Graphene Oxide

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Hollow copper sulfide nanosphere-doxorubicin/graphene oxide core-shell nanocomposite for photothermo-chemotherapy Lu Han, Yanan Hao, Xing Wei, Xuwei Chen, Yang Shu, and Jian-Hua Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00643 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 31, 2017

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ACS Biomaterials Science & Engineering

Hollow copper sulfide nanosphere-doxorubicin/graphene oxide core-shell nanocomposite for photothermo-chemotherapy

Lu Han,# Ya-Nan Hao,# Xing Wei, Xu-Wei Chen, Yang Shu* and Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110189, China #

These authors contributed equally to this work

Keywords: Copper sulfide, Graphene oxide, Controlled drug delivery, Photothermal therapy, Cancer therapy.

Abstract: A novel core-shell nanostructure, hollow copper sulfide nanosphere-doxorubicin (DOX)/graphene oxide (GO) (CuS-DOX/GO), is constructed for the purpose of controlled drug delivery and improved photothermo-chemo therapeutic effect. The CuS-DOX/GO nanocomposite is configured by employing dual photothermal agents, where the core, hollow CuS nanoparticle, acts as delivery-carrier for doxorubicin, and the shell, PEGylated GO nanosheet, prohibits the leakage of drug. DOX can be efficiently loaded onto the hollow CuS nanoparticles, and its subsequent release from CuS-DOX/GO nanocomposite is triggered in a pHand near-infrared light-dependent manner. Moreover, the integration of the two photothermal agents significantly improves the photothermal performance of this 1

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system. Ultimately, the combination of phototherapy and chemotherapy based on this system results in a much higher Hela cell killing efficacy with respect to that for single chemotherapy mode, as demonstrated by in vitro cytotoxicity tests.

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INTRODUCTION Versatile drug-delivery systems have attracted tremendous interest in the last decades.1-3 To obtain improved pharmacokinetic profile, enhanced therapeutic efficiency and reduced nonspecific side-effects of chemotherapeutic drugs, numerous nanomaterials, such as dendrimers4, liposomes5, and inorganic nanoparticles6,7, were designed as drug-delivery carriers to transport therapeutic drugs to the intended sites of action. Among the diverse nanostructures, many liposomal drug formulations, e.g., Doxil8, as well as albumin-drug formulations, e.g., Abraxane9, have been approved by US Food and Drug Administration for clinic cancer therapy. However, it is still difficult to achieve desired therapeutic outcomes, due to the low specificity and drug resistance of single chemotherapy.10,11 Therefore, an increasing number of nano-engineered multifunctional systems, which integrate multimodal therapeutic strategies, were constructed to pursue the synergistic effects for efficacy enhancement.12-14 In particular, the combination of thermotherapy and chemotherapy, i.e., thermo-chemotherapy, exhibits promise in optimizing the cancer therapeutic efficacy15, associated with hyperthermia and heat-induced vascular permeability increase as well as thermo-sensitive drug release.16,17 A variety of well-engineered nanomaterials, including gold nanostructures18, layered transition metal dichalcogenides19, copper-based semiconductors20, and carbon materials21, were capable of converting near-infrared (NIR) optical energy into heat for hyperthermia, and loading drugs for chemo treatment at the same time.

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Copper sulfide crystal, as a low-cost photothermal agent with broad absorption in NIR region derived from the d-d energy band transition of Cu2+ ions, have been utilized to fabricate multifunctional drug-delivery platforms.22,23 Copper sulfide (CuS) nanocrystals exhibit favorable photo-stability and thus have been demonstrated to be a promising alternative to the widely used gold nanostructures24-26. The hollow CuS nanocrystals, with large specific surface area and numerous mesoporous pores, were employed as intelligent photothermal vehicles for hydrophobic therapeutic agent (Camptothecin) to realize synergistic thermo-chemotherapy of cancer cells.27 In our previous work, these hollow-structured nanoparticles showed excellent loading capacity for indocyanine green, and finally achieved combined photothermal and photodynamic therapy.28 Nevertheless, the leakage of drug molecules before arriving at the lesions often brings about systemic side effects. Thus efficient encapsulation is of critical importance in configuration of drug-delivery nanoplatforms. Graphene oxide as a two-dimensional layered carbon material composed of sp2-hybridized carbon atoms29, has been exploited extensively for biomedical applications, e.g., biosensing30, molecular imaging31 and drug delivery for cancer therapy32, which is attributed to its unique optical, electrical and chemical properties. As a promising drug carrier, GO possesses high drug-loading capacity due to the ultrahigh surface area33, and stimuli-responsive (e.g., pH and temperature) release behavior.2 Moreover, its NIR-light-responsive photothermal property enables GO to be applied for efficient thermo-chemotherapy of tumors.34 Specially, GO nanosheets have

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been confirmed as a prominent shell material in the fabrication of intelligent therapeutic nanoplatforms. GO shells are competent to prevent drug leakage and improve the interfacial property of the nanostructures.35,36 In this work, we configured a stimuli-responsive nanovehicle composed of CuS nanoparticles and GO nanosheets, to deliver DOX for combined thermo-chemotherapy. Drug molecules are firstly loaded onto hollow CuS nanoparticles, and the obtained CuS-DOX composites were subsequently enwrapped with PEGylated GO nanosheets (GO-PEG) via electrostatic attraction. Then pH- and NIR-light-sensitive release behavior of DOX from the resulting CuS-DOX/GO nanocomposites was investigated. Benefiting from the excellent photothermal conversion performance of both CuS carrier and GO-PEG nanoshell, the designed drug delivery system was utilized for combinatorial photothermal and chemotherapy of cancer cells.

EXPERIMENTAL SECTION Chemicals. Ammonium sulfide ((NH4)2S) aqueous solution (20 wt%), N-hydroxysuccinimide (NHS) and N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochloride (EDC) were obtained from Aladdin Reagent (Shanghai, China). NH2-PEG-NH2 (Mw 3500) was purchased from Xing Jia Feng Science and Technology Development Co. Ltd. (Shenzhen, China). DMEM (high glucose), penicillin/streptomycin, trypsin and fetal bovine serum were received from Thermo Scientific (Logan, Utah, USA). Other chemicals used in this study were the products of Sinopharm Chemical Reagent 5



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(Shenyang, China). Deionized water of 18 MΩ cm-1 was used throughout the experiments. Preparation of CuS-DOX/GO nanocomposites. Hollow CuS nanospheres and PEGylated GO (GO-PEG) nanosheets were prepared through the methods in our previous works as described in Supplementary Information.28,37,38 Thereafter, a certain volume (20, 40, 60, 80, 120, 160 µl) of DOX (5 mg ml-1) solution was mixed with 50 µl of CuS suspension (5 mg ml-1), and the volume of the mixture was adjusted to 1.0 ml with phosphate buffer (PBS, pH 7.4, 10 mmol l-1). After shaking for 6 h at room temperature, these mixtures were centrifuged at 5000 rpm for 5 min and washed for twice with PBS to get DOX-loaded CuS nanoparticles (CuS-DOX). The supernatant was collected to determine the concentration of residual DOX by measuring the absorbance at 485 nm. The loading factor of DOX to CuS was expressed in the following 2,27, with mDOX, muDOX and mCuS as the mass of initial DOX, unabsorbed DOX and CuS, respectively. Loading factor = (mDOX - muDOX)/mCuS Subsequently, the CuS-DOX dispersion (5 mg ml-1, PBS) was mixed with 250 µl of GO-PEG and vibrated for 2 h. CuS-DOX/GO composites were obtained by centrifugation at 5000 rpm for 5 min and washing with PBS. Transmission electron microscopy (TEM) images were acquired using a JEM-2100 transmission electron microscope (JEOL, Japan). Morphological analyses were carried out on an atomic force microscope (AFM) (Dimension Icon ScanAsyst, Germany). X-ray diffraction (XRD) patterns were obtained on a D8 Advance

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diffractometer (Bruker, Germany) using CuΚα radiation (λ = 1.54 Å). The copper content is measured with inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500a, USA). The zeta potential of the nanoparticles was measured by a Zetasizer Nano ZS/ZEN3690 instrument (Malvern, England). UV-vis absorption spectra were recorded on a UH5300 spectrophotometer (Hitachi, Japan). FT-IR and Raman spectra were acquired on a Nicolet 6700 spectrophotometer (Thermo Electron, USA) and a Horiba XploRA Spectrometer (Jobin Yvon, France), respectively. Photothermal effect of the materials was determined by irradiating with a diode infrared laser (MDL-III-808 nm-2.5 W-14100192, Changchun New Industries Optoelectronics Tech. Co. Ltd, China). Photothermal effect. The photothermal behaviors of CuS, CuS-DOX and CuS-DOX/GO (the concentrations of both CuS and DOX are 200 µg ml-1) were evaluated by irradiating 0.3 ml of their solutions with a continuous wave fiber-coupled diode laser source (808 nm) for 10 min, and the temperature was recorded by a thermometer (IKA Ltd). In vitro drug release. For the evaluation of the release behavior of DOX from CuS-DOX/GO, the CuS-DOX/GO composites were dispersed in PBS buffer (with a concentration of 2 mg ml-1 and a loading factor of 1.0) at pH 5.2, 6.3 and 7.4, and the mixture was kept in a 37℃ bath for 72 h. At each time point (3, 6, 12, 24, 48, 72 h), the mixtures were centrifuged to collect the supernatant, and the amount of released DOX was estimated by UV-vis absorption spectrophotometry at 485 nm. CuS-DOX/GO sediment is then re-suspended in fresh PBS buffer.

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For the purpose of determining the release profile of DOX from CuS-DOX/GO upon exposure to NIR laser, CuS-DOX/GO composites were suspended in PBS (pH 5.2) and maintained in a 37℃ bath. At each time point (1, 3, 6, 12 h), the CuS-DOX/GO solution was irradiated by a 808 nm laser (1 W cm-2) for 5 min, followed by centrifugation to collect the supernatant. The released DOX in the supernatant was estimated by spectrophotometry at 485 nm. Afterwards, the sediment was re-suspended in fresh PBS buffer. In vitro cell experiments. HeLa cells were used for in vitro experiments. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at standard culture (37℃, 5% CO2). For observing the cellular uptake of CuS-DOX/GO nanocomposites and free DOX, the cells were seeded in glass-bottom dishes (5×104 cells/well) and cultured for 24 h. Afterwards, DMEM containing CuS-DOX/GO (50µg ml-1 for loaded DOX, at a loading factor of 0.5) was added and incubated for 0.5, 1, 2 and 4 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde and subsequently imaged by a FV 1200 confocal fluorescence microscopy by excitation at λex 488 nm (Olympus, Japan). For cell staining, the cells were grown in a 24-well plate (5×104 cells/well) and treated with CuS-DOX/GO nanocomposites (50 µg ml-1 for loaded DOX, at a loading factor of 0.5) and free DOX (50 µg ml-1) for 2 h, respectively. Thereafter, cells were exposed to NIR laser at 0.8 W cm-2 for 5 min, and incubated for an additional 1 h. After washed with PBS, the cells were treated with 0.02% trypan blue for 5 min and

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observed by a fluorescence microscope. For in vitro photothermo-chemotherapy, the cells were seeded in a 96-well plate at a density of 2×104 cells per well and incubated with various concentrations of CuS-DOX/GO or free DOX for 4 h. The cells were then irradiated by NIR laser (0.8 W cm-2) for 5 min, and incubated for an additional 20 h. The viability of cells was determined via MTT assays.

RESULTS AND DISCUSSION Characterizations. The CuS-DOX/GO nanocomposites were constructed through the strategy illustrated in Figure 1. The hollow CuS nanoparticles with an average diameter of ca.100 nm were prepared via a template-assisted approach, followed by serving as drug-delivery vehicles to load DOX. As shown in Figure 2A, the DOX loading capacity exhibited a concentration-dependent mode, and a loading factor of up to 1.75 (1.75 g DOX/1.0 g CuS) was encountered at a DOX concentration of 600 µg ml-1. With the introduction of DOX, surface charge of CuS-DOX became positive with respect to the initial negative charge of CuS nanoparticles, which enabled the negatively charged GO-PEG nanosheets to wrap around the CuS-DOX via electrostatic attraction (Figure 2B). TEM images of CuS-DOX/GO nanocomposites demonstrated the presence of GO-PEG shell with a thickness of ca.5 nm (Figure 1C). Additionally, the successful construction of CuS-DOX/GO nanocomposites was confirmed by FT-IR and Raman spectroscopy (Figure S4).

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NIR

∆T

H+ (pH) DOX CuS GO-PEG

Figure 1. Schematic illustration of the fabrication of CuS-DOX/GO nanocomposite for controlled drug delivery and photothermo-chemotherapy.

A

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1.6 Loading factor

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Figure 2. (A) The plot of DOX loading factor versus DOX concentration. (B) Zeta potentials of CuS, CuS-DOX and CuS-DOX/GO (250 µg ml-1 for CuS, 100 µg ml-1 for the loaded DOX). (C) TEM images of CuS (i), CuS-DOX (ii) and CuS-DOX/GO (iii). The parallel lines in C (iii) indicate the presence of GO-PEG shell.

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UV-vis-NIR absorption spectra of CuS, CuS-DOX and CuS-DOX/GO were recorded as illustrated in Figure 3. It was observed that CuS nanoparticles exhibited intensive absorption in the NIR region (700-900 nm), and negligible change was found in the absorption maximum after loading of DOX. With the capsulation of GO-PEG shell, the CuS-DOX/GO nanocomposites gave rise to a significantly enhanced absorbance in NIR region, indicating the promising photothermal effect of the nanocomposites. 4 CuS-DOX/GO

3 CuS-DOX Abs

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2 CuS

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600

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Figure 3. UV-vis-NIR absorption spectra of CuS, CuS-DOX and CuS-DOX/GO (250 µg ml-1 for CuS, 100 µg ml-1 for the loaded DOX).

Photothermal performance of CuS-DOX/GO. For evaluating the potential photothermal performance of CuS-DOX/GO, the heating curves of its solution upon exposure to 808 nm-laser (1.5 W cm-2) were determined. As depicted in Figure 4, the temperature elevation of the CuS-DOX/GO solution reached up to ca.27℃ for a 5-min irradiation, while both CuS and CuS-DOX exhibited lower photothermal effects at a same CuS concentration. This observation implied that the capsulation of

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GO-PEG nanosheets significantly improved the photothermal performance of the nanocomposites. Moreover, the heat generated by the nanocomposites was expected to facilitate a sudden release of DOX from the composites.

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Figure 4. The temperature curves of CuS, CuS-DOX and CuS-DOX/GO (200 µg ml-1 for CuS, 200 µg ml-1 for the loaded DOX) under 808 nm-laser irradiation at an energy level of 1.5 W cm-2.

Controlled release of DOX from CuS-DOX/GO. Previous reports have discovered that the release of DOX from drug-carriers exhibited a pH-dependent mode, which was attributed to the increase of hydrophilicity and solubility of DOX in acidic medium.2,38 In the present work, release profiles of DOX from the CuS-DOX/GO nanocomposites in PBS buffer at various pHs (7.4, 6.3 and 5.2) were illustrated in Figure 5A. It was observed that almost all of the drug molecules retained on the carriers at pH 7.4, and only 3.3% of drug leaked out after 72 h-incubation at pH 6.3. However, when the pH value of the medium was decreased to 5.2, closing to that in the endosomes/lysosomes, a rapid release of DOX was triggered. The amount of

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cumulative released drug significantly increased to 49% within 72 h. The activated release behavior of DOX from the CuS-DOX/GO nanocomposites in an acidic microenvironment was further confirmed as depicted in Figure 5B. DOX loaded on CuS-DOX/GO kept stable at pH 7.4 within the initial 6 h, while a sudden release of DOX was encountered once pH value of the medium was adjusted to 5.2. 60

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10 5 pH 7.4

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Figure 5. (A) DOX release profiles from CuS-DOX/GO at pH 7.4, 6.3 and 5.2. (B) The controlled release of DOX from CuS-DOX/GO by varying the pH value from 7.4 to 5.2.

The release profile of DOX upon NIR-laser stimulation was further investigated. As shown in Figure 6, the cumulative release of DOX was obviously enhanced under NIR irradiation at different time intervals. It was clearly seen that 62% of the loaded DOX was released by a total NIR-laser irradiation time of 20 min (4×5 min) within 12 h, which was much higher than that without irradiation (46%). These observations demonstrated the feasibility of triggering drug release from CuS-DOX/GO nanocomposites in an actively controllable manner, and this pH- and NIR

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light-responsive release behavior effectively facilitated their application in cancer therapy.

75 Drug released (%)

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60 45 30

NIR irradiation No NIR irradiation

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Figure 6. Release profiles of DOX from CuS-DOX/GO nanocomposites at pH 5.2 within 12 h. The data points in the red line are obtained by NIR laser irradiation for 5 min at 1.5 W cm-2.

Cellular uptake of CuS-DOX/GO. The cellular uptake behavior of CuS-DOX/GO was evaluated by a confocal fluorescence microscopy. It was found that the intracellular fluorescence signal of DOX increased gradually as a function of incubation time (Figure 7A), indicating the time-dependent intake of CuS-DOX/GO nanocomposites by HeLa cells. Additionally, the cellular uptake of DOX under NIR-irradiation was further monitored in consideration of the NIR-stimulated release behavior. Figure 7B illustrated that the red fluorescence was much brighter in the NIR-irradiated group in comparison to the non-irradiated group, demonstrating the enhanced internalization of DOX in cells. This observation indicated a promising potential to achieve improved chemotherapeutic efficacy upon stimulation by external

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NIR-laser.

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50 µm

Figure 7. (A) Fluorescence images of HeLa cells treated with CuS-DOX/GO at different incubation times (concentration of loaded DOX: 50 µg ml-1). (B) Confocal fluorescence images of the cells incubated with CuS-DOX/GO for 1 h, with or without NIR-irradiation (0.8 W cm-2, 5 min).

In vitro combined photothermo-chemotherapy. To evaluate the photothermo-chemotherapeutic effect of the present platform, standard MTT assays were conducted to quantify cell viability. The viabilities of HeLa cells treated with

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DOX and CuS-DOX/GO at three concentration levels were calculated as illustrated in Figure 8A. The quantitative results showed that at a concentration of 10 µg ml-1 DOX, the CuS-DOX/GO nanocomposites gave rise to a HeLa cell viability of 64% (corresponding to a killing rate of 36%), while a same concentration of free DOX produced a HeLa cell viability of 46% (corresponding to a killing rate of 54%). This might be attributed to the partial retention of loaded DOX on the nanocomposite due to an incomplete release of DOX. However, after exposing to NIR-irradiation, the photothermo-chemo treatment group exhibited significantly higher cytotoxicity with respect to that achieved by chemotherapy alone at all the tested concentration levels. The effect of NIR-irradiation were two-folds, on one hand, it promoted the release of DOX and thus improved chemotherapy, on the other hand, it caused thermal ablation of the cancer cells for photothermal therapy. In particular, the heat generated by CuS-DOX/GO nanocomposites might increase the cell membrane permeability to enhance the cellular uptake of drugs.39 As shown in Figure 8B, the thermo-chemotherapeutic effect was tested via dead cell stain by trypan blue. It was obvious that only the combined photothermo-chemo treatment can effectively kill the cancer cells within 2 h at a DOX concentration of 50 µg ml-1. The above observations illustrated the feasibility of employing the CuS-DOX/GO nanocomposites as a photothermo-chemotherapeutic agent for cancer therapy. Moreover, with the favorable therapeutic outcomes, this CuS-DOX/GO nanostructure may find promising potential applications for targeted cancer therapy in vivo with further modification by cancer-targeting ligands, such as antibodies, proteins and peptides.40,41 For this

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DOX

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purpose, further future studies will be undertaken.

50 µm

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25 0

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10 20 50 Concentration of DOX (µg ml-1)

control/+ laser

DOX

CuS-DOX/GO

Figure 8. (A) Cell viabilities by incubating with free DOX and CuS-DOX/GO nanocomposites at various concentration levels. The content of CuS is fixed at 200 µg ml-1, and concentrations of DOX are 10, 20 and 50 µg ml-1. (B) Microscopy images of trypan blue stain (DOX concentration is 50 µg ml-1 with a loading factor of 0.5). The power density of laser is fixed at 0.8 W cm-2.

CONCLUSIONS In summary, a potential core-shell structured therapeutic system (CuS-DOX/GO) based on two kinds of NIR photothermal agents (CuS and GO) and a chemotherapeutic agent (DOX) is constructed. The core component, i.e., hollow CuS nanoparticles, exhibits a high loading capacity for DOX with a loading factor of 1.75 g g-1. The GO-PEG outer shell is capable of preventing drug leakage, and meanwhile improving photothermal energy conversion efficiency. The stimuli-responsive (pH and NIR light) release of DOX from CuS-DOX/GO makes the therapeutic process 17



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more controllable and brings about high specificity. Furthermore, the combinatorial photothermo-chemotherapy efficiently enhances the cytotoxicity against HeLa cells. Overall, this CuS-DOX/GO therapeutic system provides a potential alternative for cancer treatment, and it offers a new avenue for the development of high efficiency drug delivery and therapeutic platforms.

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ASSOCIATED CONTENT *Supporting Information

AUTHOR INFORMATION *

Corresponding author.

E-mail address: [email protected] (Y. Shu), [email protected] (J.H. Wang). Tel: +86 24 83688944; Fax: +86 24 83676698

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors appreciate financial support from the Natural Science Foundation of China (21235010, 21475017, 21575020), Fundamental Research Funds for the Central Universities (N141008001, 150502001, N152004005).

Supporting Information Available: The preparation of hollow CuS nanospheres and GO-PEG nanosheets; XRD pattern and nitrogen adsorption-desorption isotherms of hollow CuS crystals; UV-vis-NIR absorption spectra and AFM images of GO-COOH and GO-PEG nanosheets; FT-IR spectra and Raman spectra of CuS, GO-PEG, CuS-DOX and CuS-DOX/GO. .

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Graphical Abstract

NIR

∆T

H+ (pH) DOX CuS GO-PEG

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Graphene Oxide

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