doxorubicin@SiO2 nanocomposites

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Available at www.sciencedirect.com

ScienceDirect journal homepage: www.elsevier.com/locate/carbon

Efficient cancer ablation by combined photothermal and enhanced chemo-therapy based on carbon nanoparticles/doxorubicin@SiO2 nanocomposites Xiaolong Tu a,1, Lina Wang a,b,1, Yuhua Cao Mengxin Zhang a,d, Zhijun Zhang a,*

a,c

, Yufei Ma a, He Shen a,

a

Key Laboratory for Nano-Bio Interface, Division of Nanobiomedicine & Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, 398 Ruoshui Road, Suzhou 215123, China b School of Life Sciences, Shanghai University, 99 Shangda Road, Shanghai 200444, China c College of Life and Health Sciences, Northeastern University, 3-11 Wenhua Road, Shenyang 110819, China d University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China

A R T I C L E I N F O

A B S T R A C T

Article history:

The combination of different treatments in cancer therapy has drawn massive attention in

Received 21 February 2015

the last decades due to its superior anticancer ability to single treatment. One of the most

Accepted 9 May 2015

useful strategies to achieve this purpose is to design and construct efficient multifunctional

Available online 21 May 2015

nanoplatforms. Here we report our effort in development of carbon nanoparticles/doxorubicin@SiO2 nanocomposites and their application for combined photothermal and chemotherapy in cancer ablation. The nanocomposites are obtained via reverse microemulsion method with controlled size and high drug loading ratio. These nanocomposites possess high heat-generating ability, pH responsive drug delivery, and heat-induced high drug release as well. In vitro experiment reveals that the combined photothermal and chemotherapy exhibits much higher toxicity to 4T1 cells than photothermal therapy or chemotherapy alone. In vivo experiment reveals that compared with single treatment, the combined photothermal and chemo-therapy can effectively inhibit tumor growth and destroy it eventually without cancer recurrence. The current research demonstrates that carbon nanoparticles/doxorubicin@SiO2 nanocomposites can be used as an efficient nanoplatform for combined cancer photothermal and chemo-therapy.  2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Cancer, a major threat to human health, has long been a torturing disease and still remains a tremendous challenge, although much effort has been made to fight against it [1–3]. Chemotherapy, which uses chemical substances as anticancer * Corresponding author. E-mail address: [email protected] (Z. Zhang). 1 These authors contributed equally to this manuscript. http://dx.doi.org/10.1016/j.carbon.2015.05.043 0008-6223/ 2015 Elsevier Ltd. All rights reserved.

drugs to destroy cancers, is an important strategy widely applied in nowadays clinical practices [4,5]. Unfortunately, due to lack of cell specificity, these drugs may kill both cancerous and normal cells, leading to serious adverse effects [6]. Chemotherapy also has its limitation that it does not always work, and even when useful, it may not completely destroy

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cancers. Drug resistance, developing over time as cancers adapt to and overcome the cytotoxicity of drugs, is a major cause of treatment failure in chemotherapy [7]. A long tough road still lies ahead before achievement of successful administration of chemotherapy. As a nascent cancer treatment, photothermal therapy (PTT), using hyperthermia generated by electromagnetic radiation (most often in near infrared wavelength) to kill cancers, has been proven to be an effective method in cancer therapy [8,9]. PTT can be applied selectively and locally at cancer sites while not affecting normal tissues, and therefore is a noninvasive and safe strategy in cancer therapy. Light sensitizers, such as noble metals, carbon materials and organic polymers and molecules, are often used to enhance the light absorption and thus improve the PTT efficacy [8–18]. Although previous studies demonstrate that with the help of the above light sensitive materials PTT is efficient in cancer therapy, single PTT cannot always eradicate cancers completely, leading to cancer recurrence [19]. The failure caused by single treatment in cancer therapy propels researchers to explore the feasibility of combined treatment such as combined photothermal and chemotherapy. Study shows that heat can enhance the effect of chemotherapy [20]. Inspired by this result, researchers pay increasing attention to the combination of PTT and chemotherapy in cancer treatments [21–26]. Chen et al. [21] demonstrated the success of mesoporous silica-coated gold nanorods for drug delivery as well as hyperthermia therapy and imaging in cancer cell lines. Guo et al. [22] reported that the doxorubicin (Dox) loaded PEGylated graphene oxide (GO) resulted in complete destruction of the tumors, while Dox alone or photothermal treatment of GO without Dox did not. Zhang et al. [23] found that the conjugation of Docetaxel to single wall carbon nanotubes together with RGD peptide showed high efficacy in suppressing tumor growth. The aforementioned work provides a useful technology for selective delivery of both heat and drugs to a tumor region, and thereby improving the overall effectiveness of cancer therapy for optimal clinical outcome. Recently, our group reported that carbon nanoparticles (CNPs) derived from clinically used activated carbon possess strong photothermal conversion ability and therefore show highly efficient cancer ablation [27]. The combined therapy based on CNPs and anticancer drugs, however, has not been investigated yet. Reverse microemulsion, which is a thermodynamically stable system of oil, water, surfactant and sometimes cosurfactant, is an effective way to synthesize functional nanoparticles [28]. In a typical reverse microemulsion, the aqueous phase is confined in uniform isolated nanoscale water droplets distributing in the continuous domain of the oil phase, the size of which can be readily controlled by adjusting the ratio of water against oil. These water droplets act as nanoreactors to produce nanoparticles with a narrow size distribution. For nanoparticles mixture, the composition can be easily controlled by adjusting the ratio of the reactants. Silica is often introduced in this method to form a stable structure by wrapping nanoparticles in it [29]. Considering the benefits of reverse microemulsion method in preparing CNPs and anticancer drug complex to explore its efficacy of combined PTT and chemotherapy, in this work, we prepared silica coated CNPs/Dox nanocomposites

(CNPs/Dox@SiO2 NCs) with controlled size and components via reverse microemulsion approach. The CNPs/Dox@SiO2 NCs possess, we revealed, strong heat-generating ability. Drug delivery experiment reveals that the CNPs/Dox@SiO2 NCs exhibit both pH and heat responsive drug release behavior. Cellular uptake experiment reveals an efficient and time-dependent cellular internalization of CNPs/Dox@SiO2 NCs. Furthermore, in vitro and in vivo experiments demonstrate the excellent cancer ablation ability of CNPs/Dox@SiO2 NCs. The strategy of material synthesis and combined PTT and chemotherapy is illustrated in Fig. 1. Our research demonstrates the as-synthesized CNPs/Dox@SiO2 NCs are promising cancer ablation nanoplatforms for combined PTT and chemotherapy.

2.

Experiment

2.1.

Materials and characterization

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tetraethoxysilane (TEOS), penicillin, and streptomycin were purchased from Sigma. Doxorubicin hydrochloride was purchased from Sagon Biotech. Activated carbon and other reagents were bought from Sinopharm Chemical Reagent Company and used as received without further purification. Transmission electron microscopy (TEM) images were obtained from an FEI Tecnai G2 F20 S-Twin transmission electron microscope. UV–Vis spectra were collected with a Shimadzu UV-2550 spectrophotometer. Fluorescence images were collected on a Nikon A1 confocal laser scanning microscope under 488-nm excitation.

2.2. Preparation Dox@SiO2 NCs

of

CNPs,

CNPs@SiO2

and

CNPs/

CNPs were prepared according to our previous work [27]. Briefly, 0.3 g of activated carbon was added in a 100 mL flask containing 15 mL of concentrated H2SO4 and 5 mL of fuming HNO3. After being sonicated for 10 min the suspension was heated to 120 C and kept for 5 min under vigorous stirring. Then the mixture was cooled down in ice water, diluted with 100 mL of deionized (DI) H2O and neutralized to pH 1–2 with NaHCO3. The mixture was centrifuged (12,000 rpm, 30 min) to collect CNPs. Eventually the obtained CNPs were washed thoroughly with DI water and further purified via spin dialysis (100 kDa cutoff, 4000 rpm). CNPs/Dox@SiO2 NCs were synthesized via a reverse microemulsion method. Briefly, 20 lL of CNPs solution (10 mg/mL) was added into a mixture containing 7.5 mL of cyclohexane, 1.8 mL of n-hexanol and 1.77 mL of Triton X100 under vigorous stirring, followed by adding 20 lL of Dox hydrochloride solution (10 mg/mL) 10 min later. After 30 min stirring, 8 lL of 25% (wt) ammonium hydroxide was added in the mixture for another 30 min stirring. 100 lL of 19:1 (v/v) cyclohexane/TEOS was added dropwise in four equal shares every 30 min, and the mixture was stirred further for 24 h before 10 mL of acetone was added. The mixture was then sonicated for 10 min and centrifuged (10,000 rpm, 5 min) to collect CNPs/Dox@SiO2 NCs which were then washed by ethanol three times and easily dissolved in DI water.

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Fig. 1 – Schematic diagram showing the strategy for the synthesis and combined photothermal and chemo-therapy of CNPs/ Dox@SiO2 NCs. (A colour version of this figure can be viewed online.)

The synthesis of CNPs@SiO2 was similar to that of CNPs/Dox@SiO2 NCs except the addition of Dox.

2.3.

Photothermal effect of CNPs/Dox@SiO2 NCs

To investigate the photothermal effect of CNPs/Dox@SiO2 NCs, 0.2 mL aqueous solutions of different concentrations (6.25, 12.5, 25 and 50 lg/mL) were irradiated under an NIR laser (808 nm, 4 W/cm2) for 5 min, respectively. A digital thermometer was used to measure the temperature during irradiation.

2.4.

Drug release of CNPs/Dox@SiO2 NCs

Drug release was conducted as follows: three dialysis bags (8– 14kD cutoff) each containing 1 mL of concentrated CNPs/Dox@SiO2 solution were put into three 1 L PBS solutions with pH 5.5, pH 7.4 and pH 5.5 +NIR irradiation, respectively. 50 lL of CNPs/Dox@SiO2 NCs from above three samples was collected at time points of 0, 0.5, 1, 2, 4, 8 and 24 h. A 4 W/cm2 808-nm laser was used to irradiate CNPs/Dox@SiO2 NCs at time points of 0.5, 2 and 8 h. After irradiation another 50 lL of CNPs/Dox@SiO2 NCs was collected. Finally the amount of Dox was determined by UV–Vis absorption at 500 nm.

2.5.

Cell culture

4T1 murine breast cancer cells were cultured in regular Roswell Park Memorial Institute media (RPMI) 1640 medium (Corning) containing 10% fetal bovine serum (FBS, Hyclone), penicillin (100 U/mL) and streptomycin (100 U/mL) under 37 C and 5% CO2 in a humidified atmosphere incubator.

2.6.

MTT assay

4T1 cells were seeded into 96-well plates at a density of 9 · 103 each well and maintained for 24 h. The cells were incubated PBS buffer (control), free Dox, CNPs@SiO2 or CNPs/Dox@SiO2 NCs of different CNPs concentrations (3, 6, 12, 25, 50 and 100 lg/mL) for 24 h in RPMI 1640 containing 10% FBS. After

that, 20 lL of MTT solution (5 mg/mL in PBS) was added to each well for 4 h, and 150 lL of dimethyl sulfoxide was then added to each well to replace the culture medium and dissolve the insoluble formazan crystals. The absorbance of each well was measured using a PerkinElmer Victor 4 microplate reader at 490 nm.

2.7.

Cellular uptake of CNPs/Dox@SiO2 NCs

4T1 cells were seeded into each well of chambered coverglass at a density of 1.5 · 104 and incubated with PBS buffer (control) and CNPs/Dox@SiO2 NCs (CNPs concentration: 50 lg/mL) after 24 h seeding. After 1, 6 and 18 h incubation, the medium was removed and the cells were washed 3 times using PBS. Fluorescence images were collected on a Nikon A1 confocal laser scanning microscope under 488-nm excitation immediately after PBS washing.

2.8. Photothermal treatment of 4T1 cells with CNPs/ Dox@SiO2 NCs 4T1 cells were seeded into each well of a 96-well plate at a density of 9 · 103. After 24 h incubation, the medium was replaced with 200 lL of fresh medium containing PBS buffer (control), CNPs@SiO2 and CNPs/Dox@SiO2 NCs of different CNPs concentrations (3, 6, 12, 25, 50 and 100 lg/mL). After 24 h incubation, the cells were then irradiated for 5 min with an NIR laser (808 nm, 4 W/cm2). The initial temperature of each well was maintained at 37 C with an electrical hotplate. After irradiation, the cell viability was evaluated using MTT assay.

2.9.

Xenograft tumor models

4T1 cells were cultured in the standard medium recommended by American type culture collection. Balb/c mice at an average age of 6–7 weeks (18–22 g) were obtained from Suzhou Industrial Park Animal Technology Co., Ltd. All animal experiments were performed under the protocols approved by Soochow University Laboratory Animal Center. The 4T1 tumor models were generated by subcutaneous

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injection of 2 · 106 4T1 cells (in 50 lL FBS free culture medium) at the right hind leg of Balb/c mice. The mice were available for further experiments once average tumor volume reached 100 mm3.

hematoxylin and eosin (H&E) staining, the slices were observed using an optical microscope.

3.

Results and discussion

2.10. NCs

3.1. NCs

The synthesis and drug loading of CNPs/Dox@SiO2

Photothermal treatment of mice with CNPs/Dox@SiO2

The 4T1 tumor bearing mice were randomly divided into six groups with five mice each. When the average tumor volume reached 100 mm3, the mice were anaesthetized and locally injected 50 lL of PBS, CNPs@SiO2 or CNPs/Dox@SiO2 NCs solution (20 mg/kg of CNPs) into their tumors. The mice were irradiated with an NIR laser (808 nm, 2 W/cm2) for 5 min after 15 min post-injection. An IR thermal camera was used to monitor the temperature change of the tumor site. The tumor size was measured by a caliper every other day and calculated as the volume = (tumor length) · (tumor width)2/2. Relative tumor volumes were calculated as V/V0 (V0 was the tumor volume when the treatment was initiated).

2.11.

Histopathological assessment

After treatment, organs (heart, liver, spleen, kidney and lung) of the mice were harvested, fixed in 4% formalin solution, processed routinely into paraffin and sectioned. After

5–10 nm spherical-like CNPs were prepared via a fast chemical oxidation method reported in our recent work [27]. CNPs/Dox@SiO2 NCs were synthesized via a reverse microemulsion method in which the size of materials can be well controlled within the confined aqueous droplets. TEM result (Fig. 2a) indicated that the size of the assynthesized CNPs/Dox@SiO2 NCs is mainly distributed in the range of 20–30 nm, much larger than the free CNPs (5– 10 nm) (Fig. 2b). Besides the size control, ratio of CNPs against Dox can be controlled by adjusting amounts of CNPs and Dox added (Fig. S1). In the UV–Vis spectrum of CNPs/Dox@SiO2 NCs (Fig. 2d), a typical absorption peak at about 500 nm assignable to Dox is observed, indicating the successful introduction of Dox. The amounts of Dox and CNPs were calculated by using home-made standard curves of Dox and CNPs, respectively. Nearly all CNPs are integrated in the NCs. 80% Dox is integrated due to the part dissolving of Dox in oil phase. The ratio of drug against photothermal

Fig. 2 – TEM images of (a) CNPs, (b) CNPs/Dox@SiO2 NCs and (c) CNPs@SiO2 and (d) UV–Vis spectra of CNPs, Dox and CNPs/ Dox@SiO2 NCs in aqueous solution. (A colour version of this figure can be viewed online.)

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agent is much higher than that in previous reports [21,30], showing that reverse microemulsion method is a good way to achieve high drug payload.

3.2. The photothermal effect and drug release behavior of CNPs/Dox@SiO2 NCs Previous study showed that coating of the SiO2 shell may spoil the heat-producing ability of gold nanorods [21]. The feasibility of CNPs/Dox@SiO2 NCs in photothermal therapy is still unknown, although we have demonstrated that CNPs exhibit strong heat-producing ability. Upon irradiation using an 808nm laser, the 200 lL of CNPs/Dox@SiO2 NCs solutions at a series of concentrations (6.25, 12.5, 25 and 50 lg/mL) experienced a temperature increase within 5 min, shown in Fig. 3a. Compared with control (PBS, irradiation) whose final temperature is less than 35 C, for CNPs/Dox@SiO2 NCs at concentrations of 6.25, 12.5, 25 and 50 lg/mL, the final temperature rises to 43, 51, 56 and 60 C, respectively. At a concentration over 12.5 lg/mL, the temperature rises over 42 C within 2 min, the critical temperature for photothermal cancer therapy. This result indicates that the CNPs/Dox@SiO2 NCs still possess high photothermal conversion ability with concentration-dependence, which is strong enough to

39

destroy cancer cells. Meanwhile, compared with CNPs@SiO2 under the same irradiation (Fig. 3b), the final temperature for CNPs/Dox@SiO2 NC is slightly higher possibly due to weak NIR absorbance of Dox. A clear understanding of drug release behavior of CNPs/Dox@SiO2 NCs is beneficial to evaluate the efficacy of chemotherapy in cancer ablation. PBS solutions at pH 7.5 and 5.5 are used to mimic microenvironments of normal body and cancers, respectively. An 808-nm laser (4 W/cm2) is used to mimic PTT. As shown in Fig. 3d, for CNPs/Dox@SiO2 NCs in PBS solution (pH 7.5), the accumulative drug release rate achieves 20.6% in which Dox escapes from CNPs/Dox@SiO2 spontaneously via Brownian movement. For CNPs/Dox@SiO2 NCs in PBS solution (pH 5.5), the accumulative drug release rate reaches 27.6%. In this case, Dox escapes from CNPs/Dox@SiO2 and dissolves quickly via a process of protonation in acidic environment, leading to a higher release rate at the same time point. Once the 808-nm laser is applied during this drug release process, a fast drug release is observed and the release rate reaches 63.4%. During this process sharp increases occur at time points of 0.5, 2 and 8 h (Fig. 3c), with the release rate being 17.7%, 10.4% and 7.7%, respectively. The irradiation of 808-nm laser causes temperature rise, which speeds up molecular movement of Dox and

Fig. 3 – Photothermal effect of (a) CNPs/Dox@SiO2 NCs and (b) CNPs@SiO2 at different concentrations (6.25, 12.5, 25 and 50 lg/mL) under NIR irradiation (808 nm, 4 W/cm2) for 5 min. (c) Drug release profile of CNPs/Dox@SiO2 at different conditions (pH 7.5, pH 5.5 and pH 5.5 +NIR) within 24 h. Dot lines refer to NIR irradiation process. (d) The cumulative release rate of CNPs/Dox@SiO2 at different conditions (pH 7.5, pH 5.5 and pH 5.5 +NIR) after 24 h. (A colour version of this figure can be viewed online.)

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Fig. 4 – Confocal fluorescence microscopic images of 4T1 cells treated with PBS and CNPs/Dox@SiO2 for 1 h, 6 h and 18 h, respectively. The arrows refer to Dox aggregates in cells. (A colour version of this figure can be viewed online.)

thus greatly accelerates its escape from CNPs/Dox@SiO2 NCs. Our results demonstrate that CNPs/Dox@SiO2 NCs possess both pH and heat responsive drug release behavior in which heat plays a dominant role. The above results provide solid evidence for feasibility of CNPs/Dox@SiO2 NCs in chemotherapy.

3.3.

The cellular uptake of CNPs/Dox@SiO2 NCs

In vitro essays were performed to further validate the efficacy of CNPs/Dox@SiO2 NCs in cancer ablation. We first studied the in vitro cellular uptake behavior of CNPs/Dox@SiO2 NCs. The CNPs/Dox@SiO2 NCs were incubated with 4T1 cells for time

periods of 1, 6 and 18 h. The Dox wrapped inside is used as a fluorescent probe. As shown in Fig. 4, intracellular fluorescence becomes brighter with the incubation time. Compared with the control (0 h), few CNPs/Dox@SiO2 NCs enter the cells, as evidenced by observation of a relatively weak fluorescence after 1 h incubation. After 6–18 h incubation, large accumulation of CNPs/Dox@SiO2 NCs occurs in the cells, leading to a dramatic increase of fluorescence. CNPs/Dox@SiO2 NCs were visible inside cells as a bright dot due to the Dox aggregation in CNPs/Dox@SiO2 NCs, as shown by arrows. The surrounding fluorescence may be attributed to free Dox released from CNPs/Dox@SiO2 NCs, existing in both the cytoplasm and the nucleus. For free Dox (Fig. S2),

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preferable accumulation of Dox in nucleus occurs during the incubation time, which indicates the ultimate fate of the Dox released from CNPs/Dox@SiO2 NCs.

3.4.

The cytotoxicity of CNPs/Dox@SiO2 NCs

The cytotoxicity of CNPs/Dox@SiO2 NCs is a key issue for in vitro essays. After incubation of 4T1 cells with CNPs/Dox@SiO2 NCs (at CNPs concentrations from 3 to 100 lg/mL) for 24 h, the MTT assay indicates that cell viability decreases gradually with the increasing dose of

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CNPs/Dox@SiO2 NCs (Fig. 5a). For comparison purpose, the toxicity of free Dox to 4T1 cells was also studied at the same concentrations in CNPs/Dox@SiO2 NCs. The dose of free Dox to kill half of total cells (IC50) is 12 lg/mL while the IC50 for CNPs/Dox@SiO2 NCs is 50 lg/mL. The result shows that CNPs/Dox@SiO2 NCs can greatly reduce the toxicity of direct administration of free Dox by wrapping Dox inside and slowing its release rate, which agrees with the drug release experiment. Without Dox inside, CNPs@SiO2 NCs do not show noticeable toxicity to 4T1 cells due to the excellent biocompatibility of both SiO2 and CNPs.

Fig. 5 – Relative viability of 4T1 cells (a) treated with Dox, CNPs@SiO2 and CNPs/Dox@SiO2 of different concentrations (3, 6, 12, 25, 50 and 100 lg/mL), respectively, for 24 h and (b) treated with PBS, CNPs@SiO2 and CNPs/Dox@SiO2 of different concentrations (3, 6, 25, 12, 24, 50 and 100 lg/mL), respectively, under NIR irradiation (808 nm, 4 W/cm2) for 5 min. (c) IR thermal images of PBS and CNPs/Dox@SiO2 NCs treated mice under 5-min NIR irradiation. (d) Relative tumor volume of different groups of mice after various treatments. (e) Weight of different groups of mice after various treatments. (A colour version of this figure can be viewed online.)

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3.5. The in vitro and in vivo cancer ablation of CNPs/ Dox@SiO2 NCs We then investigated in vitro cancer ablation efficacy of CNPs/Dox@SiO2 NCs under 808-nm laser irradiation (4 W/cm2). As showed in Fig. 5b, NIR light exposure alone causes negligible toxicity to the cells as compared to the control (PBS, dark). Increased cytotoxicity is observed as the concentration of CNPs increases. For CNPs@SiO2, the cytotoxicity increases gradually and the IC50 value is estimated to be 24 lg/mL of CNPs. For CNPs/Dox@SiO2 NCs, the cytotoxicity increases sharply and the IC50 value is estimated to be 6 lg/mL of CNPs, which is about one quarter of that in CNPs@SiO2 at the same irradiation and about half of that in PEGylated CNPs under irradiation of a 9 W/cm2 808-nm laser in our recent work [27]. The results show that, for CNPs/Dox@SiO2 NCs, the high cytotoxicity comes from both the heat via NIR irradiation and the released Dox. As can be predicted from drug release experiment, Dox release can be largely promoted by laser irradiation, leading to higher chemotherapy toxicity than that without NIR irradiation. Clearly, the combination of photothermal and chemotherapy achieves high cancer ablation efficacy with low administration dose, otherwise a higher dose or stronger laser power should be applied in either single cancer treatment. Motivated by the high efficient cancer ablation of CNPs/Dox@SiO2 NCs in vitro, we then carried out an in vivo study using the 4T1 tumor model on Balb/c mice. The tumor size was monitored every other day, as shown in Fig. 5d. For the control mice injected with saline, tumors grow fast and reach 5 times as large as the initial size after 14 days. With 808-nm laser irradiation, the size of the tumors in saline injected mice shows no noticeable difference from that of

control group, due to insufficient temperature increase (Fig. 5c). Similar tumor size also occurs in mice groups injected with free Dox or CNPs/Dox@SiO2 NCs after 14-day treatment, possibly due to the fast clearance of free Dox or the failure of CNPs/Dox@SiO2 NCs on releasing sufficient Dox to fight against tumors. For the mice injected with CNPs@SiO2 aided by 808-nm laser irradiation, tumors grew slowly and reached twice as large as the initial size finally, due to the inexhaustive cancer ablation by PTT alone. For the mice injected with CNPs/Dox@SiO2 NCs and irradiated under the 808-nm laser, tumors experienced a high temperature burning (Fig. 5c), diminished gradually and were totally eliminated on the 6th day post treatment, owing to the combination of PTT and enhanced chemotherapy. The above results demonstrate that the combination of PTT and chemotherapy show more effective and exhaustive cancer ablation than either PTT or chemotherapy alone. The weight of mice was also monitored as an important factor showing mice physical conditions. Compared with the control group, no obvious change was observed for all experimental groups (Fig. 5e). After the experiment, the mice were sacrificed and major organs (heart, liver, spleen, lung and kidney) were collected and weighed as well. No noticeable weight changes were observed in organs except spleen from experimental mice, compared with those in normal mice (Fig. 6a). An enlargement of spleen was observed in mice whose tumors were not eliminated, which is a typical syndrome in animals with cancers [31], while no such change occurs in mice group of CNPs/Dox@SiO2 NCs under NIR irradiation, indicating the total elimination of cancer. In order to make known the above change, major organs of mice were collected for histology analysis. No noticeable changes in CNPs/Dox@SiO2 NCs group were observed by standard H&E stained organ slices, compared with those from normal mice (Fig. 6b), while for other

Fig. 6 – (a) The weight index of organs from different groups of mice after various treatments. (b) H&E staining for major organs (heart, liver, spleen, lung, and kidney). (A colour version of this figure can be viewed online.)

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groups, pathological changes occur in spleen (Fig. S3), which may cause the enlargement of spleen. A few nano-carbon@SiO2 carriers have been fabricated for combined photothermal and chemo-therapy [32–35]. Compared to these previously reported nano-carbon@SiO2 carriers, the CNPs/Dox@SiO2 NCs developed by us have some distinct advantages: (1) by using the reverse microemulsion method the monodispersed CNPs/Dox@SiO2 NCs of desired sizes can be obtained by adjusting the ratio of oil against water while in other nano-carbon@SiO2 systems carbon components often spoil their size monodispersity especially in graphene oxide and carbon nanotube systems; (2) by adding different amount of drug and CNPs, the ratio of drug against CNPs in CNPs/Dox@SiO2 NCs can be readily adjusted to meet different needs, while this ratio is hard to control in the nanocarbon@SiO2 carrier systems reported previously by others where physical adsorption is used for drug loading; (3) this drug loading strategy is also flexibly applied for multiple drugs or multiple PTT agents administration and available to fabricate multifunctional systems for cancer theranostics; (4) once applied in vivo CNPs can be cleared out in short time after treatment due to their small size, while graphene oxide or carbon nanotubes may face longer time accumulation in organs [36] and cause side effects [37]. Further effort is needed to improve the tumor homing ability of CNPs/Dox@SiO2 NCs.

4.

Conclusion

In summary, we have synthesized CNPs/Dox@SiO2 NCs with uniform size, narrow size distribution and high Dox payload. We have demonstrated that the as-synthesized CNPs/Dox@SiO2 NCs possess both excellent heat-generating ability and enhanced drug release under NIR light irradiation. Both in vitro and in vivo experiments show much higher cancer ablation ability of the CNPs/Dox@SiO2 NCs than single PTT or chemotherapy. This work demonstrates the feasibility of CNPs/Dox@SiO2 NCs as a promising nanoplatform for efficient combined photothermal and chemo-therapy in cancer ablation.

Acknowledgements We greatly acknowledge financial support from National Natural Science Foundation of China (No. 51361130033), the Ministry of Science and Technology of China (No. 2014CB965003) and Collaborative Innovation Center of Suzhou Nano Science and Technology.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbon. 2015.05.043. R E F E R E N C E S

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