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Doxorubicin-conjugated CuS nanoparticles for efficient synergistic therapy triggered by near-infrared light† Huiting Bi, Yunlu Dai,* Ruichan Lv, Chongna Zhong, Fei He, Shili Gai, Arif Gulzar, Guixin Yang and Piaoping Yang* To integrate photothermal therapy (PTT) with chemotherapy for improving anticancer efficiency, we developed a novel and multifunctional doxorubicin (DOX) conjugated copper sulfide nanoparticle (CuS– DOX NP) drug delivery system using hydrazone bonds to conjugate carboxyl-functionalized copper sulfide nanoparticles (CuS NPs) and DOX. On the other hand, the hydrazone bonds could be used for improving the DOX release rate (88.0%) by cleavage in a mildly acidic environment irradiated by 808 nm laser light, which could greatly promote chemo-therapeutic efficacy. Simultaneously, CuS NPs which can absorb near infrared (NIR) light produce a clear thermal effect, giving rise to a synergistic therapeutic effect combined with enhanced chemo-therapy. The DOX-conjugated CuS NPs display an evident in vitro cytotoxicity to HeLa cancer cells under 808 nm light irradiation. High tumor inhibition efficacy has
Received 11th December 2015, Accepted 29th January 2016
been achieved after 14 day in vivo treatment, performed with intravenous administration of CuS–DOX
DOI: 10.1039/c5dt04842g
NPs with 808 nm laser irradiation on H22 tumor-bearing mice. The multifunctional system which was achieved by a facile route should be a potential candidate in the anti-cancer field due to the synergistic
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therapeutic effect, which is superior to any single approach.
Introduction Currently, surgical resection and traditional chemotherapy are still the major therapeutic approaches to cancer treatment. However, it cannot be ignored that the above two methods may cause adverse effects to normal cells and tissues, insufficient dosage to diseased regions, as well as an increased incidence of the second cancer.1–5 PTT, based on near-infrared (NIR, λ = 700–1100 nm) laser induced photothermal ablation, has shown rapid development in recent years as a minimally invasive and harmless therapeutic approach in biotechnological applications.6–14 The NIR laser can convert optical energy into thermal energy, and possesses the desirable penetration depth in biological tissues.15–18 Recently, the combined chemo- and photothermal therapeutic systems (CPTSs) have provided new ideas for cancer therapy.19,20 In particular, it has been demonstrated that PTT integrated with chemotherapy
Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin, 150001, P. R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5dt04842g
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results in improved treatment efficacy relative to the use of each approach independently. To date, photothermal therapeutic systems have been established based on several types of photothermal agents including noble metal nanostructures,21–25 carbon-based 26–29 materials and semiconductor compounds.30–32 For some gold (Au) nanostructures, including Au nanorods, nanostars and nanoshells, their NIR absorption peaks would decrease after laser radiation because of low photostability.33–37 In addition, the majority of these photothermal drugs that have been engineered based on several noble metals such as Au and Pd are very expensive, and the synthesis processes turn out to be very complicated.22 Carbon nanomaterials have shown a synergistic effect on cancer,8,38 but they have poor photothermal conversion efficiency. Hence, it is worthwhile to explore the evolution of multifunctional nanomaterials with low cost, easy synthesis and excellent PTT efficacy. Among various candidates for the carriers of anticancer agents, CuS NPs, with a unique nature such as low cost, low cytotoxicity, high stability and outstanding photothermal efficiency under an 808 nm laser, have attracted increasing attention from biomedical researchers across the globe.39–45 Furthermore, the absorption wavelength of CuS NPs is not influenced by the surrounding environment compared to the Au nanoparticles. CuS NPs possess a maximum absorbance at 900 nm located at the NIR
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range, which can be ascribed to the d–d energy band transition of Cu2+ ions, while the absorption from gold nanostructures results from surface plasmon resonance.46–48 Emission coming from the NIR laser beam at 808 nm leads to the increased temperature of CuS NPs, which could be exploited as the foundation for PTT. It is well known that a series of functional nanoparticles conjugated with an anticancer drug have achieved good results in biological applications.49–53 Numerous photothermal agents are obtained by complete surface modification and synthetic processes. Herein, we designed a chemo-photothermal therapeutic system, that is DOX conjugated CuS nanoparticles. CuS NPs synthesized by a simple method were utilized as drug vehicles. DOX, a commonly used anticancer drug,54–59 was conjugated to CuS NPs through chemical bonds. In this work, the chemo-photothermal therapeutic system has at least three outstanding features. Firstly, the therapeutic system can be easily obtained due to the facile synthetic process. Secondly, owing to the ultra-small size, CuS NPs could disperse homogenously in aqueous solution, which was beneficial for drug delivery in vivo and improved the therapeutic effect.60,61 Thirdly, the content of DOX linked to CuS NPs is as high as 88.5% for the reliability of the chemical bonding. The drug release property in vitro, cytotoxicity, cellular uptake and therapeutic effect in vivo have been investigated in detail to certify the feasible anti-cancer application of this system.
Results and discussion Synthesis, characterization and drug release in vitro The procedure for the formation of CuS–DOX NPs is illustrated in Scheme 1. Firstly, carboxylic acid-functionalized CuS NPs were synthesized at 90 °C in a water bath. Then, the carboxyl group reacts with hydrazine monohydrate to generate CuSCONHNH2, and then the –NHNH2 further reacted with the ketone group of the DOX to produce CuS–DOX NPs. The hydrazone bond (red dotted line labelled in CuS–DOX NP structure) connected CuS with DOX, which was cleavable in a weakly acidic environment to release DOX.62,63 As illustrated in Scheme 2, the CuS–DOX NPs were injected into the mice and accumulate at the tumor site by the enhanced permeability and retention (EPR) effect. In physiological terms, small molecules (such as H2O) and ions (such as Na+, K+) enter the cells by means of diffusion, and the nanoparticles enter the cells by
Scheme 1 DOX NPs.
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Schematic illustration of the preparation process of CuS–
Scheme 2 Nanoparticles enter cancer cells by endocytosis and release DOX under 808 nm NIR irradiation in a facile acid environment.
endocytosis.64,65 It can be speculated that CuS–DOX NPs enter the cancer cells by endocytosis, and release DOX in a weak acid environment by cleaving the hydrazone bond between CuS and DOX. Finally, DOX and the photothermal effect by 808 nm NIR irradiation kill the cancer cells together. The TEM images with different magnifications of CuS NPs are shown in Fig. 1a and b. It is clearly seen that the nanoparticles are relatively uniform and dispersed with an average diameter around 5 nm. After being modified with hydrazine and conjugated with DOX on the surface, the nanoparticles still maintain their morphology and the size is almost unchanged. In addition, the nanocrystals show high dispersity and have no aggregation in the solution (Fig. 1c, d and S1†). The XRD measurement is explored to understand the evolution and crystallization of CuS NPs, and the pattern is shown in Fig. 1f. All of the X-ray diffraction peaks of the sample could be indexed to the hexagonal phase of CuS and the lattice parameters are in agreement with those of the standard powder diffraction pattern of CuS (JCPDS no. 06-0464). No obvious impure peaks appear in the pattern, indicating that high purity of CuS NPs was acquired. Fig. 1e shows the high-resolution TEM (HRTEM) of CuS NPs and indicates that the CuS NPs are of high crystallization. From the image, two typical interlayer spacings of 0.30 nm and 0.25 nm could be observed, corresponding to the (102) and (103) planes of the hexagonalstructured-CuS, respectively. Fig. 2a shows the temperature change of the CuS NP aqueous solution and deionized water versus irradiation time, and a good photothermal effect is presented. After irradiation for 600 s, the temperature of the CuS aqueous solution was increased by 31.9 °C at a concentration of 60.6 μg mL−1. In contrast, the temperature of deionized water increased by only 10.3 °C. The temperature increased to 45 °C approximately after 600 s, which is in favour of PTT and can kill cancer cells after maintenance for 15 min. Fig. S2† shows the linear time
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Fig. 3 FT-IR spectra of CuS NPs (a); CuS-CONHNH2 NPs (b); and CuS– DOX NPs (c).
Fig. 1 TEM images of CuS NPs with different magnifications (a, b); CuSCONHNH2 NPs (c); CuS–DOX NPs (d); HRTEM image of CuS NPs (e); XRD pattern of the CuS NPs and the standard JCPDS card 06-0464 of CuS (f).
Fig. 2 Temperature change of CuS NP aqueous solution and deionized water versus irradiation time (a); the photothermal response of the CuS aqueous solution with NIR irradiation and then with the laser shut off (b).
data versus −ln θ obtained from the cooling period of Fig. 2b. The photothermal conversion efficiency (η) was calculated (see ESI† for details) by the following equation: η¼
hAΔTmax Qs Ið1 10Aλ Þ
According to the equation, the η value of CuS was determined to be 31.1%. The photothermal conversion efficiency is higher than that of the Au nanorods (24.0%), which are widely used for photothermal therapy.66,67
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To study the surface properties of the nanoparticles, FT-IR spectra were employed and the results are shown in Fig. 3. The bands at 3428 cm−1 and 1110 cm−1 in the spectra of CuSCOONa, CuS-CONHNH2, and CuS–DOX are assigned to the –OH stretching and bending vibrations. The peak at 1625 cm−1 can be attributed to the stretching vibrations of the carboxyl groups. After the amidation of the carboxyl group by hydrazine monohydrate, the band at 1646 cm−1 in Fig. 3b is attributed to the characteristic peak of the –CO–NH– bond. As shown in Fig. 3c, the band at 1633 cm−1 can be ascribed to the –CvN– bond from the reaction between the –NHNH2 group of nanoparticles and the ketone group at the 13-keto position of DOX, which is the signature of the grafting of DOX onto CuS. Furthermore, the peaks at 1558 cm−1 and 1412 cm−1 derived from the absorption of DOX. Zeta potentials of CuS NPs, CuSCONHNH2 NPs and CuS–DOX NPs are obtained, which show the shift of the isoelectric point (Fig. S3†). According to the FT-IR spectra and zeta potentials, we can draw the conclusion that the surface functional groups have changed and DOX has been successfully conjugated to the surface of CuS NPs. Fig. 4a shows the UV-vis spectra of CuS-COONa, CuSCONHNH2 and CuS–DOX solution with the same concentration of 62.6 μg mL−1 for Cu2+. The absorption of CuSCOONa NPs exhibits a peak at 850 nm and the absorption of CuS-CONHNH2 also shows a similar peak, indicating that the conjugation process amination does not affect the structure of CuS. Meanwhile, the broad absorption band in the NIR region is observed at 850 nm, which is attributed to a characteristic of the CuS NPs demonstrating its potential use as a PTT agent. According to the results of UV-vis spectra , when NIR excitation at 808 nm is potentially used as the radiation source, which can increase penetration depth and suppress unwanted overheating, the peak of CuS–DOX NPs at around 480 nm in the UV-vis spectrum could be attributed to the strong absorption of DOX covering up the peak of CuS. The results of UV-vis tests can further confirm that DOX has been successfully conjugated on the surface of the CuS NPs. The drug loading efficien-
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Fig. 5 The L929 fibroblast cell viability after incubating with different concentrations of CuS nanoparticles for 24 h and the quantitative assay by the standard MTT method.
Fig. 4 The UV-vis absorption spectra of CuS NPs, CuS-CONHNH2 NPs and CuS–DOX NPs (a); DOX release efficiency of CuS–DOX at pH 4.0, 5.5 and 7.4 PBS buffers (b).
cies and release abilities of the CuS–DOX NPs through the acid-dependent cleavage of the hydrazone bond were examined by the UV-vis spectrum at 480 nm at different pH values and different times. Fig. 4b exhibits the release profiles of the DOX from CuS– DOX NPs in PBS buffer solutions at pH 4.0, 5.5 and 7.4 at 37 °C. It can be seen that the drug release rate of DOX at pH 4.0 is faster than that at pH 5.0 and 7.4. The drug release rate increases by decreasing the pH value. About 88.0% and 70.0% of DOX released from the CuS–DOX NPs is at pH 4.0 and 5.5, respectively. By comparison, only about 23.0% of DOX is released under neutral ( pH 7.4) in 24 h. It can be elucidated that the behavior of pH triggered DOX release may be attributable to the cleavage of the hydrazone bond, which is stable in a normal physiological environment but is quickly cleavable in an acidic environment ( pH 4.0–5.5). Transmission and release of the DOX by CuS–DOX is through intravenous injection into blood circulation, which belongs to the internal environment, and the nanoparticles would not enter acidic tissues such as the stomach in an external experiment. It is well known that the micro-environments of tumor tissue are acidic, so CuS– DOX NPs with pH triggered drug-release properties are promising to be used as an anti-cancer platform by changing the pH, which is important and beneficial for cancer therapy.63 In vitro compatibility and cell uptake It is essential to evaluate the biocompatibility of CuS NPs before using the particles for cancer therapy both in vitro and
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in vivo. The standard MTT assay was carried out on L929 fibroblast cells and the results were monitored by using a Versamax microplate reader at 490 nm. As shown in Fig. 5, the cell viability is higher than 80% after incubation with CuS NPs in a wide range of concentrations (from 6.25 to 200 μg mL−1) for 24 h. It can be demonstrated that the nanoparticles have good biocompatibility as drug carriers for biomedical application. The cell uptake process was measured after the incubation of CuS–DOX NPs with HeLa cancer cells for 0.5 h, 1 h and 3 h with and without NIR irradiation by CLSM. The blue fluorescence from the DAPI is used to mark the nucleus and the red emission from DOX is for tracking the carrier, respectively. The merging of the two channels also occurs correspondingly. In Fig. 6a, during the first 0.5 h, only a few nanoparticles could be taken up by HeLa cells. Subsequently, it is clear that there is a stronger red emission of DOX observed in both the cytoplasm and the nucleus, suggesting that more particles appear in the cells with enhanced incubation time. In Fig. 6b, after NIR exposure for 5 min, more particles are remarkably swallowed by the cells compared to those without NIR at the same time point. The enhanced DOX fluorescence is obvious, which is due to the reason that it was easier for the particles to be taken in by the cells and release DOX after incidence of the NIR laser. This phenomenon can be attributed to the photothermal effect of CuS NPs, which can increase the temperature of the CPTSs, and promote nanoparticles entering the cells.68,69
Toxicity and photothermal effect in vitro and in vivo To explore the toxicity and photothermal effect of nanoparticles, a series of experiments were employed in vitro by incubating HeLa cells with CuS NPs, CuS–DOX, free DOX and pure DMEM respectively. Fig. 7a and b show a photograph and infrared image of the HeLa cells after being irradiated by an 808 nm laser for 5 min in a 96-well plate. The thermal image shows that the temperature of the HeLa cell supernatant is up to about 45 °C. It is well known that the cancer cells can be killed effectively when the temperature is raised to 40–60 °C within several minutes. Hence, we could infer that the photo-
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Fig. 7 The digital photograph of a 96-well HeLa cell-culture plate incubated with different concentrations of CuS NPs, CuS–DOX NPs, free DOX and pure DMEM without NIR irradiation (a); the infrared thermal photograph of a 96-well HeLa cell-culture plate incubated with CuS– DOX NPs after NIR irradiation for 5 min (b); in vitro cell viability of HeLa cells incubated with different concentrations of CuS NPs, CuS–DOX NPs, free DOX and pure DMEM for 24 h with and without NIR irradiation (c); the photograph of the mouse after intravenous injection with CuS– DOX NPs for 24 h (d); in vivo infrared thermal images of a tumorbearing mouse after injection of CuS–DOX NPs with NIR irradiation (e).
Fig. 6 Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with CuS–DOX NPs for 0.5 h, 1 h and 3 h at 37 °C without (a) and with NIR irradiation (b). Each series can be classified according to the nuclei of the cells (being dyed in blue by DAPI for visualization), DOX-conjugated CuS nanoparticles and overlay of both the channels.
thermal effects of the CuS NPs can be used to eliminate cancer cells efficiently through 808 nm laser irradiation. To evaluate and compare the cytotoxicities of CuS NPs, free DOX, CuS–DOX NPs with and without NIR irradiation in vitro, the cell viabilities were determined by MTT assay. Fig. 7c displays the in vitro HeLa cell viabilities of the above three groups with different concentrations after incubation for 24 h. As depicted, the viability of pure CuS NPs is higher than 90%, indicating the CuS NPs are nontoxic to cancer cells. After the free DOX and CuS–DOX NPs are added, the cell viabilities decrease to 38.3%–75.8% and 31.5%–66.0%, respectively. The killing cancer cell capacity of free DOX is lower than that of DOX-conjugated CuS NPs, which can be ascribed to the free DOX diffusing into cells whereas the nanoparticles are endocytosed by the cells. In order to keep the balance of the
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osmotic pressure, the extra and intracellular content of DOX are almost the same. In addition, the nanoparticles enter the cells by endocytosis, which can enhance the content and prolong residence time. To further investigate the drug delivery system for the combination of chemo-therapy and PTT, pure CuS NPs and CuS–DOX NPs were used for incubation with HeLa cells with and without NIR irradiation, respectively. It is clear that the CuS–DOX NPs show a much stronger ability against cancer cells after irradiation, indicating that the incorporative chemo-photothermal system is more effective than chemical therapy or PTT alone. The synergistic effect is probably ascribed to the elevated temperatures speeding up drug release and enhanced heat sensitivity of the cells exposed to DOX than those not. Fig. 7d shows the photograph of the mouse after intravenous injection with CuS–DOX NPs for 24 h. It can be found that the section of the tumor becomes dark after injection, indicating that the nanoparticles indeed reach the tumor, which can be ascribed to the remarkable dispersibility and homogeneity of the CuS–DOX NPs. During the treatment, the infrared thermal images of a tumor-bearing mouse are shown in Fig. 7e. The mouse was injected intravenously, and the images were acquired by using a R300SR-HD infrared camera (NEC) at different intervals under 808 nm NIR irradiation after 12 h. It is obvious that the
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corresponding irradiation tumor site changed from white to yellow, and then to red. The images reveal that the nanoparticles have an obvious photothermal effect by increasing the exposure time, by which property cancer cells could be killed effectively.
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In vivo anticancer therapy The synergistic effect of the CPTSs is studied in vivo. H22 tumor-bearing mice were injected intravenously with CuS NPs, and CuS–DOX NPs, which are treated with planned methods as given in the Experimental section. The mice were sacrificed after treatment for the 14th day. The photographs of the mice injected with CuS–DOX NPs (with and without NIR irradiation) and the tumors in different groups are given in Fig. 8a. It is remarkable that the tumor size of the mouse injected with CuS–DOX NPs and irradiated by the 808 nm laser is much smaller than the other groups, demonstrating that the combination of chemo-photothermal therapy had a good repressive effect and played a significant role during the treatment. In addition, CuS enhanced the temperature of the tumor site and accelerated the release of DOX at the same time under 808 nm NIR irradiation. Hence, the 808 nm laser was also a key factor in the treatment. According to Fig. 8b and c, it can be confirmed that the body weight of all the test groups did not decrease, which implies less systematic toxicity of the nanoparticles under different conditions. Fig. 8d presents the tumor histologic section under different treatment conditions. For the best inhibition group, the numbers of apoptotic cells and necrotic cells are much more than those of other groups.
Fig. 8 Photographs of excised tumors from representative mice after 14 day treatment and the digital photos of the mice after injection of the CuS–DOX NPs with and without NIR irradiation (a). The relative tumor volumes of H22 tumor-bearing mice (b) and the body weights in different groups after treatment (c). H&E stained tumor sections after 14 day treatment from different groups (d).
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Fig. 9 H&E stained images of the heart, liver, spleen, lung, and kidney collected from different treatment groups.
The number of cancer cells of the control group increases continually. On the basis of these obvious discrepancies, it can be assumed that the synergistic effect of chemo-photothermal therapy can kill cancer cells easily. To investigate the therapeutic effect and whether the agents result in any detrimental effect, we further carried out hematoxylin and eosin (H&E) staining analysis of the main organs, including the heart, liver, spleen, lung and kidney from different groups (Fig. 9). For the control group, an obvious inflammation appears in the liver and atrophy in the glomerulus, besides the lung appearing fibrotic. Nevertheless, compared with the control group, the organs of those with the best therapeutic effects exhibit the following phenomenon: there is no inflammation in the liver and the hepatocytes are normal without lesion. The glomerulus can be observed clearly. The other four groups show a sight inflammation in the liver, while the glomerulus is integrated. All the results confirm that the CPTSs are nontoxic to normal tissues and organs, and show its potential clinical applicability. In order to determine the biodistribution of the nanoparticles in vivo, H22 tumor-bearing mice injected with CuS– DOX NPs were euthanized at 12 h and 24 h. The concentration of Cu was measured by ICP-MS as given in Fig. 10. As the liver is the main organ of metabolism, a high concentration of Cu was detected in the liver. Nanoparticles accumulating in the spleen can be ascribed to the reason that the spleen plays the major roles in the immune system. As a crucial component of the urinary system, the kidney enriched plenty of minimal size CuS NPs. In addition, the tumor uptake was measured to be 19.4 and 12.9 μg g−1 at 24 h and 12 h, respectively. The accumulation may be attributed to the retention in the lesion location. In relative terms, the concentration of Cu in the heart and lung is much lower than in the other organs for all the times after injection. The results verify that the CuS–DOX NPs can reach and gather in the important organs, benefiting from the small size distribution and well-dispersion of the nanoparticles.
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dissolved in 180 mL of deionized water. Na2S·9H2O (20 mL, 2 mg mL−1) was added into the above solution with stirring at room temperature. After stirring for 5 min, the dark-brown reaction mixture was transferred to the water bath at a temperature of 90 °C and maintained for another 15 min until the color turned dark-green. And then, the solution was placed in ice-cold water immediately. Finally, carboxylic acid-functionalized CuS NPs (CuS-COONa) were separated by centrifugation and washed with deionized water three times, and then were dispersed in 30 mL water for further use.
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Synthesis of DOX-conjugated CuS NPs and DOX loading Fig. 10 Biodistribution of CuS–DOX NPs in mice determined by ICP-MS.
Conclusions In summary, we have successfully constructed a chemo-photothermal therapeutic system based on DOX-conjugated CuS NPs, which depend on the hydrazine bond as the cleavable link to control the DOX release under different pH values. This drug delivery system provides a high drug release rate with the aid of the photothermal effect produced by CuS under NIR irradiation in mildly acidic environments, which is beneficial for improving the effect of chemotherapy and photothermal ablation. In addition, the as-prepared nanoparticles show good dispersibility, favorable biocompatibility and high ablation efficiency to cancer cells benefiting from the ultrasmall size and the synergistic therapeutic effect of the CuS–DOX NPs. The endocytosis process of DOX-conjugated CuS NP tests on HeLa cancer cells in vitro indicates that the nanoparticles can enter into the cells. Meanwhile, NIR irradiation can promote the endocytosis process. The animal experiments and histologic section analysis verified that the CuS–DOX NPs can inhibit the growth of tumor, and avoid damage to the normal organs. Therefore, we believe that the as-synthesized DOX-conjugated CuS NPs with the combination of chemical and PTT have potential applications and a promising future in cancer therapy.
Experimental section Reagents and materials Copper(II) chloride (CuCl2·2H2O), sodium citrate, sodium sulfide (Na2S·9H2O), hydrazine monohydrate (N2H4·H2O) and methanol were purchased from Beijing Chemical Regent Co., Ltd. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Aldrich. All the above chemicals were used without further purification. Synthesis of carboxylic acid-functionalized CuS NPs CuS NPs were synthesized according to previous methods.44 Briefly, 34.1 mg of CuCl2 and 40 mg of sodium citrate were
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20 mg EDC, 20 mg NHS and 1 mL hydrazine monohydrate were added into the as-prepared CuS NP solution and stirred overnight. The products were obtained by centrifugation, washed with water three times and methanol twice and then dried at 60 °C for 12 h. After the above procedure, CuS-COONa nanoparticles were functionalized with the –NHNH2 group on the surface and then the CuS-CONHNH2 nanoparticles were obtained. Then, 20 mg of CuS-CONHNH2 NPs was dispersed in 10 mL of methanol by ultrasound. After being fully decentralized, 5 mg of DOX was added to the solution and stirred overnight in the dark. The precipitates were separated by centrifugation, washed with methanol until the solution turned colorless and were dried at 60 °C for 12 h. All the supernatants were collected for the evaluation of the drug loading efficiency by using a UV-vis spectrophotometer at 480 nm. The DOX loading capacity (DLC) was calculated by the following equation: DLC% ¼ ðT DOX SDOX Þ=T DOX 100% TDOX and SDOX are the contents of the total DOX used and the DOX in the supernatant respectively.70 Measurement of photothermal performance A 2 mL aqueous dispersion of CuS (62.6 μg mL−1) was introduced into a quartz cuvette and irradiated with an 808 nm NIR laser at a power density of 2.5 W cm−2 until the temperature did not increase. A thermocouple probe with an accuracy of 0.1 °C was inserted into the CuS aqueous solution perpendicular to the path of the laser. The temperature was recorded every 10 s. The laser was shut off when the temperature peaked, and then the solution cooled naturally. The photothermal conversion efficiency of CuS can be calculated (see ESI† for details). In vitro release of DOX-conjugated CuS NPs The resulting CuS–DOX NPs were dissolved in 5 mL of pH = 7.4, 5.5 and 4.0 phosphoric acidic buffer solution (PBS) with gentle shaking for testing the amount of drug release at 37 °C. At the selected time intervals, PBS was isolated by centrifugation to measure the concentration of the DOX in the supernatant and replaced with 5 mL of fresh buffer solution. The released content of the DOX in the supernatant was determined by using a UV-vis spectrophotometer at 480 nm.
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In vitro cell viability of CuS NPs The Dulbecco’s modified Eagle’s medium (DMEM) was prepared by adding 55 mL fetal bovine serum (FBS), 30 mg penicillin and 50 mg streptomycin to the 500 mL medium. About 8000 L929 fibroblast cells were cultured in 100 μL DMEM per well in a 96-well plate at 37 °C under a humidified 5% CO2 atmosphere for 24 h to make the cells attach to the plate. The CuS NPs were diluted at concentrations of 6.25, 12.5, 25, 50, 100 and 200 μg mL−1 respectively, and then 100 μL of solution with different densities were added to different columns of the plate, at the same time the last column was added to 100 μL DMEM free nanoparticles as control. The cells were incubated at 37 °C in 5% CO2 for 24 h. After that, 20 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (dispersed in the DMEM with a final concentration of 0.8 mg mL−1) was added into each well with continued cultivation for 4 h. And then the supernatant of incubation was removed and 150 mL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the 96-well plate was placed in a Versamax microplate reader, which is used to monitor the absorbance value at a wavelength of 490 nm (A is the absorbance value of the experimental groups and C is the absorbance value of the control group). The photoabsorbance values of the corresponding concentration for CuS NPs dissolved in DMSO solution were measured at the same time (B). The obtained results were calculated by the following equation: Cell Viability ¼ ðA BÞ=C 100% Cellular uptake assay of DOX-conjugated CuS NPs Cellular uptake was examined using a confocal laser scanning microscope (CLSM). HeLa cells were seeded into 6-well culture plates as a monolayer at 105 per well and grown overnight at 37 °C under 5% CO2. After being cultured, the cells were washed with PBS three times and then treated with the as-prepared CuS–DOX NPs (at a concentration of 500 μg mL−1) for 30 min, 1 h and 3 h, with and without 5 min NIR irradiation respectively. Thereafter, the HeLa cells were rinsed with PBS three times to remove any residual nanoparticles and fixed with 2.5% glutaraldehyde (1 mL per well) at 37 °C for 10 min, and then washed with PBS three times again. In order to perform the nucleus labeling, the nuclei were stained with DAPI solution (20 μg mL−1 in PBS, 1 mL per well) for 10 min, and then rinsed with PBS three times. The cover slips were placed on a glass microscope slide with glycerol and the samples were visualized using CLSM (Leica TCS SP8). In vitro cell cytotoxicity of DOX-conjugated CuS NPs In vitro cytotoxicity of the DOX-conjugated CuS NPs was assayed against HeLa cancer cells. The operation is very similar to the viability MTT assay. HeLa cells were cultured in a 96-well plate with a density of 8000 cells per well at 37 °C in 5% CO2 for 24 h. The 96-well HeLa cancer cells were divided into seven groups with the addition of different concentrations of CuS NPs (31.25, 62.5, 125, 250 and 500 μg mL−1), CuS–DOX NPs (31.25, 62.5, 125, 250 and 500 μg mL−1), free DOX (1.5625,
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3.125, 6.25, 12.5 and 25 μg mL−1) and pure DMEM respectively. Among them, the three substances of CuS NPs, CuS–DOX NPs and pure DMEM are treated with and without 808 nm NIR irradiation. After the cells were incubated with CuS NPs, and CuS–DOX NPs for 4 h, the groups of CuS NPs, CuS–DOX NPs and pure DMEM were irradiated with an 808 nm laser for 30 min (MW-GX-808/5000 mW, pump power of 1.0 W cm−2, 5 min break after 5 min irradiation). After that, the cells were incubated for a total of 24 h in the dark. At the end of the incubation, the cells were treated with 20 μL MTT solution (dispersed in the DMEM with a final concentration of 0.8 mg mL−1) and incubated for another 4 h. The subsequent procedure was the same as for the cell viability aforementioned. The supernatant was removed and washed with PBS, after that 150 μL was added to all the wells before the plate was examined using a microplate reader at 490 nm.
In vivo antitumor activity All animal operations were in accord with guidelines of the Institutional Animal Care and Use Committee and all anesthetization were performed by intraperitoneal injection of 10% chloral hydrate (100 μL). A suspension of H22 cells (the mouse hepatocellular carcinoma cells) was subcutaneously inoculated into the left armpit of the mice to establish tumors in mice. When the tumor size reached up to the mean volume of 100 mm3, the tumor-bearing mice were randomized into six groups (n = 6, each group). Before the treatment, the mice were weighed and the tumor dimensions were measured with a caliper. All the groups were treated by intravenous injection with saline (0.9 wt%, NaCl solution), CuS NPs and CuS–DOX NPs under different conditions. The mice in groups 1 and 2 were injected with saline, the 3rd and 5th groups were injected with CuS NPs and the 4th and 6th groups received the injection with CuS–DOX NPs. After 12 h, the 2nd, 5th and 6th groups were irradiated at the tumor site with an 808 nm laser for 15 min (1.0 W cm−2, with 5 min break after 5 min irradiation). The other groups were only injected with nanoparticles in the dark as control. The body weight and tumor size were monitored every two days during the treatment. The tumor volumes were calculated by the following equation: Tumor Volume ðVÞ ¼ ðTumor LengthÞ ðTumor WidthÞ2 =2: The relative tumor volume was calculated as V/V0 (V0 was the corresponding tumor volume when the treatment was initiated).
In vivo photothermal imaging of DOX-conjugated CuS NPs The tumor-bearing mice were intravenously injected with 100 μL of CuS–DOX NPs (500 μg mL−1). After 12 h, the thermal images at different times after exposure were recorded by using a R300SR-HD infrared camera (NEC) when the tumors were exposed to the 808 nm laser.
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Biodistribution of DOX-conjugated CuS NPs in mice In order to study the biodistribution of CuS–DOX NPs, ten tumor-bearing female Kunming mice were divided into two groups of five and intravenously injected with CuS–DOX NPs. The mice in the two groups were euthanized after 12 h and 24 h respectively. The major organs (heart, liver, spleen, lung and kidney) and tumors were collected and weighed. Then, all the tissues were dissolved in 10 mL of concentrated HCl and HNO3 (v/v = 3 : 1) at 70 °C and centrifuged to obtain a clear solution. The copper concentrations in the solutions at disparate injected intervals were determined by using an inductively coupled plasma optical emission spectrometer (ICP-MS), so that the content in each organ was calculated. Histology analysis The mice were sacrificed after two weeks from treatment. The major organs (heart, liver, spleen, lung and kidney) and tumors of the mice in all groups were dissected and dehydrated by buffer formalin, ethanol with different concentrations and xylene at room temperature for 24 h. And then, the sliced tissues were obtained after paraffin embedding and serial section. After that, 5 μm slices of the organs were stained with hematoxylin and eosin (H&E), and examined by CLSM. Characterization X-ray diffraction (XRD) measurement was examined on a Rigaku D/max-TTR-III diffractometer using monochromatic Cu Kα radiation (λ = 0.15405 nm). Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) micrographs were recorded on a FEI Tecnai G2 S-Twin transmission electron microscope equipped with a field emission gun operating at 200 kV. Fourier-transform infrared spectra were recorded on a Vertex Perkin-Elmer 580BIR spectrophotometer (Bruker) with the KBr pellet technique. The UV-vis adsorption spectral values were measured on a U-3100 spectrophotometer (Hitachi). All the measurements were performed at room temperature.
Acknowledgements Financial support from the National Natural Science Foundation of China (NSFC 21271053, 21401032, 51472058), the NCET in University (NCET-12-0622), the Natural Science Foundation of Heilongjiang Province (B201403), the Harbin Sci.Tech. Innovation Foundation (2014RFQXJ019) and the Fundamental Research Funds for the Central Universities of China is greatly acknowledged.
Notes and references 1 F. M. Kievit and M. Q. Zhang, Acc. Chem. Res., 2011, 44, 853–862.
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Paper
2 D. A. Gewirtz, M. L. Bristol and J. C. Yalowich, Curr. Opin. Invest. Drugs, 2010, 11, 612–614. 3 Z. Gu, L. Yan, G. Tian, S. Li, Z. Chai and Y. Zhao, Adv. Mater., 2013, 25, 3758–3779. 4 O. Veiseh, J. W. Gunn and M. Zhang, Adv. Drug Delivery Rev., 2010, 62, 284–304. 5 F. Lu, J. Wang, L. Yang and J. J. Zhu, Chem. Commun., 2015, 51, 9447–9450. 6 S. L. Gai, C. X. Li, P. P. Yang and J. Lin, Chem. Rev., 2014, 114, 2343–2389. 7 L. Cheng, J. Liu, X. Gu, H. Gong, X. Shi, T. Liu, C. Wang, X. Wang, G. Liu, H. Xing, W. Bu, B. Sun and Z. Liu, Adv. Mater., 2014, 26, 1886–1893. 8 J. Bai, Y. Liu and X. Jiang, Biomaterials, 2014, 35, 5805– 5813. 9 Q. W. Tian, J. Q. Hu, Y. H. Zhu, R. J. Zou, Z. G. Chen, S. P. Yang, R. W. Li, Q. Q. Su, Y. Han and X. G. Liu, J. Am. Chem. Soc., 2013, 135, 8571–8577. 10 L. Zhang, S. Gao, F. Zhang, K. Yang, Q. Ma and L. Zhu, ACS Nano, 2014, 8, 12250–12258. 11 R. C. Lv, P. P. Yang, F. He, S. L. Gai, C. X. Li, Y. L. Dai, G. X. Yang and J. Lin, ACS Nano, 2015, 9, 1630–1647. 12 C.-W. Hsiao, E.-Y. Chuang, H.-L. Chen, D. Wan, C. Korupalli, Z.-X. Liao, Y.-L. Chiu, W.-T. Chia, K.-J. Lin and H.-W. Sung, Biomaterials, 2015, 56, 26–35. 13 Y. Dai, H. Xiao, J. Liu, Q. Yuan, P. A. Ma, D. Yang, C. Li, Z. Cheng, Z. Hou, P. Yang and J. Lin, J. Am. Chem. Soc., 2013, 135, 18920–18929. 14 V. Shanmugam, S. Selvakumar and C.-S. Yeh, Chem. Soc. Rev., 2014, 43, 6254–6287. 15 W. R. Chen, R. L. Adams, R. Carubelli and R. E. Nordquist, Cancer Lett., 1997, 115, 25–30. 16 A. Vogeland and V. Venugopalan, Chem. Rev., 2003, 103, 577–644. 17 H. T. Wong, F. Vetrone, R. Naccache, H. L. W. Chan, J. H. Hao and J. A. Capobianco, J. Mater. Chem., 2011, 21, 16589–16596. 18 J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, V. Daniel and H. Dai, J. Am. Chem. Soc., 2011, 133, 6825–6831. 19 J. Chen, D. Wang, J. Xi, L. Au, A. Siekkinen, A. Warsen, Z.-Y. Li, H. Zhang, Y. Xia and X. Li, Nano Lett., 2007, 7, 1318–1322. 20 P. K. Jain, X. Huang, I. H. El-Sayed and M. A. El-Sayed, Acc. Chem. Res., 2008, 41, 1578–1586. 21 W. Dong, Y. Li, D. Niu, Z. Ma, J. Gu, Y. Chen, W. Zhao, X. Liu, C. Liu and J. Shi, Adv. Mater., 2011, 23, 5392– 5397. 22 B. Jang, J. Y. Park, C. H. Tung, I. H. Kim and Y. Choi, ACS Nano, 2011, 5, 1086–1094. 23 M. S. Yavuz, Y. Y. Cheng, J. Y. Chen, C. M. Cobley, Q. Zhang, M. Rycenga, J. W. Xie, C. Kim, K. H. Song, A. G. Schwartz, L. H. V. Wang and Y. N. Xia, Nat. Mater., 2009, 8, 935–939. 24 E. Boisselier and D. Astruc, Chem. Soc. Rev., 2009, 38, 1759– 1782.
Dalton Trans.
View Article Online
Published on 29 January 2016. Downloaded by test 3 on 09/03/2016 02:01:49.
Paper
25 X. Huang, P. K. Jain, I. H. El-Sayed and M. A. El-Sayed, Nanomedicine, 2007, 2, 681–693. 26 J. W. Fisher, S. Sarkar, C. F. Buchanan, C. S. Szot, J. Whitney, H. C. Hatcher, S. V. Torti, C. G. Rylander and M. N. Rylander, Cancer Res., 2010, 70, 9855–9864. 27 H. K. Moon, S. H. Lee and H. C. Choi, ACS Nano, 2009, 3, 3707–3713. 28 O. Akhavan, E. Ghaderi, S. Aghayee, Y. Fereydooni and A. Talebi, J. Mater. Chem., 2012, 22, 13773–13781. 29 O. Akhavan, E. Ghaderi and K. Rahimi, J. Mater. Chem., 2012, 22, 23260–23266. 30 Z. G. Chen, Q. Wang, H. L. Wang, L. S. Zhang, G. S. Song, L. L. Song, J. Q. Hu, H. Z. Wang, J. S. Liu, M. F. Zhu and D. Y. Zhao, Adv. Mater., 2013, 25, 2095–2100. 31 W. H. Li, R. Zamani, P. R. Gil, B. Pelaz, M. Ibanez, D. Cadavid, A. Shavel, R. A. Alvarez-Puebla, W. J. Parak, J. Arbiol and A. Cabot, J. Am. Chem. Soc., 2013, 135, 7098– 7101. 32 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. 33 S. B. Lakshmanan, X. J. Zou, M. Hossu, L. Ma, C. Yang and W. Chen, J. Biomed. Nanotechnol., 2012, 8, 883–890. 34 Q. H. Zeng, Y. L. Zhang, X. M. Liu, L. P. Tu, Y. Wang, X. G. Kong and H. Zhang, Chem. Commun., 2010, 46, 6479– 6481. 35 Y. L. Zhang, X. G. Kong, B. Xue, Q. H. Zeng, X. M. Liu, L. P. Tu, K. Liu and H. Zhang, J. Mater. Chem. C, 2013, 1, 6355–6363. 36 Z. Zhang, L. Wang, J. Wang, X. Jiang, X. Li, Z. Hu, Y. Ji, X. Wu and C. Chen, Adv. Mater., 2012, 24, 1418–1423. 37 Y. Xia, W. Li, C. M. Cobley, J. Chen, X. Xia, Q. Zhang, M. Yang, E. C. Cho and P. K. Brown, Acc. Chem. Res., 2011, 44, 914–924. 38 K. Yang, S. Zhang, G. Zhang, X. Sun, S.-T. Lee and Z. Liu, Nano Lett., 2010, 10, 3318–3323. 39 S. Goel, F. Chen and W. Cai, Small, 2014, 10, 631–645. 40 L. Guo, D. D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, B. Yan and W. Lu, ACS Nano, 2014, 8, 5670–5681. 41 G. Ku, M. Zhou, S. Song, Q. Huang, J. Hazle and C. Li, ACS Nano, 2012, 6, 7489–7496. 42 Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang and J. Hu, Adv. Mater., 2011, 23, 3542– 3547. 43 Q. W. Tian, F. R. Jiang, R. J. Zou, Q. Liu, Z. G. Chen, M. F. Zhu, S. P. Yang, J. L. Wang, J. H. Wang and J. Q. Hu, ACS Nano, 2011, 5, 9761–9771. 44 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. 45 X. J. Liu, F. F. Fu, K. B. Xu, R. J. Zou, J. M. Yang, Q. Wang, Q. Liu, Z. Y. Xiao and J. Q. Hu, J. Mater. Chem. B, 2014, 2, 5358–5367.
Dalton Trans.
Dalton Transactions
46 Y. Li, W. Lu, Q. Huang, M. Huang, C. Li and W. Chen, Nanomedicine, 2010, 5, 1161–1171. 47 Z. Xiao, Nanomedicine, 2014, 9, 373–375. 48 J. M. Luther, P. K. Jain, T. Ewers and A. P. Alivisatos, Nat. Mater., 2011, 10, 361–366. 49 H. R. Liu, W. Lu, H. B. Wang, L. Rao, Z. G. Yi, S. J. Zeng and J. H. Hao, Nanoscale, 2013, 5, 6023–6029. 50 X. J. Xue, F. Wang and X. G. Liu, J. Mater. Chem., 2011, 21, 13107–13127. 51 Y. Qu, X. R. Zhang, Y. Q. Wu, F. Y. Li and J. L. Hua, Polym. Chem., 2014, 5, 3396–3403. 52 K. Yang, L. Zhu, L. Nie, X. Sun, L. Cheng, C. Wu, G. Niu, X. Chen and Z. Liu, Theranostics, 2014, 4, 134–141. 53 Y. Dai, D. Yang, P. A. Ma, X. Kang, X. Zhang, C. Li, Z. Hou, Z. Cheng and J. Lin, Biomaterials, 2012, 33, 8704–8713. 54 Y. H. Tian, S. P. Li, J. Song, T. J. Ji, M. T. Zhu, G. J. Anderson, J. Y. Wei and G. J. Nie, Biomaterials, 2014, 35, 2383–2390. 55 K. Li, Q. Su, W. Yuan, B. Tian, B. Shen, Y. Li, W. Feng and F. Li, ACS Appl. Mater. Interfaces, 2015, 7, 12278–12286. 56 F. Liu, X. He, Z. Lei, L. Liu, J. Zhang, H. You, H. Zhang and Z. Wang, Adv. Healthcare Mater., 2015, 4, 559–568. 57 G. Yang, H. Gong, T. Liu, X. Sun, L. Cheng and Z. Liu, Biomaterials, 2015, 60, 62–71. 58 Y. Yang, J. Aw, K. Chen, F. Liu, P. Padmanabhan, Y. Hou, Z. Cheng and B. Xing, Chem. – Asian J., 2011, 6, 1381–1389. 59 Y. Yang, B. Velmurugan, X. Liu and B. Xing, Small, 2013, 9, 2937–2944. 60 J. Mou, P. Li, C. Liu, H. Xu, L. Song, J. Wang, K. Zhang, Y. Chen, J. Shi and H. Chen, Small, 2015, 11, 2275–2283. 61 F. Chen, H. Hong, S. Goel, S. A. Graves, H. Orbay, E. B. Ehlerding, S. Shi, C. P. Theuer, R. J. Nickles and W. Cai, ACS Nano, 2015, 9, 3926–3934. 62 N. Nasongkla, E. Bey, J. M. Ren, H. Ai, C. Khemtong, J. S. Guthi, S. F. Chin, A. D. Sherry, D. A. Boothman and J. M. Gao, Nano Lett., 2006, 6, 2427–2430. 63 S. S. Banerjee and D.-H. Chen, Nanotechnology, 2008, 19, 505104. 64 M. Mahmoudi, H. Hosseinkhani, M. Hosseinkhani, S. Boutry, A. Simchi, W. S. Journeay, K. Subramani and S. Laurent, Chem. Rev., 2011, 111, 253–280. 65 I. Canton and G. Battaglia, Chem. Soc. Rev., 2012, 41, 2718– 2739. 66 Y. Liu, K. Ai, J. Liu, M. Deng, H. He and L. Lu, Adv. Mater., 2013, 25, 1353–1359. 67 D. K. Roper, W. Ahn and M. Hoepfner, J. Phys. Chem. C, 2007, 111, 3636–3641. 68 J. Liu, C. Wang, X. Wang, X. Wang, L. Cheng, Y. Li and Z. Liu, Adv. Funct. Mater., 2015, 25, 384–392. 69 D. Yang, G. X. Yang, X. M. Wang, R. C. Lv, S. L. Gai, F. He, G. Arif and P. P. Yang, Nanoscale, 2015, 7, 12180–12191. 70 K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452–4458.
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Doxorubicin-conjugated CuS nanoparticles for efficient synergistic therapy triggered by near-infrared light† Huiting Bi, Yunlu Dai,* Ruichan Lv, Chongna Zhong, Fei He, Shili Gai, Arif Gulzar, Guixin Yang and Piaoping Yang* To integrate photothermal therapy (PTT) with chemotherapy for improving anticancer efficiency, we developed a novel and multifunctional doxorubicin (DOX) conjugated copper sulfide nanoparticle (CuS– DOX NP) drug delivery system using hydrazone bonds to conjugate carboxyl-functionalized copper sulfide nanoparticles (CuS NPs) and DOX. On the other hand, the hydrazone bonds could be used for improving the DOX release rate (88.0%) by cleavage in a mildly acidic environment irradiated by 808 nm laser light, which could greatly promote chemo-therapeutic efficacy. Simultaneously, CuS NPs which can absorb near infrared (NIR) light produce a clear thermal effect, giving rise to a synergistic therapeutic effect combined with enhanced chemo-therapy. The DOX-conjugated CuS NPs display an evident in vitro cytotoxicity to HeLa cancer cells under 808 nm light irradiation. High tumor inhibition efficacy has
Received 11th December 2015, Accepted 29th January 2016
been achieved after 14 day in vivo treatment, performed with intravenous administration of CuS–DOX
DOI: 10.1039/c5dt04842g
NPs with 808 nm laser irradiation on H22 tumor-bearing mice. The multifunctional system which was achieved by a facile route should be a potential candidate in the anti-cancer field due to the synergistic
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therapeutic effect, which is superior to any single approach.
Introduction Currently, surgical resection and traditional chemotherapy are still the major therapeutic approaches to cancer treatment. However, it cannot be ignored that the above two methods may cause adverse effects to normal cells and tissues, insufficient dosage to diseased regions, as well as an increased incidence of the second cancer.1–5 PTT, based on near-infrared (NIR, λ = 700–1100 nm) laser induced photothermal ablation, has shown rapid development in recent years as a minimally invasive and harmless therapeutic approach in biotechnological applications.6–14 The NIR laser can convert optical energy into thermal energy, and possesses the desirable penetration depth in biological tissues.15–18 Recently, the combined chemo- and photothermal therapeutic systems (CPTSs) have provided new ideas for cancer therapy.19,20 In particular, it has been demonstrated that PTT integrated with chemotherapy
Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin, 150001, P. R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5dt04842g
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results in improved treatment efficacy relative to the use of each approach independently. To date, photothermal therapeutic systems have been established based on several types of photothermal agents including noble metal nanostructures,21–25 carbon-based 26–29 materials and semiconductor compounds.30–32 For some gold (Au) nanostructures, including Au nanorods, nanostars and nanoshells, their NIR absorption peaks would decrease after laser radiation because of low photostability.33–37 In addition, the majority of these photothermal drugs that have been engineered based on several noble metals such as Au and Pd are very expensive, and the synthesis processes turn out to be very complicated.22 Carbon nanomaterials have shown a synergistic effect on cancer,8,38 but they have poor photothermal conversion efficiency. Hence, it is worthwhile to explore the evolution of multifunctional nanomaterials with low cost, easy synthesis and excellent PTT efficacy. Among various candidates for the carriers of anticancer agents, CuS NPs, with a unique nature such as low cost, low cytotoxicity, high stability and outstanding photothermal efficiency under an 808 nm laser, have attracted increasing attention from biomedical researchers across the globe.39–45 Furthermore, the absorption wavelength of CuS NPs is not influenced by the surrounding environment compared to the Au nanoparticles. CuS NPs possess a maximum absorbance at 900 nm located at the NIR
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range, which can be ascribed to the d–d energy band transition of Cu2+ ions, while the absorption from gold nanostructures results from surface plasmon resonance.46–48 Emission coming from the NIR laser beam at 808 nm leads to the increased temperature of CuS NPs, which could be exploited as the foundation for PTT. It is well known that a series of functional nanoparticles conjugated with an anticancer drug have achieved good results in biological applications.49–53 Numerous photothermal agents are obtained by complete surface modification and synthetic processes. Herein, we designed a chemo-photothermal therapeutic system, that is DOX conjugated CuS nanoparticles. CuS NPs synthesized by a simple method were utilized as drug vehicles. DOX, a commonly used anticancer drug,54–59 was conjugated to CuS NPs through chemical bonds. In this work, the chemo-photothermal therapeutic system has at least three outstanding features. Firstly, the therapeutic system can be easily obtained due to the facile synthetic process. Secondly, owing to the ultra-small size, CuS NPs could disperse homogenously in aqueous solution, which was beneficial for drug delivery in vivo and improved the therapeutic effect.60,61 Thirdly, the content of DOX linked to CuS NPs is as high as 88.5% for the reliability of the chemical bonding. The drug release property in vitro, cytotoxicity, cellular uptake and therapeutic effect in vivo have been investigated in detail to certify the feasible anti-cancer application of this system.
Results and discussion Synthesis, characterization and drug release in vitro The procedure for the formation of CuS–DOX NPs is illustrated in Scheme 1. Firstly, carboxylic acid-functionalized CuS NPs were synthesized at 90 °C in a water bath. Then, the carboxyl group reacts with hydrazine monohydrate to generate CuSCONHNH2, and then the –NHNH2 further reacted with the ketone group of the DOX to produce CuS–DOX NPs. The hydrazone bond (red dotted line labelled in CuS–DOX NP structure) connected CuS with DOX, which was cleavable in a weakly acidic environment to release DOX.62,63 As illustrated in Scheme 2, the CuS–DOX NPs were injected into the mice and accumulate at the tumor site by the enhanced permeability and retention (EPR) effect. In physiological terms, small molecules (such as H2O) and ions (such as Na+, K+) enter the cells by means of diffusion, and the nanoparticles enter the cells by
Scheme 1 DOX NPs.
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Schematic illustration of the preparation process of CuS–
Scheme 2 Nanoparticles enter cancer cells by endocytosis and release DOX under 808 nm NIR irradiation in a facile acid environment.
endocytosis.64,65 It can be speculated that CuS–DOX NPs enter the cancer cells by endocytosis, and release DOX in a weak acid environment by cleaving the hydrazone bond between CuS and DOX. Finally, DOX and the photothermal effect by 808 nm NIR irradiation kill the cancer cells together. The TEM images with different magnifications of CuS NPs are shown in Fig. 1a and b. It is clearly seen that the nanoparticles are relatively uniform and dispersed with an average diameter around 5 nm. After being modified with hydrazine and conjugated with DOX on the surface, the nanoparticles still maintain their morphology and the size is almost unchanged. In addition, the nanocrystals show high dispersity and have no aggregation in the solution (Fig. 1c, d and S1†). The XRD measurement is explored to understand the evolution and crystallization of CuS NPs, and the pattern is shown in Fig. 1f. All of the X-ray diffraction peaks of the sample could be indexed to the hexagonal phase of CuS and the lattice parameters are in agreement with those of the standard powder diffraction pattern of CuS (JCPDS no. 06-0464). No obvious impure peaks appear in the pattern, indicating that high purity of CuS NPs was acquired. Fig. 1e shows the high-resolution TEM (HRTEM) of CuS NPs and indicates that the CuS NPs are of high crystallization. From the image, two typical interlayer spacings of 0.30 nm and 0.25 nm could be observed, corresponding to the (102) and (103) planes of the hexagonalstructured-CuS, respectively. Fig. 2a shows the temperature change of the CuS NP aqueous solution and deionized water versus irradiation time, and a good photothermal effect is presented. After irradiation for 600 s, the temperature of the CuS aqueous solution was increased by 31.9 °C at a concentration of 60.6 μg mL−1. In contrast, the temperature of deionized water increased by only 10.3 °C. The temperature increased to 45 °C approximately after 600 s, which is in favour of PTT and can kill cancer cells after maintenance for 15 min. Fig. S2† shows the linear time
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Fig. 3 FT-IR spectra of CuS NPs (a); CuS-CONHNH2 NPs (b); and CuS– DOX NPs (c).
Fig. 1 TEM images of CuS NPs with different magnifications (a, b); CuSCONHNH2 NPs (c); CuS–DOX NPs (d); HRTEM image of CuS NPs (e); XRD pattern of the CuS NPs and the standard JCPDS card 06-0464 of CuS (f).
Fig. 2 Temperature change of CuS NP aqueous solution and deionized water versus irradiation time (a); the photothermal response of the CuS aqueous solution with NIR irradiation and then with the laser shut off (b).
data versus −ln θ obtained from the cooling period of Fig. 2b. The photothermal conversion efficiency (η) was calculated (see ESI† for details) by the following equation: η¼
hAΔTmax Qs Ið1 10Aλ Þ
According to the equation, the η value of CuS was determined to be 31.1%. The photothermal conversion efficiency is higher than that of the Au nanorods (24.0%), which are widely used for photothermal therapy.66,67
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To study the surface properties of the nanoparticles, FT-IR spectra were employed and the results are shown in Fig. 3. The bands at 3428 cm−1 and 1110 cm−1 in the spectra of CuSCOONa, CuS-CONHNH2, and CuS–DOX are assigned to the –OH stretching and bending vibrations. The peak at 1625 cm−1 can be attributed to the stretching vibrations of the carboxyl groups. After the amidation of the carboxyl group by hydrazine monohydrate, the band at 1646 cm−1 in Fig. 3b is attributed to the characteristic peak of the –CO–NH– bond. As shown in Fig. 3c, the band at 1633 cm−1 can be ascribed to the –CvN– bond from the reaction between the –NHNH2 group of nanoparticles and the ketone group at the 13-keto position of DOX, which is the signature of the grafting of DOX onto CuS. Furthermore, the peaks at 1558 cm−1 and 1412 cm−1 derived from the absorption of DOX. Zeta potentials of CuS NPs, CuSCONHNH2 NPs and CuS–DOX NPs are obtained, which show the shift of the isoelectric point (Fig. S3†). According to the FT-IR spectra and zeta potentials, we can draw the conclusion that the surface functional groups have changed and DOX has been successfully conjugated to the surface of CuS NPs. Fig. 4a shows the UV-vis spectra of CuS-COONa, CuSCONHNH2 and CuS–DOX solution with the same concentration of 62.6 μg mL−1 for Cu2+. The absorption of CuSCOONa NPs exhibits a peak at 850 nm and the absorption of CuS-CONHNH2 also shows a similar peak, indicating that the conjugation process amination does not affect the structure of CuS. Meanwhile, the broad absorption band in the NIR region is observed at 850 nm, which is attributed to a characteristic of the CuS NPs demonstrating its potential use as a PTT agent. According to the results of UV-vis spectra , when NIR excitation at 808 nm is potentially used as the radiation source, which can increase penetration depth and suppress unwanted overheating, the peak of CuS–DOX NPs at around 480 nm in the UV-vis spectrum could be attributed to the strong absorption of DOX covering up the peak of CuS. The results of UV-vis tests can further confirm that DOX has been successfully conjugated on the surface of the CuS NPs. The drug loading efficien-
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Fig. 5 The L929 fibroblast cell viability after incubating with different concentrations of CuS nanoparticles for 24 h and the quantitative assay by the standard MTT method.
Fig. 4 The UV-vis absorption spectra of CuS NPs, CuS-CONHNH2 NPs and CuS–DOX NPs (a); DOX release efficiency of CuS–DOX at pH 4.0, 5.5 and 7.4 PBS buffers (b).
cies and release abilities of the CuS–DOX NPs through the acid-dependent cleavage of the hydrazone bond were examined by the UV-vis spectrum at 480 nm at different pH values and different times. Fig. 4b exhibits the release profiles of the DOX from CuS– DOX NPs in PBS buffer solutions at pH 4.0, 5.5 and 7.4 at 37 °C. It can be seen that the drug release rate of DOX at pH 4.0 is faster than that at pH 5.0 and 7.4. The drug release rate increases by decreasing the pH value. About 88.0% and 70.0% of DOX released from the CuS–DOX NPs is at pH 4.0 and 5.5, respectively. By comparison, only about 23.0% of DOX is released under neutral ( pH 7.4) in 24 h. It can be elucidated that the behavior of pH triggered DOX release may be attributable to the cleavage of the hydrazone bond, which is stable in a normal physiological environment but is quickly cleavable in an acidic environment ( pH 4.0–5.5). Transmission and release of the DOX by CuS–DOX is through intravenous injection into blood circulation, which belongs to the internal environment, and the nanoparticles would not enter acidic tissues such as the stomach in an external experiment. It is well known that the micro-environments of tumor tissue are acidic, so CuS– DOX NPs with pH triggered drug-release properties are promising to be used as an anti-cancer platform by changing the pH, which is important and beneficial for cancer therapy.63 In vitro compatibility and cell uptake It is essential to evaluate the biocompatibility of CuS NPs before using the particles for cancer therapy both in vitro and
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in vivo. The standard MTT assay was carried out on L929 fibroblast cells and the results were monitored by using a Versamax microplate reader at 490 nm. As shown in Fig. 5, the cell viability is higher than 80% after incubation with CuS NPs in a wide range of concentrations (from 6.25 to 200 μg mL−1) for 24 h. It can be demonstrated that the nanoparticles have good biocompatibility as drug carriers for biomedical application. The cell uptake process was measured after the incubation of CuS–DOX NPs with HeLa cancer cells for 0.5 h, 1 h and 3 h with and without NIR irradiation by CLSM. The blue fluorescence from the DAPI is used to mark the nucleus and the red emission from DOX is for tracking the carrier, respectively. The merging of the two channels also occurs correspondingly. In Fig. 6a, during the first 0.5 h, only a few nanoparticles could be taken up by HeLa cells. Subsequently, it is clear that there is a stronger red emission of DOX observed in both the cytoplasm and the nucleus, suggesting that more particles appear in the cells with enhanced incubation time. In Fig. 6b, after NIR exposure for 5 min, more particles are remarkably swallowed by the cells compared to those without NIR at the same time point. The enhanced DOX fluorescence is obvious, which is due to the reason that it was easier for the particles to be taken in by the cells and release DOX after incidence of the NIR laser. This phenomenon can be attributed to the photothermal effect of CuS NPs, which can increase the temperature of the CPTSs, and promote nanoparticles entering the cells.68,69
Toxicity and photothermal effect in vitro and in vivo To explore the toxicity and photothermal effect of nanoparticles, a series of experiments were employed in vitro by incubating HeLa cells with CuS NPs, CuS–DOX, free DOX and pure DMEM respectively. Fig. 7a and b show a photograph and infrared image of the HeLa cells after being irradiated by an 808 nm laser for 5 min in a 96-well plate. The thermal image shows that the temperature of the HeLa cell supernatant is up to about 45 °C. It is well known that the cancer cells can be killed effectively when the temperature is raised to 40–60 °C within several minutes. Hence, we could infer that the photo-
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Fig. 7 The digital photograph of a 96-well HeLa cell-culture plate incubated with different concentrations of CuS NPs, CuS–DOX NPs, free DOX and pure DMEM without NIR irradiation (a); the infrared thermal photograph of a 96-well HeLa cell-culture plate incubated with CuS– DOX NPs after NIR irradiation for 5 min (b); in vitro cell viability of HeLa cells incubated with different concentrations of CuS NPs, CuS–DOX NPs, free DOX and pure DMEM for 24 h with and without NIR irradiation (c); the photograph of the mouse after intravenous injection with CuS– DOX NPs for 24 h (d); in vivo infrared thermal images of a tumorbearing mouse after injection of CuS–DOX NPs with NIR irradiation (e).
Fig. 6 Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with CuS–DOX NPs for 0.5 h, 1 h and 3 h at 37 °C without (a) and with NIR irradiation (b). Each series can be classified according to the nuclei of the cells (being dyed in blue by DAPI for visualization), DOX-conjugated CuS nanoparticles and overlay of both the channels.
thermal effects of the CuS NPs can be used to eliminate cancer cells efficiently through 808 nm laser irradiation. To evaluate and compare the cytotoxicities of CuS NPs, free DOX, CuS–DOX NPs with and without NIR irradiation in vitro, the cell viabilities were determined by MTT assay. Fig. 7c displays the in vitro HeLa cell viabilities of the above three groups with different concentrations after incubation for 24 h. As depicted, the viability of pure CuS NPs is higher than 90%, indicating the CuS NPs are nontoxic to cancer cells. After the free DOX and CuS–DOX NPs are added, the cell viabilities decrease to 38.3%–75.8% and 31.5%–66.0%, respectively. The killing cancer cell capacity of free DOX is lower than that of DOX-conjugated CuS NPs, which can be ascribed to the free DOX diffusing into cells whereas the nanoparticles are endocytosed by the cells. In order to keep the balance of the
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osmotic pressure, the extra and intracellular content of DOX are almost the same. In addition, the nanoparticles enter the cells by endocytosis, which can enhance the content and prolong residence time. To further investigate the drug delivery system for the combination of chemo-therapy and PTT, pure CuS NPs and CuS–DOX NPs were used for incubation with HeLa cells with and without NIR irradiation, respectively. It is clear that the CuS–DOX NPs show a much stronger ability against cancer cells after irradiation, indicating that the incorporative chemo-photothermal system is more effective than chemical therapy or PTT alone. The synergistic effect is probably ascribed to the elevated temperatures speeding up drug release and enhanced heat sensitivity of the cells exposed to DOX than those not. Fig. 7d shows the photograph of the mouse after intravenous injection with CuS–DOX NPs for 24 h. It can be found that the section of the tumor becomes dark after injection, indicating that the nanoparticles indeed reach the tumor, which can be ascribed to the remarkable dispersibility and homogeneity of the CuS–DOX NPs. During the treatment, the infrared thermal images of a tumor-bearing mouse are shown in Fig. 7e. The mouse was injected intravenously, and the images were acquired by using a R300SR-HD infrared camera (NEC) at different intervals under 808 nm NIR irradiation after 12 h. It is obvious that the
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corresponding irradiation tumor site changed from white to yellow, and then to red. The images reveal that the nanoparticles have an obvious photothermal effect by increasing the exposure time, by which property cancer cells could be killed effectively.
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In vivo anticancer therapy The synergistic effect of the CPTSs is studied in vivo. H22 tumor-bearing mice were injected intravenously with CuS NPs, and CuS–DOX NPs, which are treated with planned methods as given in the Experimental section. The mice were sacrificed after treatment for the 14th day. The photographs of the mice injected with CuS–DOX NPs (with and without NIR irradiation) and the tumors in different groups are given in Fig. 8a. It is remarkable that the tumor size of the mouse injected with CuS–DOX NPs and irradiated by the 808 nm laser is much smaller than the other groups, demonstrating that the combination of chemo-photothermal therapy had a good repressive effect and played a significant role during the treatment. In addition, CuS enhanced the temperature of the tumor site and accelerated the release of DOX at the same time under 808 nm NIR irradiation. Hence, the 808 nm laser was also a key factor in the treatment. According to Fig. 8b and c, it can be confirmed that the body weight of all the test groups did not decrease, which implies less systematic toxicity of the nanoparticles under different conditions. Fig. 8d presents the tumor histologic section under different treatment conditions. For the best inhibition group, the numbers of apoptotic cells and necrotic cells are much more than those of other groups.
Fig. 8 Photographs of excised tumors from representative mice after 14 day treatment and the digital photos of the mice after injection of the CuS–DOX NPs with and without NIR irradiation (a). The relative tumor volumes of H22 tumor-bearing mice (b) and the body weights in different groups after treatment (c). H&E stained tumor sections after 14 day treatment from different groups (d).
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Fig. 9 H&E stained images of the heart, liver, spleen, lung, and kidney collected from different treatment groups.
The number of cancer cells of the control group increases continually. On the basis of these obvious discrepancies, it can be assumed that the synergistic effect of chemo-photothermal therapy can kill cancer cells easily. To investigate the therapeutic effect and whether the agents result in any detrimental effect, we further carried out hematoxylin and eosin (H&E) staining analysis of the main organs, including the heart, liver, spleen, lung and kidney from different groups (Fig. 9). For the control group, an obvious inflammation appears in the liver and atrophy in the glomerulus, besides the lung appearing fibrotic. Nevertheless, compared with the control group, the organs of those with the best therapeutic effects exhibit the following phenomenon: there is no inflammation in the liver and the hepatocytes are normal without lesion. The glomerulus can be observed clearly. The other four groups show a sight inflammation in the liver, while the glomerulus is integrated. All the results confirm that the CPTSs are nontoxic to normal tissues and organs, and show its potential clinical applicability. In order to determine the biodistribution of the nanoparticles in vivo, H22 tumor-bearing mice injected with CuS– DOX NPs were euthanized at 12 h and 24 h. The concentration of Cu was measured by ICP-MS as given in Fig. 10. As the liver is the main organ of metabolism, a high concentration of Cu was detected in the liver. Nanoparticles accumulating in the spleen can be ascribed to the reason that the spleen plays the major roles in the immune system. As a crucial component of the urinary system, the kidney enriched plenty of minimal size CuS NPs. In addition, the tumor uptake was measured to be 19.4 and 12.9 μg g−1 at 24 h and 12 h, respectively. The accumulation may be attributed to the retention in the lesion location. In relative terms, the concentration of Cu in the heart and lung is much lower than in the other organs for all the times after injection. The results verify that the CuS–DOX NPs can reach and gather in the important organs, benefiting from the small size distribution and well-dispersion of the nanoparticles.
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dissolved in 180 mL of deionized water. Na2S·9H2O (20 mL, 2 mg mL−1) was added into the above solution with stirring at room temperature. After stirring for 5 min, the dark-brown reaction mixture was transferred to the water bath at a temperature of 90 °C and maintained for another 15 min until the color turned dark-green. And then, the solution was placed in ice-cold water immediately. Finally, carboxylic acid-functionalized CuS NPs (CuS-COONa) were separated by centrifugation and washed with deionized water three times, and then were dispersed in 30 mL water for further use.
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Synthesis of DOX-conjugated CuS NPs and DOX loading Fig. 10 Biodistribution of CuS–DOX NPs in mice determined by ICP-MS.
Conclusions In summary, we have successfully constructed a chemo-photothermal therapeutic system based on DOX-conjugated CuS NPs, which depend on the hydrazine bond as the cleavable link to control the DOX release under different pH values. This drug delivery system provides a high drug release rate with the aid of the photothermal effect produced by CuS under NIR irradiation in mildly acidic environments, which is beneficial for improving the effect of chemotherapy and photothermal ablation. In addition, the as-prepared nanoparticles show good dispersibility, favorable biocompatibility and high ablation efficiency to cancer cells benefiting from the ultrasmall size and the synergistic therapeutic effect of the CuS–DOX NPs. The endocytosis process of DOX-conjugated CuS NP tests on HeLa cancer cells in vitro indicates that the nanoparticles can enter into the cells. Meanwhile, NIR irradiation can promote the endocytosis process. The animal experiments and histologic section analysis verified that the CuS–DOX NPs can inhibit the growth of tumor, and avoid damage to the normal organs. Therefore, we believe that the as-synthesized DOX-conjugated CuS NPs with the combination of chemical and PTT have potential applications and a promising future in cancer therapy.
Experimental section Reagents and materials Copper(II) chloride (CuCl2·2H2O), sodium citrate, sodium sulfide (Na2S·9H2O), hydrazine monohydrate (N2H4·H2O) and methanol were purchased from Beijing Chemical Regent Co., Ltd. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were obtained from Aldrich. All the above chemicals were used without further purification. Synthesis of carboxylic acid-functionalized CuS NPs CuS NPs were synthesized according to previous methods.44 Briefly, 34.1 mg of CuCl2 and 40 mg of sodium citrate were
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20 mg EDC, 20 mg NHS and 1 mL hydrazine monohydrate were added into the as-prepared CuS NP solution and stirred overnight. The products were obtained by centrifugation, washed with water three times and methanol twice and then dried at 60 °C for 12 h. After the above procedure, CuS-COONa nanoparticles were functionalized with the –NHNH2 group on the surface and then the CuS-CONHNH2 nanoparticles were obtained. Then, 20 mg of CuS-CONHNH2 NPs was dispersed in 10 mL of methanol by ultrasound. After being fully decentralized, 5 mg of DOX was added to the solution and stirred overnight in the dark. The precipitates were separated by centrifugation, washed with methanol until the solution turned colorless and were dried at 60 °C for 12 h. All the supernatants were collected for the evaluation of the drug loading efficiency by using a UV-vis spectrophotometer at 480 nm. The DOX loading capacity (DLC) was calculated by the following equation: DLC% ¼ ðT DOX SDOX Þ=T DOX 100% TDOX and SDOX are the contents of the total DOX used and the DOX in the supernatant respectively.70 Measurement of photothermal performance A 2 mL aqueous dispersion of CuS (62.6 μg mL−1) was introduced into a quartz cuvette and irradiated with an 808 nm NIR laser at a power density of 2.5 W cm−2 until the temperature did not increase. A thermocouple probe with an accuracy of 0.1 °C was inserted into the CuS aqueous solution perpendicular to the path of the laser. The temperature was recorded every 10 s. The laser was shut off when the temperature peaked, and then the solution cooled naturally. The photothermal conversion efficiency of CuS can be calculated (see ESI† for details). In vitro release of DOX-conjugated CuS NPs The resulting CuS–DOX NPs were dissolved in 5 mL of pH = 7.4, 5.5 and 4.0 phosphoric acidic buffer solution (PBS) with gentle shaking for testing the amount of drug release at 37 °C. At the selected time intervals, PBS was isolated by centrifugation to measure the concentration of the DOX in the supernatant and replaced with 5 mL of fresh buffer solution. The released content of the DOX in the supernatant was determined by using a UV-vis spectrophotometer at 480 nm.
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In vitro cell viability of CuS NPs The Dulbecco’s modified Eagle’s medium (DMEM) was prepared by adding 55 mL fetal bovine serum (FBS), 30 mg penicillin and 50 mg streptomycin to the 500 mL medium. About 8000 L929 fibroblast cells were cultured in 100 μL DMEM per well in a 96-well plate at 37 °C under a humidified 5% CO2 atmosphere for 24 h to make the cells attach to the plate. The CuS NPs were diluted at concentrations of 6.25, 12.5, 25, 50, 100 and 200 μg mL−1 respectively, and then 100 μL of solution with different densities were added to different columns of the plate, at the same time the last column was added to 100 μL DMEM free nanoparticles as control. The cells were incubated at 37 °C in 5% CO2 for 24 h. After that, 20 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) solution (dispersed in the DMEM with a final concentration of 0.8 mg mL−1) was added into each well with continued cultivation for 4 h. And then the supernatant of incubation was removed and 150 mL of dimethyl sulfoxide (DMSO) was added to each well. Finally, the 96-well plate was placed in a Versamax microplate reader, which is used to monitor the absorbance value at a wavelength of 490 nm (A is the absorbance value of the experimental groups and C is the absorbance value of the control group). The photoabsorbance values of the corresponding concentration for CuS NPs dissolved in DMSO solution were measured at the same time (B). The obtained results were calculated by the following equation: Cell Viability ¼ ðA BÞ=C 100% Cellular uptake assay of DOX-conjugated CuS NPs Cellular uptake was examined using a confocal laser scanning microscope (CLSM). HeLa cells were seeded into 6-well culture plates as a monolayer at 105 per well and grown overnight at 37 °C under 5% CO2. After being cultured, the cells were washed with PBS three times and then treated with the as-prepared CuS–DOX NPs (at a concentration of 500 μg mL−1) for 30 min, 1 h and 3 h, with and without 5 min NIR irradiation respectively. Thereafter, the HeLa cells were rinsed with PBS three times to remove any residual nanoparticles and fixed with 2.5% glutaraldehyde (1 mL per well) at 37 °C for 10 min, and then washed with PBS three times again. In order to perform the nucleus labeling, the nuclei were stained with DAPI solution (20 μg mL−1 in PBS, 1 mL per well) for 10 min, and then rinsed with PBS three times. The cover slips were placed on a glass microscope slide with glycerol and the samples were visualized using CLSM (Leica TCS SP8). In vitro cell cytotoxicity of DOX-conjugated CuS NPs In vitro cytotoxicity of the DOX-conjugated CuS NPs was assayed against HeLa cancer cells. The operation is very similar to the viability MTT assay. HeLa cells were cultured in a 96-well plate with a density of 8000 cells per well at 37 °C in 5% CO2 for 24 h. The 96-well HeLa cancer cells were divided into seven groups with the addition of different concentrations of CuS NPs (31.25, 62.5, 125, 250 and 500 μg mL−1), CuS–DOX NPs (31.25, 62.5, 125, 250 and 500 μg mL−1), free DOX (1.5625,
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3.125, 6.25, 12.5 and 25 μg mL−1) and pure DMEM respectively. Among them, the three substances of CuS NPs, CuS–DOX NPs and pure DMEM are treated with and without 808 nm NIR irradiation. After the cells were incubated with CuS NPs, and CuS–DOX NPs for 4 h, the groups of CuS NPs, CuS–DOX NPs and pure DMEM were irradiated with an 808 nm laser for 30 min (MW-GX-808/5000 mW, pump power of 1.0 W cm−2, 5 min break after 5 min irradiation). After that, the cells were incubated for a total of 24 h in the dark. At the end of the incubation, the cells were treated with 20 μL MTT solution (dispersed in the DMEM with a final concentration of 0.8 mg mL−1) and incubated for another 4 h. The subsequent procedure was the same as for the cell viability aforementioned. The supernatant was removed and washed with PBS, after that 150 μL was added to all the wells before the plate was examined using a microplate reader at 490 nm.
In vivo antitumor activity All animal operations were in accord with guidelines of the Institutional Animal Care and Use Committee and all anesthetization were performed by intraperitoneal injection of 10% chloral hydrate (100 μL). A suspension of H22 cells (the mouse hepatocellular carcinoma cells) was subcutaneously inoculated into the left armpit of the mice to establish tumors in mice. When the tumor size reached up to the mean volume of 100 mm3, the tumor-bearing mice were randomized into six groups (n = 6, each group). Before the treatment, the mice were weighed and the tumor dimensions were measured with a caliper. All the groups were treated by intravenous injection with saline (0.9 wt%, NaCl solution), CuS NPs and CuS–DOX NPs under different conditions. The mice in groups 1 and 2 were injected with saline, the 3rd and 5th groups were injected with CuS NPs and the 4th and 6th groups received the injection with CuS–DOX NPs. After 12 h, the 2nd, 5th and 6th groups were irradiated at the tumor site with an 808 nm laser for 15 min (1.0 W cm−2, with 5 min break after 5 min irradiation). The other groups were only injected with nanoparticles in the dark as control. The body weight and tumor size were monitored every two days during the treatment. The tumor volumes were calculated by the following equation: Tumor Volume ðVÞ ¼ ðTumor LengthÞ ðTumor WidthÞ2 =2: The relative tumor volume was calculated as V/V0 (V0 was the corresponding tumor volume when the treatment was initiated).
In vivo photothermal imaging of DOX-conjugated CuS NPs The tumor-bearing mice were intravenously injected with 100 μL of CuS–DOX NPs (500 μg mL−1). After 12 h, the thermal images at different times after exposure were recorded by using a R300SR-HD infrared camera (NEC) when the tumors were exposed to the 808 nm laser.
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Biodistribution of DOX-conjugated CuS NPs in mice In order to study the biodistribution of CuS–DOX NPs, ten tumor-bearing female Kunming mice were divided into two groups of five and intravenously injected with CuS–DOX NPs. The mice in the two groups were euthanized after 12 h and 24 h respectively. The major organs (heart, liver, spleen, lung and kidney) and tumors were collected and weighed. Then, all the tissues were dissolved in 10 mL of concentrated HCl and HNO3 (v/v = 3 : 1) at 70 °C and centrifuged to obtain a clear solution. The copper concentrations in the solutions at disparate injected intervals were determined by using an inductively coupled plasma optical emission spectrometer (ICP-MS), so that the content in each organ was calculated. Histology analysis The mice were sacrificed after two weeks from treatment. The major organs (heart, liver, spleen, lung and kidney) and tumors of the mice in all groups were dissected and dehydrated by buffer formalin, ethanol with different concentrations and xylene at room temperature for 24 h. And then, the sliced tissues were obtained after paraffin embedding and serial section. After that, 5 μm slices of the organs were stained with hematoxylin and eosin (H&E), and examined by CLSM. Characterization X-ray diffraction (XRD) measurement was examined on a Rigaku D/max-TTR-III diffractometer using monochromatic Cu Kα radiation (λ = 0.15405 nm). Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) micrographs were recorded on a FEI Tecnai G2 S-Twin transmission electron microscope equipped with a field emission gun operating at 200 kV. Fourier-transform infrared spectra were recorded on a Vertex Perkin-Elmer 580BIR spectrophotometer (Bruker) with the KBr pellet technique. The UV-vis adsorption spectral values were measured on a U-3100 spectrophotometer (Hitachi). All the measurements were performed at room temperature.
Acknowledgements Financial support from the National Natural Science Foundation of China (NSFC 21271053, 21401032, 51472058), the NCET in University (NCET-12-0622), the Natural Science Foundation of Heilongjiang Province (B201403), the Harbin Sci.Tech. Innovation Foundation (2014RFQXJ019) and the Fundamental Research Funds for the Central Universities of China is greatly acknowledged.
Notes and references 1 F. M. Kievit and M. Q. Zhang, Acc. Chem. Res., 2011, 44, 853–862.
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Paper
2 D. A. Gewirtz, M. L. Bristol and J. C. Yalowich, Curr. Opin. Invest. Drugs, 2010, 11, 612–614. 3 Z. Gu, L. Yan, G. Tian, S. Li, Z. Chai and Y. Zhao, Adv. Mater., 2013, 25, 3758–3779. 4 O. Veiseh, J. W. Gunn and M. Zhang, Adv. Drug Delivery Rev., 2010, 62, 284–304. 5 F. Lu, J. Wang, L. Yang and J. J. Zhu, Chem. Commun., 2015, 51, 9447–9450. 6 S. L. Gai, C. X. Li, P. P. Yang and J. Lin, Chem. Rev., 2014, 114, 2343–2389. 7 L. Cheng, J. Liu, X. Gu, H. Gong, X. Shi, T. Liu, C. Wang, X. Wang, G. Liu, H. Xing, W. Bu, B. Sun and Z. Liu, Adv. Mater., 2014, 26, 1886–1893. 8 J. Bai, Y. Liu and X. Jiang, Biomaterials, 2014, 35, 5805– 5813. 9 Q. W. Tian, J. Q. Hu, Y. H. Zhu, R. J. Zou, Z. G. Chen, S. P. Yang, R. W. Li, Q. Q. Su, Y. Han and X. G. Liu, J. Am. Chem. Soc., 2013, 135, 8571–8577. 10 L. Zhang, S. Gao, F. Zhang, K. Yang, Q. Ma and L. Zhu, ACS Nano, 2014, 8, 12250–12258. 11 R. C. Lv, P. P. Yang, F. He, S. L. Gai, C. X. Li, Y. L. Dai, G. X. Yang and J. Lin, ACS Nano, 2015, 9, 1630–1647. 12 C.-W. Hsiao, E.-Y. Chuang, H.-L. Chen, D. Wan, C. Korupalli, Z.-X. Liao, Y.-L. Chiu, W.-T. Chia, K.-J. Lin and H.-W. Sung, Biomaterials, 2015, 56, 26–35. 13 Y. Dai, H. Xiao, J. Liu, Q. Yuan, P. A. Ma, D. Yang, C. Li, Z. Cheng, Z. Hou, P. Yang and J. Lin, J. Am. Chem. Soc., 2013, 135, 18920–18929. 14 V. Shanmugam, S. Selvakumar and C.-S. Yeh, Chem. Soc. Rev., 2014, 43, 6254–6287. 15 W. R. Chen, R. L. Adams, R. Carubelli and R. E. Nordquist, Cancer Lett., 1997, 115, 25–30. 16 A. Vogeland and V. Venugopalan, Chem. Rev., 2003, 103, 577–644. 17 H. T. Wong, F. Vetrone, R. Naccache, H. L. W. Chan, J. H. Hao and J. A. Capobianco, J. Mater. Chem., 2011, 21, 16589–16596. 18 J. T. Robinson, S. M. Tabakman, Y. Liang, H. Wang, H. S. Casalongue, V. Daniel and H. Dai, J. Am. Chem. Soc., 2011, 133, 6825–6831. 19 J. Chen, D. Wang, J. Xi, L. Au, A. Siekkinen, A. Warsen, Z.-Y. Li, H. Zhang, Y. Xia and X. Li, Nano Lett., 2007, 7, 1318–1322. 20 P. K. Jain, X. Huang, I. H. El-Sayed and M. A. El-Sayed, Acc. Chem. Res., 2008, 41, 1578–1586. 21 W. Dong, Y. Li, D. Niu, Z. Ma, J. Gu, Y. Chen, W. Zhao, X. Liu, C. Liu and J. Shi, Adv. Mater., 2011, 23, 5392– 5397. 22 B. Jang, J. Y. Park, C. H. Tung, I. H. Kim and Y. Choi, ACS Nano, 2011, 5, 1086–1094. 23 M. S. Yavuz, Y. Y. Cheng, J. Y. Chen, C. M. Cobley, Q. Zhang, M. Rycenga, J. W. Xie, C. Kim, K. H. Song, A. G. Schwartz, L. H. V. Wang and Y. N. Xia, Nat. Mater., 2009, 8, 935–939. 24 E. Boisselier and D. Astruc, Chem. Soc. Rev., 2009, 38, 1759– 1782.
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25 X. Huang, P. K. Jain, I. H. El-Sayed and M. A. El-Sayed, Nanomedicine, 2007, 2, 681–693. 26 J. W. Fisher, S. Sarkar, C. F. Buchanan, C. S. Szot, J. Whitney, H. C. Hatcher, S. V. Torti, C. G. Rylander and M. N. Rylander, Cancer Res., 2010, 70, 9855–9864. 27 H. K. Moon, S. H. Lee and H. C. Choi, ACS Nano, 2009, 3, 3707–3713. 28 O. Akhavan, E. Ghaderi, S. Aghayee, Y. Fereydooni and A. Talebi, J. Mater. Chem., 2012, 22, 13773–13781. 29 O. Akhavan, E. Ghaderi and K. Rahimi, J. Mater. Chem., 2012, 22, 23260–23266. 30 Z. G. Chen, Q. Wang, H. L. Wang, L. S. Zhang, G. S. Song, L. L. Song, J. Q. Hu, H. Z. Wang, J. S. Liu, M. F. Zhu and D. Y. Zhao, Adv. Mater., 2013, 25, 2095–2100. 31 W. H. Li, R. Zamani, P. R. Gil, B. Pelaz, M. Ibanez, D. Cadavid, A. Shavel, R. A. Alvarez-Puebla, W. J. Parak, J. Arbiol and A. Cabot, J. Am. Chem. Soc., 2013, 135, 7098– 7101. 32 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. 33 S. B. Lakshmanan, X. J. Zou, M. Hossu, L. Ma, C. Yang and W. Chen, J. Biomed. Nanotechnol., 2012, 8, 883–890. 34 Q. H. Zeng, Y. L. Zhang, X. M. Liu, L. P. Tu, Y. Wang, X. G. Kong and H. Zhang, Chem. Commun., 2010, 46, 6479– 6481. 35 Y. L. Zhang, X. G. Kong, B. Xue, Q. H. Zeng, X. M. Liu, L. P. Tu, K. Liu and H. Zhang, J. Mater. Chem. C, 2013, 1, 6355–6363. 36 Z. Zhang, L. Wang, J. Wang, X. Jiang, X. Li, Z. Hu, Y. Ji, X. Wu and C. Chen, Adv. Mater., 2012, 24, 1418–1423. 37 Y. Xia, W. Li, C. M. Cobley, J. Chen, X. Xia, Q. Zhang, M. Yang, E. C. Cho and P. K. Brown, Acc. Chem. Res., 2011, 44, 914–924. 38 K. Yang, S. Zhang, G. Zhang, X. Sun, S.-T. Lee and Z. Liu, Nano Lett., 2010, 10, 3318–3323. 39 S. Goel, F. Chen and W. Cai, Small, 2014, 10, 631–645. 40 L. Guo, D. D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, B. Yan and W. Lu, ACS Nano, 2014, 8, 5670–5681. 41 G. Ku, M. Zhou, S. Song, Q. Huang, J. Hazle and C. Li, ACS Nano, 2012, 6, 7489–7496. 42 Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang and J. Hu, Adv. Mater., 2011, 23, 3542– 3547. 43 Q. W. Tian, F. R. Jiang, R. J. Zou, Q. Liu, Z. G. Chen, M. F. Zhu, S. P. Yang, J. L. Wang, J. H. Wang and J. Q. Hu, ACS Nano, 2011, 5, 9761–9771. 44 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. 45 X. J. Liu, F. F. Fu, K. B. Xu, R. J. Zou, J. M. Yang, Q. Wang, Q. Liu, Z. Y. Xiao and J. Q. Hu, J. Mater. Chem. B, 2014, 2, 5358–5367.
Dalton Trans.
Dalton Transactions
46 Y. Li, W. Lu, Q. Huang, M. Huang, C. Li and W. Chen, Nanomedicine, 2010, 5, 1161–1171. 47 Z. Xiao, Nanomedicine, 2014, 9, 373–375. 48 J. M. Luther, P. K. Jain, T. Ewers and A. P. Alivisatos, Nat. Mater., 2011, 10, 361–366. 49 H. R. Liu, W. Lu, H. B. Wang, L. Rao, Z. G. Yi, S. J. Zeng and J. H. Hao, Nanoscale, 2013, 5, 6023–6029. 50 X. J. Xue, F. Wang and X. G. Liu, J. Mater. Chem., 2011, 21, 13107–13127. 51 Y. Qu, X. R. Zhang, Y. Q. Wu, F. Y. Li and J. L. Hua, Polym. Chem., 2014, 5, 3396–3403. 52 K. Yang, L. Zhu, L. Nie, X. Sun, L. Cheng, C. Wu, G. Niu, X. Chen and Z. Liu, Theranostics, 2014, 4, 134–141. 53 Y. Dai, D. Yang, P. A. Ma, X. Kang, X. Zhang, C. Li, Z. Hou, Z. Cheng and J. Lin, Biomaterials, 2012, 33, 8704–8713. 54 Y. H. Tian, S. P. Li, J. Song, T. J. Ji, M. T. Zhu, G. J. Anderson, J. Y. Wei and G. J. Nie, Biomaterials, 2014, 35, 2383–2390. 55 K. Li, Q. Su, W. Yuan, B. Tian, B. Shen, Y. Li, W. Feng and F. Li, ACS Appl. Mater. Interfaces, 2015, 7, 12278–12286. 56 F. Liu, X. He, Z. Lei, L. Liu, J. Zhang, H. You, H. Zhang and Z. Wang, Adv. Healthcare Mater., 2015, 4, 559–568. 57 G. Yang, H. Gong, T. Liu, X. Sun, L. Cheng and Z. Liu, Biomaterials, 2015, 60, 62–71. 58 Y. Yang, J. Aw, K. Chen, F. Liu, P. Padmanabhan, Y. Hou, Z. Cheng and B. Xing, Chem. – Asian J., 2011, 6, 1381–1389. 59 Y. Yang, B. Velmurugan, X. Liu and B. Xing, Small, 2013, 9, 2937–2944. 60 J. Mou, P. Li, C. Liu, H. Xu, L. Song, J. Wang, K. Zhang, Y. Chen, J. Shi and H. Chen, Small, 2015, 11, 2275–2283. 61 F. Chen, H. Hong, S. Goel, S. A. Graves, H. Orbay, E. B. Ehlerding, S. Shi, C. P. Theuer, R. J. Nickles and W. Cai, ACS Nano, 2015, 9, 3926–3934. 62 N. Nasongkla, E. Bey, J. M. Ren, H. Ai, C. Khemtong, J. S. Guthi, S. F. Chin, A. D. Sherry, D. A. Boothman and J. M. Gao, Nano Lett., 2006, 6, 2427–2430. 63 S. S. Banerjee and D.-H. Chen, Nanotechnology, 2008, 19, 505104. 64 M. Mahmoudi, H. Hosseinkhani, M. Hosseinkhani, S. Boutry, A. Simchi, W. S. Journeay, K. Subramani and S. Laurent, Chem. Rev., 2011, 111, 253–280. 65 I. Canton and G. Battaglia, Chem. Soc. Rev., 2012, 41, 2718– 2739. 66 Y. Liu, K. Ai, J. Liu, M. Deng, H. He and L. Lu, Adv. Mater., 2013, 25, 1353–1359. 67 D. K. Roper, W. Ahn and M. Hoepfner, J. Phys. Chem. C, 2007, 111, 3636–3641. 68 J. Liu, C. Wang, X. Wang, X. Wang, L. Cheng, Y. Li and Z. Liu, Adv. Funct. Mater., 2015, 25, 384–392. 69 D. Yang, G. X. Yang, X. M. Wang, R. C. Lv, S. L. Gai, F. He, G. Arif and P. P. Yang, Nanoscale, 2015, 7, 12180–12191. 70 K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452–4458.
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