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Acta Biomaterialia 49 (2017) 402–413

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Acta Biomaterialia

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Programmed near-infrared light-responsive drug delivery system for combined magnetic tumor-targeting magnetic resonance imaging and chemo-phototherapy

http://dx.doi.org/10.1016/j.actbio.2016.11.035 1742-7061/� 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding authors at: School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China.

E-mail addresses: [email protected] (L. Hou), [email protected] (Z. Zhang).

Qianhua Feng a,b,c, Yuanyuan Zhang a, Wanxia Zhang a, Yongwei Hao a,b,c, Yongchao Wang a,b,c, Hongling Zhang a,b,c, Lin Hou a,b,c,⇑, Zhenzhong Zhang a,b,c,⇑ a School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China b Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, China c Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, Zhengzhou 450001, China

a r t i c l e i n f o

Article history: Received 15 July 2016 Received in revised form 3 November 2016 Accepted 15 November 2016 Available online 24 November 2016

Keywords: Hollow mesoporous copper sulfide Magnetic targeting Controlled release Theranostics

a b s t r a c t

In this study, an intelligent drug delivery system was developed by capping doxorubicin (DOX)-loaded hollow mesoporous CuS nanoparticles (HMCuS NPs) with superparamagnetic iron oxide nanoparticles (IONPs). Under near infrared (NIR) light irradiation, the versatile HMCuS NPs could exploit the merits of both photothermal therapy (PTT) and photodynamic therapy (PDT) simultaneously. Herein, the mul- tifunctional IONPs as gatekeeper with the enhanced capping efficiency were supposed to realize ‘‘zero premature release” and minimize the adverse side effects during the drug delivery in vivo. More impor- tantly, the hybrid metal nanoplatform (HMCuS/DOX@IONP-PEG) allowed several emerging exceptional characteristics. Our studies have substantiated the hybrid nanoparticles possessed an enhanced PTT effect due to coupled plasmonic resonances with an elevated heat-generating capacity. Notably, an effec- tive removal of IONP-caps occurred after NIR-induced photo-hyperthermia via weakening of the coordi- nation interactions between HMCuS-NH2 and IONPs, which suggested the feasibility of sophisticated controlled on-demand drug release upon exposing to NIR stimulus with spatial/temporal resolution. Benefiting from the favorable magnetic tumor targeting efficacy, the in vitro and in vivo experiments indi- cated a remarkable anti-tumor therapeutic efficacy under NIR irradiation, resulting from the synergistic combination of chemo-phototherapy. In addition, T2-weighted magnetic resonance imaging (MRI) con- trast performance of IONPs provided the identification of cancerous lesions. Based on these findings, the well-designed drug delivery system via integration of programmed functions will provide knowledge for advancing multimodality theranostic strategy.

Statement of Significance

As we all know, a series of shortcomings of conventional chemotherapy such as limited stability, rapid clearing and non-specific tumor targeting ability remain a significant challenge to achieve successful clin- ical therapeutic efficiency in cancer treatments. Fortunately, developing drug delivery system under the assistance of multifunctional nanocarries might be a great idea. For the first time, we proposed an intel- ligent drug delivery system by capping DOX-loaded hollow mesoporous CuS nanoparticles (HMCuS NPs) with multifunctional IONPs to integrate programmed functions including enhanced PTT effect, sophisti- cated controlled drug release, magnetic targeting property and MR imaging. The results showed HMCuS/ DOX@IONP-PEG could significantly enhance anti-tumor therapeutic efficacy due to the synergistic com- bination of chemo-phototherapy. By this delicate design, we believe such smart and extreme versatile all- in-one drug delivery platform could arouse broad interests in the fields of biomaterials, nanotechnology, and drug delivery system.

� 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Notwithstanding its pharmacological effect for cancer therapy, conventional chemotherapy has been compromised by a series of

Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413 403

shortcomings such as limited stability, rapid clearing and non- specific tumor targeting ability, which brought out serious side effects [1,2]. In order to address this, developing drug delivery sys- tem under the assistance of nanocarries is an increasingly recog- nized alternative method for cancer treatment [3,4]. Moreover, allowing for the complexity and variability of cancers, it is indis- pensable to require synergistic combination of several therapeutic approaches in a coordinated way. From this viewpoint, realizing multifunctional nanoparticles with distinct mechanisms is particu- larly anticipated to optimize cancer therapy.

Recently, copper sulfide nanoparticles (CuS NPs) have received tremendous attention for their unique characteristics of NIR local- ized surface plasmon resonances (LSPR). Unlike other NIR resonant materials, which usually kill cancer cells relying on one single prin- ciple such as heat or reactive oxygen species (ROS) generation, the versatile CuS NPs could exploit the merits of both photothermal therapy (PTT) and photodynamic therapy (PDT) simultaneously [5,6]. Among numerous CuS nanomaterials reported so far [7,8], hollow mesoporous CuS NPs (HMCuS NPs) were considered as an intelligent drug-delivery vehicle preferable to solid nanoparticles due to their uniform pore structure and high surface area for drug encapsulation [9–11]. Based on these advantages, a promising paradigm combining chemotherapy with phototherapy (including PTT and PDT) based on HMCuS NPs was spontaneously obtained for synergistic cancer therapy. Nevertheless, with regard to meso- porous materials without gatekeeper modification, the undesirable premature drug leakage in circulation should be taken into account prior to their biomedical application [12,13]. Thus, it might be a great copping strategy to cap the HMCuS NPs with a multifunc- tional smart gatekeeper, which would remedy the drug leakage defect to prevent any complications.

The FDA-approved superparamagnetic iron oxide nanoparticles (IONPs), which featured inherent compatibility and high magneti- zation values, have been taken advantages in biomedical fields related to drug delivery, diagnostics and hyperthermia therapy [14–16]. Herein, IONPs with ultrafine particle sizes could act as a gatekeeper through capping onto HMCuS NPs to realize ‘‘zero pre- mature release”. In the meanwhile, the hybrid nanoplatform makes it possible to obtain some emerging exceptional characteristics. According to our interparticle coupling effects on the surface plas- mon resonances of metallic complex structures could generate highly enhanced local electromagnetic field and further enhance the NIR absorption as reported in literatures such as Au-Fe3O4, Au-CuS and r-GO-Au [17–20]. Encouragingly, our results substanti- ated that the hybrid HMCuS@IONP nanoparticles with enhanced SPR effect could generate elevated photothermal transduction effi- ciency by lower power laser irradiation in a short time. In addition, a quantity of IONP-caps could be remotely removed from the sur- face of HMCuS by NIR-induced photo-hyperthermia, probably resulting from the weakening of the coordination interactions between HMCuS-NH2 and IONPs [21]. Thus, it was more than critical that the NIR stimulus would make progress on controlled on-demand drug release with spatial/temporal resolution. Further- more, the acknowledged intrinsic magnetic properties of IONPs would endow the nanoplatform with magnetic targeted therapeu- tic effect and T2-weighted MR imaging contrast performance [22,23]. As a matter of course, the integration of IONPs on the HMCuS NPs surface certainly would offer great advantages in cancer treatment and diagnosis.

Herein, we proposed a programmed NIR-responsive drug deliv- ery system for combined magnetic tumor targeting MR imaging and chemo-phototherapy. As illustrated in Scheme 1, HMCuS NPs was utilized to encapsulate the chemotherapeutical drug doxoru- bicin (DOX), and then capped with multifunctional IONPs to inte- grate programmed functions including enhanced PTT effect, sophisticated controlled drug release, magnetic targeting property

and MR imaging, which indicated a smart and extreme versatile all-in-one drug delivery platform. In addition, hydrophilic PEG modification allowed the nanoparticles for biocompatibility and prolonged circulation characteristics [24,25]. The photothermal effect under NIR irradiation was evaluated by using a thermal cam- era. Next, the tunable drug release upon exposing to NIR stimulus was explored on MCF-7 cells. Based on the noticeable magnetic tumor targeting effect, it was envisioned that the synergistic com- bined chemo-phototherapy would significantly improve anti- tumor therapeutic efficacy with minimal side effects. Besides, the MR imaging was also tested in vivo. By this delicate design, such a versatile hybrid nanoplatform of HMCuS/DOX@IONP-PEG with multi-functional characteristics will show great promising poten- tial in multimodality theranostic applications in cancer treatment.

2. Experimental section

2.1. Materials

Doxorubicin hydrochloride (DOX HCl) was purchased from Aladdin Reagent Database Inc. (Shanghai, China). PEG2000-COOH, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) and 1-Hydroxy-2,5-pyrrolidinedione (NHS) were obtained from Sigma-Aldrich (Boston, MA, USA). All other chemi- cals acquired from the suppliers were at analytical grade. All ani- mal experiments were carried out in accordance with institutional guidelines and approved by the local ethical committee.

2.2. Synthesis of HMCuS/DOX@IONP-PEG

Synthesis of HMCuS NPs. Briefly, CuCl2 (8.5 mg), poly- (vinylpyrrolidone) (PVP-K30, 240 mg) and hydrazine anhydrous solution (6.4 lL) were added to 50 mL deionized water under mag- netic stirring at room temperature to form Cu2O nanoparitcles. Subsequently, Na2S (64 mg) was quickly added into the above solution. The mixture was maintained at 60 �C under strong stir- ring for 2 h. Then the resulting products (HMCuS NPs) were puri- fied by washing three times with water. To obtain amination derivative of HMCuS, HMCuS NPs (1 mg/mL) was mixed with 2-aminoethanethiol (2 mg/mL) in PBS buffer under strong stirring for 24 h. As an end, the mixture was concentrated to obtain HMCuS-NH2.

DOX loading. 1 mL of DOX in PBS (1 mg/mL) were added into 3 mL of HMCuS-NH2 in PBS (1 mg/mL). After stirring for 24 h, the retrieved HMCuS/DOX was collected by centrifugation at 15,000 r/min for 5 min. The redundant DOX collected from super- natants was quantified by UV–vis spectroscopy measurements. DOX loading capacity (LC) was calculated in the following formula:

LC ¼ MDOX�prep � MDOX�supernatant MDOX�prep

� 100%

Synthesis of IONPs and IONPs capping onto HMCuS/DOX. The individual aqueous IONPs were synthesized by chemical co- precipitation method [26]. FeCl2 (0.86 g) and FeCl3 (2.35 g) were added into 40 mL of deionized water and then the mixture was heated at 60 �C under N2 atmosphere. Subsequently, aqueous ammonia (5 mL) was slowly dropped into the above solution. After reaction for 30 min, citric acid (0.5 g/mL, 2 mL) was added. The as- prepared mixture was then maintained at 95 �C for 80 min and cooled to room temperature under continuous stirring. The result- ing IONPs were collected with an external magnet. Finally, the resulting product (HMCuS/DOX@IONP) was obtained by mixing IONPs and HMCuS/DOX (1:5) under stirring via noncovalent interaction.

Scheme 1. Schematic representation of the synthesis of the drug delivery system (HMCuS/DOX@IONP-PEG) for combining MR imaging with chemo-phototherapy.

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Conjugation of PEG to HMCuS/DOX@IONP. In brief, a mixture of EDC HCl, NHS and PEG2000-COOH with molar ratio of 1:1:1 was dis- solved in 20 mL of PBS, and the reaction was carried out for 30 min. Then the mixed solution was added to HMCuS/DOX@IONP in PBS and stirred at room temperature for another 3 h. At last, the as- prepared HMCuS/DOX@IONP-PEG was collected by centrifugation. According to the same manner, another product of HMCuS@IONP- PEG NPs as blank vehicles for in vitro and in vivo experiments was also achieved without DOX loading.

2.3. Characterization

The morphology of the as-prepared samples was observed by transmission electron microscopy (TEM, FEI Tecnai G20). N2 adsorption-desorption isotherms were recorded on a Micromerit- ics ASAP2020 sorptometer (Micromeritics, USA). X-ray diffraction (XRD) measurements were conducted on an X-ray diffractometer (Model: XD-3X, Beijing, China). Ultra-violet-visible (UV–vis) spec- tra were recorded on an UV–vis spectrophotometer (Shimadzu). FT-IR spectra were performed on a Nicolet iS10 spectrometer (Thermo, USA). A vibrating sample magnetometer (VSM) was uti- lized for magnetic characterization of HMCuS/DOX@IONP-PEG. The leakage experiment was performed by incubating HMCuS/ DOX-PEG and HMCuS/DOX@IONP-PEG with the PBS (pH 7.4) con- taining 10% of FBS at 37 �C and monitoring leakage of free DOX continuously.

2.4. Photothermal effect of NIR on HMCuS/DOX@IONP-PEG

For photothemal effect, a continuous-wave NIR laser (Chang- chun New Industries Optoelectronics Technology, China; wave-

length: 808 nm, power density: 2 W/cm2, spot size: 5 mm) was used. Samples of different concentrations were irradiated by 808 nm laser for 3 min. And the photothermal images were taken by a thermal camera (FLIR, T330).

2.5. NIR light triggered release of DOX from HMCuS/DOX@IONP-PEG

In vitro DOX release from HMCuS/DOX@IONP-PEG was per- formed in PBS buffer at pH 7.4. In brief, 1.5 mg DOX loaded nanoparticles were dialyzed in 100 mL PBS and shaken gently at 37 �C. To evaluate NIR sensitivity of the formulation, samples were repeatedly exposed to a NIR irradiation (808 nm, 2 W/cm2, 3 min). Subsequently, the released free DOX was taken at designed time point and quantified by UV–vis spectra.

2.6. Cellular experiments

Cell culture. MCF-7 human breast cancer cells were cultured in normal RPMI-1640 culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 �C in a humidi- fied atmosphere of 5% CO2 and 95% air. Cells were harvested by the use of trypsin and resuspended in fresh absolute medium before plating.

NIR laser controlled DOX release in vitro. MCF-7 cells (5 � 104 - cells per well) were co-cultured with HMCuS/DOX@IONP-PEG (DOX concentration: 5 lg/mL) in 6-well plates. After the internal- ization of formations for 4 h, cells were exposed to a NIR laser irra- diation (808 nm, 2 W/cm2, 3 min). Then cell nucleus were stained with DAPI for another 30 min. At last, cell imaging was conducted by using confocal laser scanning microscopy (CLSM, Olympus FV1100) after washing cells with PBS.

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Intracellular ROS detection. DCFH-DA Reactive Oxygen Species Assay Kit was used to detected intracellular ROS generation. MCF-7 cells were plated at 5 � 104 cells per well in a 6-well plate. Following incubation with free DOX (5 lg/mL), HMCuS@IONP-PEG (12 lg/mL) and HMCuS/DOX@IONP-PEG (17 lg/mL) for 4 h, the cells were washed three times with PBS and then DCFH-DA was entrapped into the cells. After 30 min incubation, the cells were irradiated with a NIR laser (808 nm, 2 W/cm2) for 3 min. Then flu- orescence images of treated cells were obtained by using a fluores- cence microscope (Nikon Eclipse 50i, Japan).

In vitro cytotoxicity assay. MCF-7 cells were plated at 5 � 103 cells per well in 96-well plates and cultured for 24 h. Then serial dilutions of different formulations at the indicated concentrations were added to the culture medium. Subsequently, the cells were irradiated with a NIR irradiation (808 nm, 2 W/ cm2, 3 min). After incubation for 24 h, cell samples were treated with a standard sulforhodamine B (SRB) assay to determine cell viabilities.

Magnetic targeting effect in vitro. MCF-7 cells were placed into 6-well plates (5 � 104 cells per well) and then treated with HMCuS/DOX@IONP-PEG (17 lg/mL) for 4 h with a magnet (mag- netic field intensity: 0.3 T) placed under the center of the culture dish. Then intracellular ROS were detected under NIR irradiation according to the method described above. Finally, cell imaging in green and red field was carried out by using a fluorescence micro- scope (Nikon Eclipse 50i, Japan).

MR imaging of cells. MCF-7 cells were treated with different concentrations of HMCuS/DOX@IONP-PEG for 4 h, and then were rinsed with PBS for several times. Subsequently, T2-weighted MR images in vitro were collected on a 3-T clinical MRI scanner (SIEMENS).

2.7. In vivo experiments

In vivo optical imaging. The near-infrared dye (IR783) was used to mark the vehicle as a fluorescent probe. The tumor- bearing mice received intravenous administration of HMCuS/ IR783@IONP-PEG (100 lg IR783/kg). The magnet group were treated with an external magnet (magnetic field intensity: 0.3 T) laying above the tumor. The real-time optical imaging was performed at the indicated time point (1, 3, 6, 8, 12 and 24 h) by using a noninvasive optical imaging system (Bruker, Germany) with an excitation band pass filter at 730 nm and an emission at 790 nm. Then mice were then sacrificed, with their major organs (heart, liver, spleen, lung, kidney and tumor) har- vested for ex vivo imaging.

MR imaging in vivo. The tumor-bearing mice were intra- venously injected with HMCuS/DOX@IONP-PEG (13 mg/kg). A magnet (magnetic field intensity: 0.3 T) was placed onto the tumor of the magnet group. At 6 h after injection, MR imaging was per- formed on a 3-T clinical MRI scanner.

In vivo anti-tumor activity. The tumor bearing mice were assigned to seven groups randomly (n = 6 per group). The mice were intravenously injected every other day with (1) saline, (2) HMCuS@IONP-PEG, (3) HMCuS@IONP-PEG + NIR laser, (4) DOX, (5) HMCuS/DOX@IONP-PEG, (6) HMCuS/DOX@IONP-PEG + NIR laser, (7) HMCuS/DOX@IONP-PEG + NIR laser + magnet (DOX dose: 4 mg/kg, HMCuS@IONP-PEG: 9 mg /kg), respectively. The laser- treated groups were irradiated with an 808 nm laser at a power density of 2 W/cm2 for 0.5 min at 6 h after injection. And the tumor size was measured with a caliper and calculated by using the fol- lowing formula: Volume = (tumor length) � (tumor width)2/2. After treatment, blood samples were collected for chemical analy- sis and tissues were excised for hematoxylin and eosin (H&E) staining.

2.8. Statistical analysis

Quantitative data shown in this article were presented as the mean ± SD and analyzed by using Student’s t test. A P-value <0.05 was considered statistically significant.

3. Results and discussion

3.1. Synthesis and characterization of HMCuS/DOX@IONP-PEG

Highly uniform hollow mesoporous CuS NPs (HMCuS NPs) with spherical shape and good monodispersity were first synthesized with an ion-exchanging process and characterized with TEM. As depicted in Fig. 1A, a, the transparency core of HMCuS NPs revealed their hollow interior with particle size of 100 nm, while the shell with a thickness of about 20 nm exhibited distinct mesoporous characteristics. Moreover, the EDS spectrum (Fig. S1) illustrated a strong correlation of Cu and S elements in HMCuS NPs. The valence states of HMCuS were determined by XPS measurement (Fig. S2). The doublet features of Cu 2p spectrum arose because of spin orbit splitting. As depicted in Fig. S2, the Cu 2p peaks at 933.6 and 954.1 eV were associated with Cu(II), while the peaks 932.6 and 952.5 eV were assigned to Cu(I). This indicated that Cu(I) was also existed in the products.

The features of hollow mesoporous CuS NPs, such as their large surface area and great pore volume, made them candidates for nanocarriers in which small DOX molecules could be loaded as guest molecules inside the pores in doses sufficient for high thera- peutic efficacy [9,27]. Despite the high drug storage capacity (336 lg/mg) of HMCuS/DOX, DOX leakage through mesoporous without gatekeeper modification might take place during drug delivery in vivo [12,13]. Herein, the individual aqueous IONPs with size ranging from 4 to 10 nm were prepared as gatekeepers for drug controlled release (Fig. 1A, b), and the dynamic light scatter- ing analysis exhibited an average size of IONPs was 8.7 ± 0.6 nm (Fig. S3). Afterwards, the outer surface of HMCuS/DOX was capped with multifunctional IONPs to obtain HMCuS/DOX@IONP mainly through two possible interactions between IONPs and HMCuS- NH2: 1) the electrostatic interaction between the positively charged HMCuS-NH2 (16.8 ± 1.4 mV) and negatively charged IONPs (�8.5 ± 0.8 mV) (Fig. S4); 2) the coordination interactions between the amino groups on the surface of HMCuS-NH2 and Fe atoms. Nev- ertheless, along with the increase of metal content in the hybrid nanoplatform during this reaction, HMCuS/DOX@IONP with poor dispersion was not suitable for biological systems (Fig. 1B, b). In order to tackle this issue, hydrophilic PEG modification endowed the platform with biocompatibility and high aqueous solubility of 5 mg/mL (Fig. 1B, c). Moreover, TEM images of HMCuS/ DOX@IONP-PEG (Fig. 1A, c) provided direct evidence of IONPs dis- tribution on the outer surface of HMCuS due to an evident coating morphology by forming an uniform and dense layer, though PEG modification barely influenced the structure. As depicted in Fig. S5, the dynamic light scattering analysis showed an average size of HMCuS/DOX@IONP-PEG was 124.5 ± 3.8 nm, which was consistent with the TEM results. The surface charge of HMCuS/ DOX@IONP-PEG was further examined to be around � 20.3 ± 2.1 mV (Fig. S4), which was beneficial for individual disper- sion and long blood circulation property of the nanoparticles [28]. In this system, the conjugating ratio of IONPs and PEG were inves- tigated. The amount of modified IONPs was about 11.8% with an ICP-MS quantitative analysis of Fe. Then TGA was performed to determine the amount of organic PEG grafted onto the nanoparti- cles (Fig. S6). Pure PEG degraded completely at about 617 �C, HMCuS@IONP and HMCuS@IONP-PEG showed about 33.9% and 42.5% weight losses at 617 �C, respectively, thus the relative

Fig. 1. Characterization of the nanoplatform. (A) TEM images of HMCuS NPs (a), IONPs (b), HMCuS/DOX@IONP-PEG (c) and HMCuS/DOX@IONP-PEG after NIR irradiation (808 nm, 2 W/cm2, 10 min) (d); (B) The stability of HMCuS/DOX (a), HMCuS/DOX@IONP (b) and HMCuS/DOX@IONP-PEG (c) dispersed in PBS buffer; (C) Leakage of DOX from HMCuS/DOX-PEG and HMCuS/DOX@IONP-PEG stored at 37 �C; (D) X-ray diffraction (XRD) pattern; (E) FT-IR spectra; (F) UV–vis absorption spectra; (G) N2 sorption isotherms and pore size distribution plots of HMCuS, HMCuS/DOX@IONP-PEG and HMCuS/DOX@IONP-PEG after NIR irradiation (808 nm, 2 W/cm2, 10 min).

406 Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413

amount of PEG grafted onto nanoparticles was about 8.6%. The loading efficiency of DOX on HMCuS with or without IONPs were measured to be 336 and 118 lg/mg, respectively. And the gate- keeper of IONPs with admirable capping efficiency should be responsible for the higher loading capacity of HMCuS/

DOX@IONP-PEG. Then the capping efficiency of IONPs was further assessed by leakage experiment (Fig. 1C). In the case of HMCuS/ DOX-PEG group, about 31.4% of DOX leakage was observed after 7 days of storage. While little DOX leaked (5.2%) in the formation of HMCuS/DOX@IONP-PEG and IONPs. Thus, the gatekeeper of

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IONPs should be responsible for the admirable capping efficiency and was supposed to minimize premature drug release during the drug delivery in vivo.

Subsequently, to obtain more insight into the nanoparticles, various characterization of HMCuS@IONP-PEG without drug were carried out to verify the successful IONPs capping and PEG modifi- cation. According to the XRD patterns in Fig. 1D, the three strong peaks of as-prepared individual HMCuS [(102), (103) and (110)] matched exactly those of the covellite CuS (JCPDS No. 06-0464) while peaks of IONPs [(311), (511) and (440)] matched those of face-centered cubic Fe3O4 (JCPDS No. 75-1609) [29,30]. The sam- ples of HMCuS@IONP showed the typical patterns of both CuS and Fe3O4, which could be indexed to the successful conjugation of the two. Then the reaction was further supported by FT-IR results in Fig. 1E. The appearance of strong N–H peak at 1640 cm�1 substantiated the formation of amination derivative of HMCuS (HMCuS-NH2). Additionally, the new peak at 570 cm

�1

owing to of Fe–O lattice mode of IONPs and peak at 1650 cm�1

of the characteristic –NH–CO– stretching vibration further revealed the successful IONPs capping and PEG modification, respectively. Next, the optical property of HMCuS@IONP-PEG was investigated. As Fig. 1F showed, both bare HMCuS and IONPs exhibited broad absorption throughout the visible and NIR region. The strong absorption of HMCuS in NIR region (k = 700–1000 nm) was consistent with previous reports due to SPR arising from p- type carriers [31]. And the absorption band of IONPs was mainly attributable to the transitions from atomic 3d states of the Fe2+

and Fe3+ ions to the 4s band with an onset near 2 eV [17]. More- over, it was noteworthy that HMCuS@IONP-PEG showed a remark- able enhancement of absorption in NIR region, together with a redshift of adsorption edge in comparison with IONPs as well as HMCuS alone. Such phenomenon could be explained by the fact

Fig. 2. Photothermal heating effect and in vitro release profiles. (A) Infrared thermal im after irradiation by an 808 nm laser for 3 min; (B) The photothermal heating curves of H laser; (C) Release curves of DOX from HMCuS/DOX@IONP-PEG with or without NIR irra

that surface plasmon coupling effect in hybrid metallic nanoparti- cles generated highly enhanced local electromagnetic field, thus extending beyond the resonant plasmon excitation energy and fur- ther increasing effective absorption cross sections [32,33].

Encouraged by the enhanced absorption capacity of our mate- rial in NIR region as mentioned above, we moved on to investigate its photothermal effect. As depicted in Fig. 2A, the heat-generating capacity of HMCuS (4T = 29.7 �C, 100 lg/mL) was higher than that of IONPs (4T = 8.2 �C, 20 lg/mL) after NIR irradiation for 3 min, and this was in sharp contrast to that of PBS (4T = 2.1 �C). Surpris- ingly, the temperature of HMCuS/DOX@IONP-PEG (200 lg/mL, containing 100 lg/mL HMCuS and 20 lg/mL IONPs) increased by even 43.0 �C, indicating a favorable photothermal transduction efficiency of IONPs capped HMCuS owing to the local field enhanced absorption intensity. Even though the formation sub- 10 nm Fe3O4@Cu2-xS core-shell nanoparticles have been reported by Tian et al., their local field enhancement of PTT was rarely explored [34]. Then Fig. 2B revealed that the photothermal effect of HMCuS/DOX@IONP-PEG occurred in both concentration- and time-dependent manners. Then the photothermal conversion effi- ciency of HMCuS/DOX@IONP-PEG was measured. Briefly, NPs dis- persions were continuously illuminated by an 808 nm laser with a power of 2 W/cm2 until the temperature reached a plateau. The irradiation source was then switched off while the temperature was monitored to determine the rate of heat transfer from the sys- tem (Fig. S7). Following a previously reported procedure (See also the detailed calculation of photothermal conversion efficiency in the Supplementary data) [35], the photothermal conversion effi- ciency (g) of HMCuS/DOX@IONP-PEG was calculated to be 42.12%. This value was higher than that of recently reported Cu9S5 NCs (25.7%) [36]. Therefore, such extraordinary photother- mal effect of HMCuS/DOX@IONP-PEG suggested it could serve as

ages of centrifuge tubes with PBS, HMCuS NPs, IONPs and HMCuS/DOX@IONP-PEG MCuS/DOX@IONP-PEG with different concentrations after irradiation by an 808 nm diation.

Fig. 3. Magnetic properties of HMCuS/DOX@IONP-PEG. (A) Magnetization loops of IONPs and HMCuS/DOX@IONP-PEG, the inset showed that the HMCuS/DOX@IONP- PEG was rapidly attracted to one side of the cuvette after adding external magnetic field for 5 min; (B) T2-weighted MR photographs of HMCuS/DOX@IONP-PEG in aqueous solution with different Fe concentrations; (C) Corresponding relaxation rate r2 (1/T2) of HMCuS/DOX@IONP-PEG as a function of Fe concentration.

408 Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413

a promising photothermal agent for thermal ablation in cancer therapy.

A previous study explained that copper ions leaking from cop- per sulfide nanocrystals under NIR irradiation would be subjected to similar redox reactions with surrounding medium primarily via a modified Haber-Weiss cycle [37], and then proved the enhanced �OH levels (up to 83.5%) generation under NIR irradiation compared to the no-laser-treated group by electron spin resonance spec- troscopy (ESR) [5]. To further provide the evidence of the PDT effect in this system, the �OH generation by HMCuS/DOX@IONP- PEG was measured by degradation reaction of the methylene blue (MB) molecules due to the quenching of �OH in a cell-free experi- ment. The UV–vis spectra in Fig. S8 showed that the characteristic absorption peaks of MB at 290 and 680 nm decreased rapidly trea- ted with the NPs in an irradiation time-dependent manner, which demonstrated the �OH generation under NIR irradiation. According to the above results, we believe the HMCuS/DOX@IONP-PEG as photodynamic agents would offer the opportunity for PDT to attain a more effective antitumor nanotherapy.

Besides, the TEM image after NIR irradiation was also taken, with results exhibiting an appreciable removal of IONPs to a cer- tain extent from the outer surface of HMCuS (Fig. 1A, d). To the best of our knowledge, the coordinate bonds are commonly considered to be comparable to, but weaker than covalent bonds [38]. Most interestingly, heating up the system gradually leads to thermally induced cleavage of coordinate bond [39]. And this phenomenon has been reported in a published literature [21], which substanti- ated the photo-hyperthermia induced by Pd nanosheets as pho- tothermal agents resulted in coordinate bond cleavage. Thus, the removal of IONPs-caps could be probably interpreted by the fact that the photothermal effect weakened the coordination between HMCuS-NH2 and IONPs under NIR irradiation. Next, the NIR- induced removal of IONPs was further demonstrated by nitrogen adsorption-desorption isotherms (Fig. 1G). HMCuS itself displayed a type of V isotherm with a well-defined hysteresis loop revealing well-developed mesoporous characteristics. As listed in Table S1, the specific Brunauer–Emmett–Teller (BET) surface area of HMCuS was measured to be 155.8 m2/g and the average pore size was around 5.1 nm, allowing small drug molecules, DOX (size of 1.53–1.19 nm), to spread into the hollow interior through the mesoporous shells. In contrast, after DOX loading and IONPs cap- ping, the striking decreases in the surface area (22.3 m2/g) and pore size (almost to zero) were observed, which indicated an unex- ceptionable pore blocking to realize ‘‘zero premature release”. Whereas, the surface area and pore size of HMCuS/DOX@IONP- PEG after NIR irradiation increased to 96.4 m2/g and 3.2 nm, respectively, mainly due to the effective removal of IONPs-caps triggered by NIR stimulus.

In light of these desirable properties of HMCuS/DOX@IONP-PEG under NIR irradiation, it was interesting to explore its drug release behavior. Fig. 2C showed that the no-laser-treated group displayed a sustained-release property along with the time progress and only a small amount of DOX (24.4%) released within 14 h. By contrast, a burst release of DOX occurred with NIR irradiation and an increased release amount about 57.8% was observed, indicating a NIR-responsive controlled drug release profile in an impulsive manner. On the basis of the NIR-induced removal of IONP-caps as mentioned above, it was not surprised that the NIR stimulus could trigger the encapsulated DOX release from nanoparticles. Given the fact that the normal body temperature was about 37 �C, far lower to cleave the coordinate bond, few IONPs may detached from CuS during the delivery process. Therefore, the attachment of IONPs on HMCuS would be stable during the drug delivery in vivo, and the drug would release in a burst manner at the tumor site under a locally applied external NIR irradiation. In

this sense, it was plausible to believe that such smart controllable on-demand cargo delivery (HMCuS/DOX@IONP-PEG) with spatial/ temporal resolution was expected to minimize the adverse side effects together with enhancing antitumor efficacy in vivo.

The magnetic properties of HMCuS/DOX@IONP-PEG were then evaluated. As shown in Fig. 3A, similar to IONPs, the S-shape mag-

Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413 409

netization loop of HMCuS/DOX@IONP-PEG exhibited typical super- paramagnetic characteristic. The lower saturation magnetization (Ms) of HMCuS/DOX@IONP-PEG (44.8 emu/g) than IONPs (69.1 emu/g) might be mainly due to the presence of the nonmag- netic components (HMCuS, PEG and DOX etc.). Meanwhile, it was clearly seen that HMCuS/DOX@IONP-PEG could be rapidly manip- ulated and controlled by an external magnetic field, showing an effective magnetic response. Then, the magnetic resonance signal enhancing capability of HMCuS/DOX@IONP-PEG was further assessed by T2-weighted MR images (Fig. 3B), revealing a remark- able concentration-dependent darkening effect. With the high transverse relaxivity (r2 = 29.5 mM

�1 s�1) (Fig. 3C), HMCuS/ DOX@IONP-PEG as a promising T2 MRI contrast agent merited its usage for theranostic biomedical applications.

Fig. 4. In vitro cell experiments. (A) Confocal images of MCF-7 cells incubated with HMCu in MCF-7 cells of control group (a), DOX (b), HMCuS@IONP-PEG (c) and HMCuS/DOX@IO NIR irradiation (808 nm, 2 W/cm2, 3 min), the free DOX group used an equivalent DOX c MCF-7 cells treated with different HMCuS/DOX@IONP-PEG concentrations; (E) Photo of HMCuS/DOX@IONP-PEG treated MCF-7 cells in the presence of a magnet (right). DCFH fo 1, 2, 3 represented above the magnet, the edge of the magnet and outside the magnet, re figure legend, the reader is referred to the web version of this article.)

3.2. Cellular experiments

Keeping in mind the appreciable cell-free in vitro NIR- responsive controlled release profile of HMCuS/DOX@IONP-PEG, it was highly urgent to test its practicability in MCF-7 cells. Confo- cal laser scanning microscopy (CLSM) images of HMCuS/ DOX@IONP-PEG in Fig. 4A showed that an insignificant DOX fluo- rescence intensity was observed in the perinuclear cytoplasm owing to the fluorescence quenching effect of metallic nanomate- rials [40]. While a stronger red fluorescence accumulated in nucleus after NIR irradiation in comparison with the no-laser- treated group as expected. These results provided direct evidence of NIR-responsive controlled DOX release in vitro, which was required to realize higher cytotoxicity to tumor cells. In addition,

S/DOX@IONP-PEG without or with NIR irradiation; (B) Detection of intracellular ROS NP-PEG (d); (C) Cytotoxicity assays of MCF-7 cells after different treatments under oncentration to the HMCuS/DOX@IONP-PEG group; (D) T2-weighted MR images of the cell culture plate in the presence of a magnet (left) and fluorescence images of r ROS detection (green) and released DOX fluorescence (red) were recorded. Position spectively. *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this

410 Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413

the level of intracellular ROS induced by HMCuS as photosensitizer was further investigated by using DCFH-DA fluorescent probe. For HMCuS@IONP-PEG group (Fig. 4B, c), a negligible fluorescence of DCFH was observed without NIR irradiation, while the laser- treated group displayed an enhanced green fluorescence with sig- nal increasing by almost seven times (Fig. S9). Generally, The ROS generation by HMCuS under NIR irradiation could be explained by the fact that copper ions leaking from HMCuS would be subjected to similar redox reactions with surrounding medium primarily via a modified Haber-Weiss cycle [5]. Besides, DOX group also exhib- ited a dim fluorescence intensity (Fig. 4B, b). And it was also note- worthy that HMCuS/DOX@IONP-PEG displayed the highest fluorescence intensity under NIR irradiation, indicating a great deal of ROS generation (Fig. 4B, d). To the best of our knowledge, ROS certainly would induce irreversibly oxidation damage to DNA and proteins, as well as cause strong pro-inflammatory and pro- apoptotic effects to some extend [41]. From this viewpoint, ROS generation by HMCuS/DOX@IONP-PEG would offer the opportu- nity for PDT to attain a more effective antitumor nanotherapy.

Next, in vitro cytotoxic effects of various formulations were evaluated on MCF-7 cells by SRB assay in this section. It was found that the cell viability of carrier was unaffected even at high concen- trations up to 500 lg/mL (Fig. S10), suggesting that HMCuS@IONP- PEG possessed good biocompatibility for biological applications. Under such circumstances, a NIR irradiation was conducted and a concentration-dependent cytotoxicity induced by HMCuS@IONP- PEG was observed with a decreased cell viability of 61.7 ± 1.9% at 24 lg/mL (Fig. 4C), which could be explained by the effective pho- totherapy (enhanced PTT as well as PDT) of the hybrid platform. In addition, HMCuS@IONP-PEG as a photosensitizer and drug carrier simultaneously could offer a platform for combined chemo- phototherapy. As illustrated in Fig. 4C, all groups displayed the concentration-dependent cytotoxicity. It was of great importance to note that only HMCuS@IONP-PEG under NIR irradiation (pho- totherapy) or DOX (chemotherapy) alone could not realize the ideal therapeutic efficacy. Nevertheless, the cell viability of HMCuS/DOX@IONP-PEG (HMCuS@IONP-PEG concentration: 24 lg/mL) under NIR irradiation declined significantly to 7.8 ± 1.3%, considerably lower than that of DOX (42.2 ± 2.5%) or HMCuS@IONP-PEG (61.7 ± 1.9%) group. Thus, the synergistic com- bination of chemo-phototherapy with enhanced cytotoxic effect occurred and was bound to improve the therapeutic index of drugs with high efficiency. Moreover, the enhanced DOX release upon exposing to NIR stimulus was also responsible for the prominent cell-killing effect.

In the end, the magnetic properties of HMCuS/DOX@IONP-PEG were further investigated in vitro. In agreement with Fig. 4B and A, green or red fluorescence for ROS detection and DOX release under NIR irradiation, respectively, were detected in Fig. 4E. Strong fluorescence in cells was observed above the magnet (position 1) in the whole vision, while the cells outside the magnet (position 3) showed a dim fluorescence intensity. At the edge of the magnet (position 2), both red fluorescence of released DOX and green fluorescence of DCFH had obvious demarcation in the half vision. The above results demonstrated the promising potential of HMCuS/DOX@IONP-PEG in magnetic targeted theranostic appli- cations. Meanwhile, cells treated with HMCuS/DOX@IONP-PEG displayed a significant negative contrast enhancement with the increase of the [Fe] concentration (Fig. 4D), successfully suggesting it as an promising T2 MRI contrast agent for cell labeling.

3.3. In vivo experiments

Prior to investigating the anti-tumor efficacy, the in vivo biodis- tribution of nanoparticles labeled with the near-infrared dye (IR783) would be made clear in tumor-bearing mice by a non-

invasive optical imaging technique (Fig. 5A, a). And semi- quantitative biodistribution analysis of the tumor site was per- formed over time (Fig. S11). Compared to IR783 group, evident flu- orescence signals of HMCuS/IR783@IONP-PEG were widely distributed throughout the whole body within 3 h, indicating their prolonged circulation features due to PEG modification. Intrigu- ingly, the fluorescence intensity preferentially accumulated at the tumor region as a function of time and reached a maximum at 6 h post-injection, probably resulting from the enhanced perme- ability and retention effect (EPR effect). When a magnet was glued onto the tumor site, elevated fluorescence signals were observed at the tumor site and persisted for more than 24 h post-injection, demonstrating the favorable magnetic tumor targeting efficiency. Next, the inspiring tumor targeting efficiency was further con- firmed by ex vivo fluorescence imaging (Fig. 5A, b) and analysis (Fig. 5B). The fluorescence intensity of HMCuS/IR783@IONP-PEG + magnet group at the tumor region was �4.8 and �1.7 times as compared to that of free IR783 and HMCuS/IR783@IONP-PEG group, respectively. Because of the combined function of prolonged circulation characteristics and tumor targeting ability (EPR effect and magnetic tumor targeting effect), this HMCuS@IONP-PEG based drug delivery system should play an important role in its therapeutic performance. Moreover, considering the maximum accumulation time of fluorescence, HMCuS@IONP-PEG mediated phototherapy or MR imaging would be conducted at 6 h after injection.

Furthermore, the DOX distribution of each group was investi- gated after exposure to a NIR irradiation at 6 h post-injection (Fig. S12). Compared to the DOX group, HMCuS/DOX@IONP-PEG group exhibited a superior tumor targeting ability and NIR- responsive controlled drug release behavior, which was consistent with the in vivo optical imaging results. In the meanwhile, the rel- atively high drug distribution of nanoparticles in liver and lung could be probably interpreted by the sessile macrophages present there [42]. Furthermore, Fig. S12 showed that HMCuS/DOX@IONP- PEG group could significantly decreased the distributions of DOX in heart, even though higher fluorescence signals of NPs in heart was found in optical imaging. Thus the distribution of IR873 could not represent the distribution behavior of DOX completely.

As compared with optical imaging modality, MR imaging with high spatial resolution is one of the most widely used clinical imag- ing tools in cancer diagnosis and prognosis [43]. In light of this, the in vivo T2-weighted MRI contrast performance of the nanoparticles was carried out as a proof-of-concept trial (Fig. 5C). In the case of HMCuS/DOX@IONP-PEG group, an appreciable darkening effect in the tumor site was observed in comparison with the control group. What’s more, the T2-weighted signals in tumor region of magnet- treated group were extraordinarily lower than HMCuS/ DOX@IONP-PEG group, showing a 2.5-fold reduction in the signal intensity due to magnetic targeting effect (Fig. 5D), which was in consistent with the in vivo optical imaging data. Reasonably, the contrast enhancing effect of HMCuS/DOX@IONP-PEG as an MRI contrast agent offered compelling chances for multimodality ther- anostic applications in cancer treatments.

Encouraged by the notable tumor accumulation of HMCuS@IONP-PEG described in imaging data, we moved on to evaluate the anti-tumor efficacy of our system in tumor-bearing mice. The changes of relative tumor volumes (V/V0) after various treatments were recorded as a function of time in Fig. 5E. The HMCuS@IONP-PEG treatment exhibited no decent tumor regres- sion with tumor volumes increasing rapidly. While exposed to a NIR irradiation, the tumor growth was partially inhibited to some extent with V/V0 of 4.01 ± 0.28. And a moderate growth inhibition effect with V/V0 of 3.13 ± 0.21 was observed in the case of DOX group. Unfortunately, neither purely phototherapy nor chemother- apy at the study endpoint could ideally clean up the tumors. In

Fig. 5. In vivo experiments. (A) (a) Time-dependent in vivo optical imaging of tumor bearing mice in different treatment groups, the tumor regions were marked by red circle; (b) Ex vivo optical imaging of the dissected organs at 24 h post-injection. Note: (I) free IR783; (II) HMCuS/IR783@IONP-PEG; (III) HMCuS/IR783@IONP-PEG + magnet. (B) Biodistribution of IR783 in each tumor and organ; (C) In vivo T2-weighted MR images of tumor-bearing mice (top row) and tumor site (bottom row) under different treatments: (a) Control group, (b) HMCuS/DOX@IONP-PEG and (c) HMCuS/DOX@IONP-PEG + magnet, the tumor regions were marked by red ellipse; (D) Average MRI signal intensity of tumor measured from MR images shown in (C); (E) Tumor growth curves in different treatment groups of tumor-bearing mice; (F) H&E stained tumor tissues harvested from different groups of mice: (a) Control group, (b) HMCuS@IONP-PEG, (c) HMCuS@IONP-PEG + NIR, (d) DOX, (e) HMCuS/DOX@IONP-PEG, (f) HMCuS/DOX@IONP-PEG + NIR and (g) HMCuS/ DOX@IONP-PEG + NIR + magnet. *p < 0.05, **p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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contrast, the HMCuS/DOX@IONP-PEG under NIR irradiation reduced the tumor growth at the initial level with V/V0 of 0.97 ± 0.15. Impressively, the tumors were almost completely sup- pressed without recurrence with V/V0 of 0.41 ± 0.13 after adding the magnetic field, displaying an unexceptionable therapeutic effect. Pathology changes of tumors were further investigated through H&E staining to reveal the therapeutic mechanism. Fig. 5F showed that typical pathological characteristics such as intact shape and tight arrangement were observed in the control and HMCuS@IONP-PEG treated group. As expected, HMCuS/ DOX@IONP-PEG group under NIR irradiation and magnetic field exhibited the most serious malignant necrosis with intercellular blank and fragmentation to a significant extent, although apparent cell apoptosis in view was also found in other groups.

Such striking anti-tumor therapeutic efficacy with multi- mechanisms for HMCuS/DOX@IONP-PEG + NIR + magnet group might be contributed to the following factors: 1) the prolonged cir- culation characteristics and favorable magnetic tumor targeting ability reinforced the significant accumulation in tumor site; 2) the combination of chemo-phototherapy inevitably resulted in a remarkably synergistic therapeutic effect for cancer treatment. Moreover, the elevated temperature by PTT effect might enhance the reactivity of ROS for tumor apoptosis according to a previous report [5]; 3) the enhanced DOX release upon exposing to NIR stimulus was also responsible for the higher therapeutic effect.

In spite of the excellent anti-tumor efficacy of the multifunc- tional drug delivery system, the detailed long-term toxicity in vivo should be put into consideration. As can be seen, no obvious body weight decrease was observed during the treatment (Fig. S13). Then blood biochemistry studies of tumor-bearing mice were performed. As depicted in Fig. S14, both the liver function marker AST and the kidney function marker BUN were measured to be normal, suggesting no obvious hepatic and kidney disorder of mice after the treatment. Comparatively, there was a noticeable elevation of CK and LDH associated with the physiological status of heart in DOX group, while biochemical parameters in other groups remained normal levels. This finding implied that the injection of DOX possibly caused certain long-term toxic effect to heart. Then the histological analysis (Fig. S15) of heart tissue in DOX group showed a serious cardiotoxicity with extensive muscle fiber break- age and cell nucleus gather. By contrast, rare structural disturbance in heart was found in HMCuS/DOX@IONP-PEG group. Hence, all the results from anticancer activity and systematic toxicity studies implied that HMCuS/DOX@IONP-PEG with remarkable tumor tar- geting efficiency could dramatically reduce side effect of DOX to other organs and was considered a promising candidate for cancer treatments.

In our study, this system have demonstrated their abilities to enhance therapeutic efficacy and reduce unwanted side effects in the experiments. What’s more, there were many other reported theranostics nano-agents such as IONPs decorated MoS2 nanosheets for imaging guided photothermal therapy which had shown a number of advantages compared with conventional chemotherapy [44]. However, there are still many challenges ahead toward further clinical applications. (i) The potential long term toxicity of the nanomaterials. Although no obvious toxicities of most nanoagents were observed in the tested dose ranges, it could still be tough for clinical use. Thus, the development of bio- compatible and biodegradable nanoagents for photothermal ther- apy has a much higher clinical value. (ii) The limited light penetration depth of NIR light. At present, phototherapy has made effective treatment for superficial cancerous lesions but generally failed to treat deep localized tumor. Encouragingly, phototherapy in areas with tumor invasion following the tumor removal as an additional intraoperative treatment might be an alternative approach [45]. (iii) The development of multifunctional nanocarri-

ers that enable different therapeutic mechanisms for cancer com- bination therapy may bring great opportunities to the new generation of cancer therapy [46].

4. Conclusions

In summary, the well-designed magnetic tumor targeted and NIR-responsive drug delivery system (HMCuS/DOX@IONP-PEG) developed here offered compelling highlights for multimodality theranostic applications in cancer treatments. Encouragingly, the enhanced absorption of IONPs capped HMCuS NPs in NIR region could generate elevated photothermal transduction efficiency in short time. With the spatiotemporally control of NIR irradiation, on-demand DOX release taken place due to the photo- hyperthermia. Moreover, the unexceptionable therapeutic efficacy and MR imaging in vitro and in vivo of this versatile nanoplatform will open exciting opportunities for theranostic biomedical appli- cations. Such versatile HMCuS/DOX@IONP-PEG, which confirmed significant advantages in unexceptionable therapeutic efficacy and MR imaging in vitro and in vivo, held a great promising in can- cer diagnosis and therapy.

Acknowledgements

This work was supported by Grants from the National Natural Science Foundation of China (81573364 and 81572991), Science and Technology Project of Henan Province (162102310510) and Outstanding young Talent Research Fund of Zhengzhou University (51099255).

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.actbio.2016.11. 035.

References

[1] L. Hou, X. Yang, J. Ren, Y. Wang, H. Zhang, Q. Feng, Y. Shi, X. Shan, Y. Yuan, Z. Zhang, A novel redox-sensitive system based on single-walled carbon nanotubes for chemo-photothermal therapy and magnetic resonance imaging, Int. J. Nanomed. 11 (2016) 607–624.

[2] Y. Hao, L. Wang, Y. Zhao, D. Meng, D. Li, H. Li, B. Zhang, J. Shi, H. Zhang, Z. Zhang, Y. Zhang, Targeted imaging and chemo-phototherapy of brain cancer by a multifunctional drug delivery system, Macromol. Biosci. 15 (2015) 1571– 1585.

[3] Y. Hao, B. Zhang, C. Zheng, R. Ji, X. Ren, F. Guo, S. Sun, J. Shi, H. Zhang, Z. Zhang, L. Wang, Y. Zhang, The tumor-targeting core-shell structured DTX-loaded PLGA@Au nanoparticles for chemo-photothermal therapy and X-ray imaging, J. Control. Release 220 (2015) 545–555.

[4] J. Shi, Z. Chen, L. Wang, B. Wang, L. Xu, L. Hou, Z. Zhang, A tumor-specific cleavable nanosystem of PEG-modified C60@Au hybrid aggregates for radio frequency-controlled release, hyperthermia, photodynamic therapy and X-ray imaging, Acta Biomater. 29 (2016) 282–297.

[5] S. Wang, A. Riedinger, H. Li, C. Fu, H. Liu, L. Li, T. Liu, L. Tan, M.J. Barthel, G. Pugliese, F.D. Donato, M.S. D’Abbusco, X. Meng, L. Manna, H. Meng, T. Pellegrino, Plasmonic copper sulfide nanocrystals exhibiting near-infrared photothermal and photodynamic therapeutic effects, ACS Nano 9 (2015) 1788–1800.

[6] K. Ding, J. Zeng, L. Jing, R. Qiao, C. Liu, M. Jiao, Z. Li, M. Gao, Aqueous synthesis of PEGylated copper sulfide nanoparticles for photoacoustic imaging of tumors, Nanoscale 7 (2015) 11075–11081.

[7] M. Zhou, J. Li, S. Liang, A. Sood, D. Liang, C. Li, CuS nanodots with ultrahigh efficient renal clearance for positron emission tomography imaging and image-guided photothermal therapy, ACS Nano 9 (2015) 7085–7096.

[8] Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang, J. Hu, Hydrophilic flower-like CuS superstructures as an efficient 980 nm laser- driven photothermal agent for ablation of cancer cells, Adv. Mater. 23 (2011) 3542–3547.

[9] K. Dong, Z. Liu, Z. Li, J. Ren, X. Qu, Hydrophobic anticancer drug delivery by a 980 nm laser-driven photothermal vehicle for efficient synergistic therapy of cancer cells in vivo, Adv. Mater. 25 (2013) 4452–4458.

Q. Feng et al. / Acta Biomaterialia 49 (2017) 402–413 413

[10] L. Guo, D. Yan, D. Yang, Y. Li, X. Wang, O. Zalewski, B. Yan, W. Lu, Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles, ACS Nano 8 (2014) 5670–5681.

[11] Q. Feng, Y. Zhang, W. Zhang, X. Shan, Y. Yuan, H. Zhang, L. Hou, Z. Zhang, Tumor-targeted and multi-stimuli responsive drug delivery system for near- infrared light induced chemo-phototherapy and photoacoustic tomography, Acta Biomater. 38 (2016) 129–142.

[12] J. Shi, Z. Chen, B. Wang, L. Wang, T. Lu, Z. Zhang, Reactive oxygen species- manipulated drug release from a smart envelope-type mesoporous titanium nanovehicle for tumor sonodynamic-chemotherapy, ACS Appl. Mater. Interfaces 7 (2015) 28554–28565.

[13] S. Giri, B.G. Trewyn, M.P. Stellmaker, V.S.Y. Lin, Stimuli-responsive controlled-release delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles, Angew. Chem. Int. Ed. 44 (2005) 5038–5044.

[14] Q. Gan, X. Lu, Y. Yuan, J. Qian, H. Zhou, X. Lu, J. Shi, C. Liu, A magnetic, reversible pH-responsive nanogated ensemble based on Fe3O4 nanoparticles-capped mesoporous silica, Biomaterials 32 (2011) 1932–1942.

[15] L. Wang, P. Zhang, J. Shi, Y. Hao, D. Meng, Y. Zhao, Y. Yin, D. Li, J. Chang, Z. Zhang, Radiofrequency-triggered tumor-targeting delivery system for theranostics application, ACS Appl. Mater. Interfaces 7 (2015) 5736–5747.

[16] S. Shen, S. Wang, R. Zheng, X. Zhu, X. Jiang, D. Fu, W. Yang, Magnetic nanoparticle clusters for photothermal therapy with near-infrared irradiation, Biomaterials 39 (2015) 67–74.

[17] B. Wang, S. Qu, Absorption spectra and near-electric field enhancement effects of Au- and Ag-Fe3O4 dimers, Appl. Surf. Sci. 292 (2014) 1002–1008.

[18] S. Lakshmanan, X. Zou, M. Hossu, L. Ma, C. Yang, W. Chen, Local field enhanced Au/CuS nanocomposites as efficient photothermal transducer agents for cancer treatment, J. Biomed. Nanotechnol. 8 (2012) 883–890.

[19] H. Moon, D. Kumar, H. Kim, C. Sim, J. Chang, J. Kim, H. Kim, D. Lim, Amplified photoacoustic performance and enhanced photothermal stability of reduced graphene oxide coated gold nanorods for sensitive photo acoustic imaging, ACS Nano 9 (2015) 2711–2719.

[20] D. Lim, A. Barhoumi, R. Wylie, G. Reznor, R. Langer, D. Kohane, Enhanced photothermal effect of plasmonic nanoparticles coated with reduced graphene oxide, Nano Lett. 13 (2013) 4075–4079.

[21] W. Fang, S. Tang, P. Liu, X. Fang, J. Gong, N. Zheng, Pd nanosheet-covered hollow mesoporous silica nanoparticles as a platform for the chemo- photothermal treatment of cancer cells, Small 8 (2012) 3816–3822.

[22] N. Lee, D. Yoo, D. Ling, M. Cho, T. Hyeon, J. Cheon, Iron oxide based nanoparticles for multimodal imaging and magnetoresponsive therapy, Chem. Rev. 115 (2015) 10637–10689.

[23] J. Park, G. Maltzahn, L. Zhang, M. Schwartz, E. Ruoslahti, S. Bhatia, M. Sailor, Magnetic iron oxide nanoworms for tumor targeting and imaging, Adv. Mater. 20 (2008) 1630–1635.

[24] G. Prencipe, S. Tabakman, K. Welsher, Z. Liu, A. Goodwin, L. Zhang, J. Henry, H. Dai, PEG branched polymer for functionalization of nanomaterials with ultralong blood circulation, J. Am. Chem. Soc. 131 (2009) 4783–4787.

[25] S. Kooijmans, L. Fliervoet, R. Meel, M. Fens, H. Heijnen, P. Henegouwen, P. Vader, R. Schiffelers, PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time, J. Control. Release 224 (2016) 77–85.

[26] Y. Sahoo, A. Goodarzi, M. Swihart, T. Ohulchanskyy, N. Kaur, E. Furlani, P. Prasad, Aqueous ferrofluid of magnetite nanoparticles: fluorescence labeling and magnetophoretic control, J. Phys. Chem. B 109 (2005) 3879–3885.

[27] L. Han, Y. Zhang, X. Chen, Y. Shu, J. Wang, Protein-modified hollow copper sulfide nanoparticles carrying indocyanine green for photothermal and photodynamic therapy, J. Mater. Chem. B 4 (2016) 105–112.

[28] X. Guo, C. Shi, G. Yang, J. Wang, Z. Cai, S. Zhou, Dual-responsive polymer micelles for target-cell-specific anticancer drug delivery, Chem. Mater. 26 (2014) 4405–4418.

[29] Z. Zha, S. Zhang, Z. Deng, Y. Li, C. Li, Z. Dai, Enzyme-responsive copper sulphide nanoparticles for combined photoacoustic imaging, tumor-selective chemotherapy and photothermal therapy, Chem. Commun. 49 (2013) 3455– 3457.

[30] P. Kucheryavy, J. He, V. John, P. Maharjan, L. Spinu, G. Goloverda, V. Kolesnichenko, Superparamagnetic iron oxide nanoparticles with variable size and an iron oxidation state as prospective imaging agents, Langmuir 29 (2013) 710–716.

[31] X. Tan, X. Pang, M. Lei, M. Ma, F. Guo, J. Wang, M. Yu, F. Tan, N. Li, An efficient dual-loaded multifunctional nanocarrier for combined photothermal and photodynamic therapy based on copper sulfide and chlorin e6, Int. J. Pharm. 503 (2016) 220–228.

[32] J. Lee, E. Shevchenko, D. Talapin, Au-PbS core-shell nanocrystals: plasmonic absorption enhancement and electrical doping via intra-particle charge transfer, J. Am. Chem. Soc. 130 (2008) 9673–9675.

[33] E. Cho, C. Kim, F. Zhou, C. Cobley, K. Song, J. Chen, Z. Li, L. Wang, Y. Xia, Measuring the optical absorption cross sections of Au-Ag nanocages and Au nanorods by photoacoustic imaging, J. Phys. Chem. C 113 (2009) 9023–9028.

[34] Q. Tian, J. Hu, Y. Zhu, R. Zou, Z. Chen, S. Yang, R. Li, Q. Su, Y. Han, X. Liu, Sub- 10 nm Fe3O4@Cu2-xS core-shell nanoparticles for dual-modal imaging and photothermal therapy, J. Am. Chem. Soc. 135 (2013) 8571–8577.

[35] D. Roper, W. Ahn, M. Hoepfner, Microscale heat transfer transduced by surface plasmon resonant gold nanoparticles, J. Phys. Chem. C 111 (2007) 3636–3641.

[36] I. Kriegel, C. Jiang, J. Rodriguez-Fernandez, R. Schaller, D. Talapin, E. Como, J. Feldmann, Tuning the excitonic and plasmonic properties of copper chalcogenide nanocrystals, J. Am. Chem. Soc. 134 (2012) 1583–1590.

[37] M. Kadiiska, P. Hanna, L. Hernandez, R. Mason, In vivo evidence of hydroxyl radical formation after acute copper and ascorbic acid intake: electron spin resonance spin-trapping investigation, Mol. Pharmacol. 42 (1992) 723–729.

[38] Z. Xu, Mechanics of metal-catecholate complexes: the roles of coordination state and metal types, Sci. Rep. UK 3 (2013).

[39] M. Konopka, R. Turansky, J. Reichert, H. Fuchs, D. Marx, I. Stich, Mechanochemistry and thermochemistry are different: stress-induced strengthening of chemical bonds, Phys. Rev. Lett. 100 (2008).

[40] Z. Wang, Z. Chen, Z. Liu, P. Shi, K. Dong, E. Ju, J. Ren, X. Qu, A multi-stimuli responsive gold nanocage-hyaluronic platform for targeted photothermal and chemotherapy, Biomaterials 35 (2014) 9678–9688.

[41] X. Fu, M. Yang, M. Cao, D. Li, X. Yang, J. Sun, Z. Zhang, L. Mao, S. Zhang, F. Wang, F. Zhang, C. Fan, B. Fan, Strategy to suppress oxidative damage-induced neurotoxicity in PC12 cells by curcumin: the role of ROS-mediated DNA damage and the MAPK and AKT pathways, Mol. Neurobiol. 53 (2016) 369–378.

[42] R. Patil, J. Yu, S. Banerjee, Y. Ren, D. Leong, X. Jiang, M. Poper, B. Tsui, D. Kraitchman, H. Mao, Probing in vivo trafficking of polymer/DNA micellar nanoparticles using SPECT/CT imaging, Mol. Ther. 19 (2011) 1626–1635.

[43] M. Mertens, S. Koch, P. Schuster, J. Wehner, Z. Wu, F. Gremse, V. Schulz, L. Rongen, F. Wolf, J. Frese, V. Geshé, M. Zandvoort, P. Mela, S. Jockenhoevel, F. Kiessling, T. Lammers, USPIO-labeled textile materials for non-invasive MR imaging of tissue-engineered vascular grafts, Biomaterials 39 (2015) 155–163.

[44] T. Liu, S. Shi, C. Liang, S. Shen, L. Cheng, C. Wang, X. Song, S. Goel, T. Barnhart, W. Cai, Z. Liu, Iron oxide decorated MoS2 nanosheets with double PEGylation for chelator-free radio labeling and multimodal imaging guided photothermal therapy, ACS Nano 9 (2015) 950–960.

[45] Q. Chen, H. Ke, Z. Dai, Z. Liu, Nanoscale theranostics for physical stimulus- responsive cancer therapies, Biomaterials 73 (2015) 214–230.

[46] L. Cheng, C. Wang, L. Feng, K. Yang, Z. Liu, Functional nanomaterials for phototherapies of cancer, Chem. Rev. 114 (2014) 10869–10939.