min. Then the suspension was removed and the cells were washed with PBS twice. Cell morphology of the adherent cells in PBS (500 μL) was observed by an inverted optical microscope (Olympus, IX71, Japan) with a magnification of 20×. Cells stained by Trypan blue were counted as dead cells.
2.9
Laser scanning confocal microscopy
Confocal microscopic imaging was performed with a Leica TCS SP5 II inverted microscopy (Leica DMI 6000B, Solms, Germany). HeLa cells were seeded into a 12-well cell culture plate at a density of 5 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. Then, the culture medium was replaced with PEGylated Ni3C NCs (50 μg mL−1) in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh complete medium (800 μL) was added to the wells, followed by the 808 nm laser irradiation for 10 min with a power density of 0.50 W cm−2. After laser irradiation, the cells were co-stained by a mixture solution containing Calcein-AM (2 μmol L−1) and propidium iodide (PI) (4 μmol L−1) to differentiate live (green) and dead (red) cells for 15 min. Calcein-AM and PI was excited by 488 nm and 543 nm lasers, respectively. Labeled HeLa cells were observed on a laser scanning confocal microscope.
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2.10 Flow cytometry HeLa cells were seeded into a 12-well cell culture plate at a density of 5 × 104 per well and cultured in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin at 37 °C and 5% CO2 for 24 h. The culture medium was then replaced without or with PEGylated Ni3C NCs (50 μg mL−1) dispersed in RPMI 1640 medium. After incubation for 4 h, excess unbound materials were removed by rinsing two times with PBS. Fresh complete medium (800 μL) was added to the wells followed by the 808 nm laser irradiation for 10 min with the power density of 0.25 and 0.50 W cm−2, respectively. After the laser irradiation, HeLa cells were washed twice with PBS and harvested by treatment with 0.25% trypsin-EDTA solution. After centrifugation, the obtained HeLa cells were suspended in PBS solution and analyzed with a flow cytometer (Beckman Coulter, Quanta SC, USA). The collected data were analyzed using Flow Jo software 7.6.5. 2.11 Animal experiments ICR rats (∼40 g body weight) and HeLa tumor-bearing nude mice (∼20 g body weight) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Animal care and handling procedures were in agreement with the guidelines of the Regional Ethics Committee for Animal Experiments. The nude mice were used for further experiments when the tumor volume reached about 360 mm3. 2.12 In vivo cytotoxicity assay Three healthy ICR rats (∼40 g body weight) were intravenously injected with a dose of 10 mg kg−1 PEGylated Ni3C NCs in the saline solution. For a control group, three other ICR rats were treated with saline (200 μL). Over a one month period, ICR rats were sacrificed to collect the blood (1 mL) and the tissues, including the heart, liver, lung, kidney, and spleen. For the short term in vivo cytotoxicity, another six rats were used (three rats for each group) as previously described. The only difference was that the rats were sacrificed 24 h after the injection instead of a month. The blood analysis data were measured in Shanghai SLAC Laboratory Animal Co., Ltd. All the tissues were stained with hematoxylin and eosin (H & E) to assess tissue and cellular morphology. 2.13 Photothermal imaging in vivo HeLa tumor-bearing nude mice were intratumorally or intravenously injected with a dose of 10 mg kg−1 of PEGylated Ni3C NCs in the saline solution or saline (200 μL). After 1 h, the tumors in the mice were irradiated with an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co., Ltd) for 5 min with a power density of 0.25 and 0.50 W cm−2, respectively, then imaged and recorded by a FLIR A300 (USA, spectral band: 7.5–13 μm). 2.14 Photothermal therapy of HeLa cells in vivo HeLa tumor-bearing nude mice were intratumorally or intravenously injected with a dose of 10 mg kg−1 of PEGylated Ni3C
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NCs or saline (200 μL). After 1 h, the tumors in the mice were irradiated with an 808 nm laser (Shanghai Xilong Optoelectronics Technology Co., Ltd) for 5 min with a power density of 0.25 and 0.50 W cm−2, respectively, then imaged and recorded by a FLIR A300 (USA). The mice were euthanized after the laser treatment. The tumors were removed, fixed in 4% paraformaldehyde at 4 °C overnight, and embedded in paraffin for H & E staining and Tunel staining. 2.15 H & E and TUNEL staining Serial 5 μm thick tumor sections were prepared and stained with haematoxylin/eosin (HE, Beyotime, China). The histology and morphology of the tumors were observed under the Eclipse E800 microscope (Nikon, Japan). The DeadEnd Colorimetric TUNEL System from Promega (Mannheim, Germany) was used. Biotinylated nucleotides were incorporated at the 3′-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase (TdT). Horseradish peroxidase-labeled streptavidin was then bound to these biotinylated nucleotides. Peroxidase activity was visualized using the liquid DAB substrate chromogen system (Dako, Hamburg, Germany). In case of antibodyTUNEL double staining, Cy2-labeled streptavidin (Dianova, Hamburg, Germany) was used. The number and percentage of
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TUNEL-positive cells were counted and determined by counting 1 × 103 cells from five random selected fields.
3
Results and discussion
3.1
Synthesis and characterization of Ni3C NCs
The prerequisite Ni3C NCs were synthesized by the high-temperature pyrolysis method from the precursor of nickel acetylacetonate in the mixture of octadecylene and oleylamine at 285 °C. The resulting organic ligand-capped Ni3C NCs show excellent dispersibility and stability in chloroform (Fig. 1b inset), with an average diameter of 42.7 ± 5.0 nm without obvious aggregation as depicted in the field of view from a representative transmission electron microscope (TEM) image (Fig. 1b and S1a†). A high-resolution TEM image (Fig. S1b†) of the nanoparticles reveals the high crystalline nature of Ni3C NCs. The lattice spacing between two adjacent planes is 0.203 nm, which corresponds very well to the d-spacing for the (113) lattice plane. The crystallography of NCs was verified by powder X-ray diffraction (XRD) (Fig. 1c). All the diffraction peaks of the as-prepared Ni3C NCs are well indexed to the hexagonal Ni3C phase (JCPDS card no. 06-0697). The mean
Fig. 1 (a) Illustration of the synthesis and in vivo near-infrared laser driven photothermal therapy of PEGylated Ni3C NCs. (b) TEM image of Ni3C NCs and a photograph in chloroform solution (2 mg mL−1, inset). (c) XRD pattern of Ni3C NCs. (d) XPS spectra of Ni 2p3/2 region. (e) Absorption spectra of PEGylated Ni3C aqueous solution at different concentrations in water (Inset: the absorbance of PEGylated Ni3C aqueous solution at 808 nm as a function of concentration related to (e).) (f ) Temperature changes of the aqueous solution of PEGylated Ni3C NCs at different concentration under the 808 nm laser irradiation with a power density 0.50 W cm−2 for 5 min. (g) Temperature changes of the aqueous solution of PEGylated Ni3C NCs (100 μg mL−1) over four Laser on/off cycles (0.50 W cm−2).
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crystalline size is estimated to be ∼45 nm from the halfmaximum full width of (113) peak according to the Scherrer equation, which matches the size observed from TEM analysis. Considering that the crystal structure of Ni3C phase is almost identical to that of hcp Ni phase, X-ray photoelectron spectroscopy (XPS) was performed (Fig. 1d). In the Ni 2p region, the Ni 2p3/2 peak is observed at 853.1 eV, which is consistent with the binding energy of nickel carbide reported in the literature.41 In addition, the very weak magnetization properties of Ni3C NCs from the hysteresis loops further confirms the Ni3C phase, but not the hcp-Ni phase (Fig. S1e†).42 To make the Ni3C NCs water soluble and biocompatible, PEG chains were anchored on the surface of Ni3C NCs via coordination between carboxylate ions and the Ni3C surface. The immobilization is confirmed by the absorption band at 1105 cm−1 in the Fourier transform infrared spectroscopy (FT-IR) spectra (Fig. S1f†). Moreover, such modification does not change the morphology of NCs with a diameter of 44.4 ± 4.8 nm verified by TEM (Fig. S1c and S1d†). The hydrodynamic diameter measured by dynamic light scattering (DLS) is 107 ± 4.5 nm, which is larger than that observed by TEM owing to the slight aggregation. The PEGylated Ni3C NCs display the high dispersity in water, PBS and RPMI with 10% fetal bovine calf serum (FBS) as simulated in vivo plasma (Fig. S1c† inset). The property is essential for the biological studies. 3.2
Photothermal effect of PEGylated Ni3C NCs
The absorption spectra of PEGylated Ni3C NCs (Fig. 1e) exhibit a broad absorption ranging from visible to NIR region, and there is a good linear correlation between the absorbance at 808 nm and the concentration of PEGylated Ni3C NCs (Fig. 1e inset). The absorption co-efficient of PEGylated Ni3C NCs was ∼4.0 L g−1 cm−1. The absorption of PEGylated Ni3C NCs in the NIR region is probably attributed to the charge transfer transition between Ni atom and C atom in the Ni3C phase, which is confirmed by the increased Ni 2p3/2 peak compared to that of the metallic Ni (852.7 eV).41 The absorption in the NIR region of PEGylated Ni3C NCs encouraged us to demonstrate the possibility as a photothermal agent for cancer therapy. Different concentrations of PEGylated Ni3C NCs (100, 200, 500 and 1000 μg mL−1) were irradiated by an 808 nm NIR laser with a power density of 0.50 W cm−2 for 5 min (Fig. 1f ). Notably, the PEGylated Ni3C NCs solution showed a rapid rise in temperature during laser irradiation while no obvious temperature change was observed for pure water in the absence of the material under the same experimental conditions. Even at the concentration of 100 μg mL−1, the temperature was raised by 16.3 °C. Furthermore, higher concentration of NCs leads to a more prominent temperature increase. For example, an increase of 30 °C was obtained at the concentration of 1000 μg mL−1 in 5 min. According to a literature reported method,43 the photothermal conversion efficiency was calculated to be ∼16.9% (Fig. S1g and S1h†). To evaluate the NIR photostability of PEGylated Ni3C NCs, four on/off laser cycles were performed by irradiating the aqueous solution of PEGylated Ni3C NCs via an 808 nm
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laser for 5 min (laser on, Fig. 1g), followed by cooling down to room temperature without NIR laser irradiation for 20 min (laser off, Fig. 1g). The temperature increase of 16.3 °C was achieved after the first laser on at the NCs concentration of 100 μg mL−1. No significant change in the temperature increase was observed after four cycles. These findings indicate that PEGylated Ni3C NCs have the good NIR photostability. 3.3
In vitro and in vivo cytotoxicity assays
As discussed above, in addition to high photothermal conversion efficiency and NIR photostability, an ideal photothermal agent should be of low toxicity for biological applications.44,45 To evaluate the cytotoxicity of PEGylated Ni3C NCs, a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with HeLa cells was used. The concentrationdependent effect of PEGylated Ni3C NCs on the cell viability incubated for 12 and 24 h was determined (Fig. S2†). These studies clearly showed that the high cell viability (>80%) can still be attained within the concentration of 100 μg mL−1 incubated for 12 h, which provides the basis for in vivo photothermal therapy. Meanwhile, in order to further investigate the potential in vivo toxicity, healthy ICR rats were intravenously injected with a dose of PEGylated Ni3C NCs (10 mg kg−1 of body weight). The rats were sacrificed for blood analysis and histological examination after 24 h and 30 d, respectively. The liver function index (Fig. 2a1, 2a2, S3a1, and S3a2†) including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), kidney function index (Fig. 2b1, 2b2, S3b1, and S3b2†) including urea nitrogen (BUN), creatinine (CREA) and a complete blood panel were tested (Fig. 2c1–2c8 and Fig. S3c1– S3c8†). According to the analysis of blood indexes with the control groups, except for the white blood cells value that was likely caused by inflammation from the PEGylated Ni3C NCs, there was no significant difference and all measured parameters showed only slight variations within normal ranges, suggesting no obvious hepatic and kidney damage. In addition, organs were excised, including the heart, liver, lung, kidney, and spleen, and stained with hematoxylin and eosin (H&E) for a histology analysis (Fig. 2d and Fig. S3d†). Compared with the control group, few differences were observed for mice injected with PEGylated Ni3C NCs. Therefore, PEGylated Ni3C NCs showed no noticeable toxicology in vivo experiments under our experimental conditions and dosage. 3.4
Photothermal therapy of HeLa cells in vitro
We then evaluated the in vitro photothermal therapeutic capability of the PEGylated Ni3C NCs to cancer cells under the laser irradiation by optical microscopy. HeLa cells were incubated with or without PEGylated Ni3C NCs at a concentration of 50 μg mL−1 for 4 h and then were irradiated by an 808 nm laser of 0.50 W cm−2 for 10 min. After NIR laser exposure, the cells were stained blue by treating with 0.4% Trypan blue for 5 min. As shown in the optical microscope images in Fig. 3a1–3a3, there were no obvious changes for the control
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Fig. 2 Blood analysis data of ICR rats intravenously injected with PEGylated Ni3C NCs (10 mg kg−1 body weight). Untreated rats were used as the control group. Liver function index includes alanine aminotransferase (ALT, a1) and aspartate aminotransferase (AST, a2). Kidney function index includes urea nitrogen (BUN, b1) and creatinine (CREA, b2). The complete blood analysis includes red blood cells (RBC, c1), white blood cells (WBC, c2), haemoglobin (HGB, c3), hematocrit (HCT, c4), mean corpuscular volume (MCV, c5) mean corpuscular hemoglobin concentration (MCHC, c6), mean corpuscular hemoglobin (MCH, c7) and platelets (PLT, c8). Statistics were based on three mice per data point. (d) Histological changes in the heart, liver, spleen, lung and kidney of ICR rats without or with intravenous injection of PEGylated Ni3C NCs (10 mg kg−1 body weight) after 30 days. These organs were stained with H & E and observed under an optical microscope at a 40× magnification.
groups of HeLa cells cultured without or with PEGylated Ni3C NCs (50 μg mL−1, Fig. 3a1 and 3a2). Moreover, negligible cell destruction was also observed for HeLa cells with 808 nm laser irradiation alone (Fig. 3a3). In contrast, nearly all HeLa cells incubated with PEGylated Ni3C NCs after the 808 nm laser irradiation were stained blue (Fig. 3a4), indicating that cancer cells were damaged by the irradiation. The photothermal therapeutic effect of PEGylated Ni3C NCs on cancer cells was also confirmed by laser confocal fluorescence microscopy. Following laser irradiation, HeLa cells were co-stained by CalceinAM and propidium iodide (PI) to differentiate live (green) and dead (red) cells, respectively. These studies indicate that the PEGylated Ni3C NCs are able to kill HeLa cells via photothermal destruction (Fig. 3b4). In contrast, HeLa cells without treatment (Fig. 3b1), incubated with PEGylated Ni3C NCs alone (Fig. 3b2) and irradiated by an 808 nm laser only (Fig. 3b3) were not affected under similar experimental conditions. These results also corroborated well with the above optical microscopic imaging data. We further quantitatively investigated the photothermal effect of PEGylated Ni3C NCs to HeLa cells using a MTT assay and a fluorescence activating cell sorter (FACS) at a concen-
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tration of 50 μg mL−1 PEGylated Ni3C NCs. It was determined that the viability of HeLa cells significantly dropped as an 808 nm laser power density of PEGylated Ni3C NCs increased. As shown in Fig. 3d, while the cell viability remained 74.5 ± 1.7% by the MTT assay (∼74.5% by FACS analysis, Fig. S4b†) with a power density of 0.25 W cm−2, the viability decreased rapidly to 13.7 ± 0.7% by the MTT assay (∼7.2% by FACS analysis, Fig. 3c3) with a power density of 0.50 W cm−2. As expected, neither PEGylated Ni3C NCs (50 μg mL−1) nor an 808 nm laser treatment showed obvious destructive effect on HeLa cells in the control experiments (Fig. 3c1, 3c2 and Fig. S4a†). These experimental findings demonstrated that the combination of PEGylated Ni3C NCs and an 808 nm laser illumination of 0.50 W cm−2 could kill localized tumor cells efficiently. To clarify the cell death mode upon photothermal treatment with a power density of 0.50 W cm−2, annexin V-FITC/propidium iodide (PI) double staining analysis was used. The quantity of apoptotic and necrotic cells was determined by the percentage of AV+/PI−, while the quantity of necrotic cells was determined by the percentages of AV+/PI+. Statistical analysis of the data showed that the laser irradiation plus PEGylated Ni3C NCs treatment induced ∼6.1% cell apoptosis and ∼84.6% cell
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Fig. 3 Trypan blue staining (a1–a4) and confocal fluorescence images (b1–b4) of HeLa cells. Control HeLa cells (a1, b1), HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1, a2, b2), HeLa cells treated with the 808 nm laser irradiation for 10 min (0.5 W cm−2, a3, b3), HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1) and irradiated by an 808 nm laser for 10 min (0.50 W cm−2, a4, b4). (c) Representative FACS plots. (c1) HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1), (c2) HeLa cells after 808 nm laser irradiation for 10 min with a power density of 0.50 W cm−2, (c3) HeLa cells cultured with PEGylated Ni3C NCs (50 μg mL−1) and then irradiated by an 808 nm laser for 10 min with a power density of 0.50 W cm−2. (d) Cell viability of HeLa cells with different treatments.
necrosis (Fig. 3c3). Therefore, these results show that the main mechanism of in vitro photothermal therapy induced cell death is necrosis rather than apoptosis. 3.5
Photothermal experiments in vivo
Encouraged by the effective photothermal therapeutic capability of the newly developed PEGylated Ni3C NCs to cancer cells, we then turned our attention on the in vivo studies of cancer PTT using HeLa tumor mouse model. HeLa tumorbearing nude mice were intratumorally injected with PEGylated Ni3C NCs (10 mg kg−1 of body weight) and then exposed to an 808 nm laser for 5 min. The temperature change was monitored by an infrared thermal camera (Fig. 4a). To our delight, we found that the temperature injected with PEGylated Ni3C NCs under an 808 nm laser irradiation with a power density of 0.25 and 0.50 W cm−2 rapidly increased from ∼30 to
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∼36.5 and ∼44 °C within 5 min, respectively (Fig. 4b). It is established that photothermal therapy can destruct cancer cells when the temperature is greater than 43 °C.46,47 In contrast, the temperature at the tumor site without PEGylated Ni3C NCs treatment was marginally affected even after being exposed to the laser irradiation with a power density of 0.50 W cm−2, showing only a temperature increase of 3 °C after 5 min. More importantly, no obvious temperature increase was observed on other parts near the tumor, which effectively minimizes the damage of normal tissues. Although a significant accumulation of PEGylated Ni3C NCs in the tumor was observed due to the enhanced permeability and retention (EPR) effect (Fig. S7†), the low absolute amount of NCs in the tumor did not measurably increase the temperature of the tumor under the 808 nm laser irradiation after the intravenous injection with the same dose (Fig. S5†).
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Fig. 4 (a) Thermal infrared images of tumor area intratumorally injected with PEGylated Ni3C NCs (10 mg kg−1) recorded at different time intervals. Mice injected saline was taken as the control group. The tumor was exposed to an 808 nm laser with a power density of 0.25 and 0.5 W cm−2, respectively. (b) Plots of tumor temperature as a function of time that corresponds to (a).
Finally, to assess the in vivo therapeutic effect in a HeLa tumor mouse model, hematoxylin and eosin (H & E) staining, and terminal deoxynucleotidyl transferase biotin-dUTP nickend labelling (TUNEL) staining studies of tumor slices were carried out. H & E staining of tumours treated with PEGylated Ni3C NCs (10 mg kg−1 of body weight) under the 808 nm laser
irradiation with a power density of 0.50 W cm−2 showed significant thermal damage, including shrinkage and nuclear chromatin that disintegrated into small fragments. Nevertheless, tumor tissues of the control cells remained normal without changes (Fig. 5a and 5b). TUNEL staining is a method for probing of DNA fragmentation, which results from apoptotic
Fig. 5 H&E (a) and TUNEL (b) stained histological images of tumor sections. (c) The percentage of TUNEL-positive cells in tumor sections with different experiment groups.
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signalling cascades by labelling the terminal end of nucleic acids. Therefore, TUNEL enables to identify extensive thermonecrotic or apoptotic cells, in which dead cells are stained in brown.48,49 As expected, there were more brown cells than in the control groups (Fig. 5a and 5b). Moreover, as shown in Fig. 5c, 18.5% ± 1.7% and 21.7% ± 3.0% TUNEL positive cells were found only cultured with PEGylated Ni3C NCs and under the laser alone, respectively. However, after laser irradiation, the quantity of TUNEL positive cells increased to 44.1% ± 1.7% after treatment with the NCs. Under such a short irradiation time, a significant tumor killing effect is achieved. However, no obvious damage of the tumor tissue under the similar 808 nm laser irradiation was observed after the intravenous injection with the same dose, which was probably attributed to the accumulation with this low absolute amount in tumor (Fig. S6 and S7†).
4 Conclusions In summary, we have developed PEGylated transition-metal carbide Ni3C NCs as a new class of PTT agents. The Ni3C NCs can be conveniently prepared via a cost-effective thermal decomposition and ligand exchange method to give uniformly small-sized NCs. The PEGylated Ni3C NCs had a photothermal conversion efficiency of ∼16.9% at a desired NIR irradiation wavelength and NIR photostability in the aqueous solution. As demonstrated, cancer cells in vitro and in vivo photothermal treatment can be efficiently destroyed under the 808 nm laser irradiation with a power density of 0.50 W cm−2. Therefore, the features of their small size, high photothermal conversion efficiency, low cost and low cytotoxicity enable the PEGylated Ni3C NCs as a promising tool for cancer treatment. Furthermore, the work described here opens a new avenue for the application of transition metal carbides in the field of biomedical science.
Acknowledgements This work was partially supported by National Natural Science Foundation of China (No. 21271130 and 21371122), program for Changjiang Scholars and Innovative Research Team in University (no. IRT1269), Shanghai Natural Science Fund Project (no. 12ZR1421800 and 13520502800), Shanghai Pujiang Program (13PJ1406600), Shanghai Municipal Education Commission (no. 13ZZ110) and Shanghai Normal University (no. DXL122 and SK201339).
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