Albumin-Bioinspired Gd:CuS Nanotheranostic Agent for In Vivo

Oct 24, 2016 - Herein, a biocompatible Gd-integrated CuS nanotheranostic agent ... The immune response triggered by Gd:CuS@BSA-mediated PTT is ...
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Albumin-Bioinspired Gd:CuS Nanotheranostic Agent for In Vivo Photoacoustic/Magnetic Resonance Imaging-Guided Tumor-Targeted Photothermal Therapy Weitao Yang,†,‡,⊥ Weisheng Guo,§,⊥ Wenjun Le,‡ Guoxian Lv,† Fuhe Zhang,‡ Lei Shi,‡ Xiuli Wang,‡ Jun Wang,‡ Sheng Wang,† Jin Chang,*,† and Bingbo Zhang*,‡ †

School of Materials Science and Engineering, School of Life Science, Tianjin Engineering Center of Micro-Nano Biomaterials and Detection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China ‡ Institute of Photomedicine, Shanghai Skin Disease Hospital, The Institute for Biomedical Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200443, China § CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190, China S Supporting Information *

ABSTRACT: Photothermal therapy (PTT) is attracting increasing interest and becoming more widely used for skin cancer therapy in the clinic, as a result of its noninvasiveness and low systemic adverse effects. However, there is an urgent need to develop biocompatible PTT agents, which enable accurate imaging, monitoring, and diagnosis. Herein, a biocompatible Gd-integrated CuS nanotheranostic agent (Gd:CuS@BSA) was synthesized via a facile and environmentally friendly biomimetic strategy, using bovine serum albumin (BSA) as a biotemplate at physiological temperature. The as-prepared Gd:CuS@BSA nanoparticles (NPs) with ultrasmall sizes (ca. 9 nm) exhibited high photothermal conversion efficiency and good photostability under near-infrared (NIR) laser irradiation. With doped Gd species and strong tunable NIR absorbance, Gd:CuS@BSA NPs demonstrate prominent tumor-contrasted imaging performance both on the photoacoustic and magnetic resonance imaging modalities. The subsequent Gd:CuS@BSA-mediated PTT result shows high therapy efficacy as a result of their potent NIR absorption and high photothermal conversion efficiency. The immune response triggered by Gd:CuS@BSA-mediated PTT is preliminarily explored. In addition, toxicity studies in vitro and in vivo verify that Gd:CuS@BSA NPs qualify as biocompatible agents. A biodistribution study demonstrated that the NPs can undergo hepatic clearance from the body. This study highlights the practicality and versatility of albumin-mediated biomimetic mineralization of a nanotheranostic agent and also suggests that bioinspired Gd:CuS@BSA NPs possess promising imaging guidance and effective tumor ablation properties, with high spatial resolution and deep tissue penetration. KEYWORDS: biomimetic mineralization, CuS, photothermal therapy, photoacoustic, MR imaging

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PTT, imaging-guided strategies are in development to realize real-time visualization of PTT to assist with cancer therapy. Magnetic resonance (MR) imaging, as a general but powerful imaging technology, has been widely applied in disease

ancer has been recognized as one of the leading causes of mortality worldwide for decades.1,2 Among various strategies for tumor treatment, photothermal therapy (PTT) has attracted particular attention.3 Agent-mediated PTT employs photothermal agents to effectively thermally ablate cancer cells and tissues by converting absorbed laser energy into heat with minimal invasiveness and tumor-specific localization.4 To further improve the therapeutic efficiency of © 2016 American Chemical Society

Received: August 26, 2016 Accepted: October 24, 2016 Published: October 24, 2016 10245

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Scheme 1. Schematic Illustration of Gd:CuS@BSA Hybrid Theranostic Agents for In Vivo PA/MR Imaging-Guided Tumor Photothermal Therapy

bioapplications. Our group has reported a bioinspired strategy to synthesize gadolinium-based hybrid nanoparticles as a positive blood pool contrast agent with high relaxivity and a prolonged imaging time window.37 On this basis, Gd2O3/Au nanoclusters and Ag2S NIR QDs were developed by Yan’s group for in vivo imaging.38,39 Very recently, Wang et al. prepared cypate-grafted gadolinium oxide nanocrystals (CyGdNCs) for PA/MR/near-infrared imaging-guided pH-responsive PTT and illustrated the process of albumin biomineralization.40 Herein, we develop a bioinspired and straightforward strategy for biomimetic mineralization of Gd:CuS@BSA nanotheranostic hybrid NPs (Gd:CuS@BSA NPs) using an albumin-mediated strategy. The obtained Gd:CuS@BSA exhibits a pronounced increase of temperature under NIR laser irradiation with intense PA signals and acceptable longitudinal relaxivity (r1 = 16.032 mM−1·s−1), favoring PA/ MR bimodal imaging and subsequent PTT treatment. In addition, the potential immunogenic response induced by Gd:CuS@BSA is also discussed.

diagnosis because of its high spatial resolution for soft tissues, noninvasiveness, and unlimited depth of tissue penetration.5−8 However, poor sensitivity may hinder its application for accurate tumor diagnosis.9−11 Photoacoustic (PA) imaging, as a recent and promising imaging modality, integrates the advantages of optical imaging and ultrasonic imaging and has some distinguishing superiorities, including nonionization, high sensitivity, and background-free detection.12−15 Consequently, MR/PA dual-modal imaging is expected to be a promising approach for accurate cancer diagnosis with good imaging sensitivity and spatial resolution.16−18 High-performing, biocompatible contrast agents are urgently needed for imaging guidance during cancer PTT. Copper chalcogenides and, in particular, CuS nanoparticles (CuS NPs), with strong near-infrared (NIR) absorbance, photostability, and low toxicity, are ideal candidates for in vivo PA imaging and PTT.2,19−23 To date, CuS NPs have been widely synthesized in organic or aqueous phases. Although CuS NPs produced by an organic synthetic strategy have a regular morphology and high crystallinity, harsh reaction conditions are usually required, such as increased temperatures (150−180 °C), an oxygen-free atmosphere, and toxic organic solvents.21,24−27 An additional phase transfer process is required prior to biomedical applications. In contrast, an aqueous synthetic strategy can simplify the synthesis but mostly involves a relatively high reflux temperature (∼90 °C)28−30 and toxic chemical ligands.31 Consequently, it is imperative to develop a facile, mild, biocompatible, and cost-effective strategy to prepare CuS NPbased nanotheranostic agents for in vivo imaging-guided PTT. Recently, albumin-mediated biomimetically mineralized inorganic nanoparticles have attracted considerable interest due to a multitude of advantages including much milder reaction conditions (near room temperature, in aqueous solutions), “green” processing, good reproducibility, biocompatibility, and robust stability.32 Bovine serum albumin (BSA), as a commercially available protein, has been frequently reported to assist in the preparation of various inorganic nanoparticles (e.g., fluorescent Au nanoclusters,29,33 Cu nanoclusters,34 CdSe,35 and HgS36 quantum dots (QDs)) for in vivo

RESULTS AND DISCUSSION Characterization of Size, Morphology, Structure, and Composition. In this work, multifunctional Gd:CuS@BSA NPs as theranostic agents were synthesized via a bioinspired synthetic route (Scheme 1), where BSA acted as the stabilizer and reaction scaffold. Due to the great affinity of carboxyl groups to ions, Cu and Gd species were intensely anchored by BSA during the reaction. At the initial stage, the yellowish green turbid solution was observed and implied the formation of the Gd3+−BSA−Cu2+ complex. This mixed solution rapidly became transparent and a purple-blue color upon adjustment to approximately pH 12, which may be ascribed to the conformational transformation of BSA from a three-dimensional (3D) folding structure into an unfolded configuration under alkaline conditions. Upon the injection of Na2S·9H2O solution, the solution turned brown immediately, suggesting the nucleation of NPs (Figure S1 in the Supporting Information). Dark green cotton-like powder was collected by lyophilization and can be redispersed well in deionized water (Figure 1A). 10246

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Figure 1. Characterization of the main physicochemical properties of the nanoparticles. (A) Digital photos of lyophilized powder and redispersed solution. (B) TEM and HRTEM (inset) images. (C) EDS of the nanoparticles. (D) FESEM image and the corresponding elemental mapping images (S, Cu, Gd, and O) of a collection of Gd:CuS@BSA blocks. (E) CD spectra of pure BSA and Gd:CuS@BSA. (F) Size distribution and (G) UV−vis absorption spectra of the Gd:CuS@BSA NPs.

diameter, a slight increase in hydrodynamic diameter (HD) of the obtained Gd:CuS@BSA NPs (Figure 1F) indicates the presence of individual nanoparticles in the solution. Due to the adequate carboxyl groups in BSA, the obtained Gd:CuS@BSA NPs were granted a negatively charged surface (−22 mV, Figure S7 in the Supporting Information). Investigation of the optical properties (Figure 1G) revealed that the Gd:CuS@BSA NPs showed pronounced absorbance in a wide NIR range (650−1000 nm). In particular, it is demonstrated that the 980 nm laser has a better penetration ability than that at 808 nm, and the NPs showed more intense absorbance at 980 nm than at 808 nm (Figure 1G). This absorption property favors better PA imaging and PTT in vivo. To optimize the reaction conditions, the effect of the Cu/S molar ratio, reaction time, and ion concentrations on the resulting NPs were investigated. As presented in Figure 2A, more intense absorption at 980 nm was achieved when Gd:CuS@BSA NPs were synthesized at higher feeding ratio of Cu/S, and 1/8 was selected as the optimized ratio of Cu/S for typical synthesis. With a fixed Cu/S feeding ratio, Gd:CuS@ BSA NPs with a maximum absorption were obtained after reacting at 37 °C for 4 h (Figure 2B). As depicted in the UV−vis absorption spectra of Gd:CuS@ BSA (Figure 2C), the spectrum demonstrates a steady increase and higher absorbance in the NIR range (600−1000 nm) with the increasing Cu ion concentration. Moreover, the good linear fit (0.994) further reveals a significant positive correlation between the Cu2+ concentration and absorbance at 980 nm (Figure 2C, inset). The strong absorption in the NIR range demonstrates that Gd:CuS@BSA can act as a potential theranostic agent for NIR laser-induced photoacoustic imaging-guided PTT.

Notably, the size distribution and the UV−vis absorption spectrum demonstrated no noticeable changes when the NP powder was redispersed in an aqueous phase (Figure S2 in the Supporting Information). On the basis of the TEM observation, energy-dispersive spectrometry (EDS) analysis, and the X-ray photoelectron spectroscopy (XPS) spectrum (Figure 1B,C and Figure S3 in the Supporting Information), the Gd:CuS@BSA NPs are monodisperse with an average size of ∼9 nm and composition of O, Gd, Cu, and S elements. In addition, elemental mapping images (Figure 1D) and line scanning data of a Gd:CuS@BSA bulk further indicate the existence and the homogeneous distribution of S, Cu, Gd, and O elements (Figure S4 in the Supporting Information). Additionally, by means of XPS Peak 4.1 software, the XPS spectra of Gd (4d) and O (1s) are analyzed. It can be claimed that peaks at 143.737 and 141.286 eV in the Gd 4d spectrum are allocated to the Gd2O3 and Gd(OH)3, respectively. Peaks at 532.096 and 530.909 eV are the characteristics of oxygen in Gd(OH)3 and Gd2O3, while the peak at 532.6 eV is attributed to the oxygen in −COOH and −OH present in BSA (Figure S5 in the Supporting Information).37 Circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) characterizations were adopted to examine the conformation of BSA and Gd:CuS@BSA. As shown in Figure S6 in the Supporting Information, the characteristic bands of Gd:CuS@BSA NPs were very consistent with those of pure BSA, suggesting the presence of BSA on the surface of Gd:CuS NPs. As displayed in Figure 1E, compared to the CD spectra of pure BSA, the peak at 210 nm in the CD spectra of Gd:CuS@BSA NPs showed a slight blue shift, and the peak at 221 nm remarkably vanished, suggesting an increase in random coil structures. It is believed that this change in secondary structure is beneficial in controlling the synthesis of NPs.38 With respect to the TEM 10247

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Figure 2. Optimization of experimental conditions and photothermal properties of Gd:CuS@BSA NPs. (A) Absorbance spectra of Gd:CuS@ BSA NPs synthesized at Cu/S ratios of 1:1, 1:4, 1:8, and 1:16. Inset: Corresponding absorption at 980 nm. (B) Time-dependent UV−vis absorption spectra of Gd:CuS@BSA. Inset: Corresponding absorption at 980 nm. (C) UV−vis absorption spectra with increased Cu2+ concentrations (from 0 to 0.75 mM). Inset: Linear fitting plots of absorbance at 980 nm versus Cu2+ concentrations. (D) Temperature increase of Gd:CuS@BSA NPs at varying Cu2+ concentrations (0−0.96 mM, 800 μL) under laser irradiation (980 nm, 0.6 W/cm2) as a function of time (0−300 s). (E) Plot of the temperature increase (ΔT) over a period of 300 s versus Cu2+ concentration. (F) Infrared thermal images of an aqueous Gd:CuS@BSA NP droplet (Cu2+ concentration = 1.56 mM) and DI water droplet irradiated with a 980 nm laser for 60 s at varied power densities of 0.3 and 0.6 W/cm2, respectively. (G) Photothermal effect of a Gd:CuS@BSA NP aqueous solution irradiated with a 980 nm laser, and the laser was turned off after irradiation for 600 s. (H) Obtained time constant for heat transfer of this system (τs = 212.2 s) by applying linear time data versus ln θ from the cooling stage.

Figure 3. In vitro MR/PA imaging of Gd:CuS@BSA NPs. Plots of 1/T1 (A) and 1/T2 (B) versus Gd3+ concentration. (C) T1-weighted and false-color-mapped MR images of Gd:CuS@BSA and Magnevist at varying Gd3+ concentrations ranging from 0 to 1.0 mM in H2O. (D) Corresponding T1 signal enhancement. (E) Photoacoustic signal spectrum of Gd:CuS@BSA NPs at various concentrations (0−5 mg/mL). (F) Linear relationship between PA signal intensity and concentration of Gd:CuS@BSA NPs. Inset: PA imaging phantoms, consisting of various concentrations of Gd:CuS@BSA NPs embedded in agar gel cylinders.

Photothermal Effect of Gd:CuS@BSA. Encouraged by the significant absorption of the Gd:CuS@BSA NPs in the NIR

window, we further investigated their photothermal properties. A 980 nm laser was employed throughout the whole 10248

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Figure 4. Stability assessment. (A−E) Colloidal stability of Gd:CuS@BSA NPs measured using changes in UV−vis absorption. Gd:CuS@BSA was dispersed in different solutions and incubated over a period of 7 days, including (A) H2O, (B) PBS (1× , pH 7.4), (C) borate buffer (50 mM, pH 8.2), and (D) DMEM (10% fetal bovine serum, 1% PS). The insets of A−D show the variation of absorption at 980 nm. (E) Variation of size distribution of Gd:CuS@BSA NPs (inset: digital photographs). (F−I) Photostability of Gd:CuS@BSA: (F) variation in UV−vis absorption spectrum (inset: corresponding absorption at 980 nm), (G) size distribution (inset: digital photographs before and after laser irradiation), (H) photothermal conversion curves for four cycles, and (I) relaxation time stability.

In Vitro MR/PA Dual-Modal Imaging Performance. To evaluate the capacity of Gd:CuS@BSA NPs as effective T1weighted MR imaging contrast agents, their longitudinal (T1) and transverse (T2) relaxation times were measured using a 1.41 T NMR analyzer. As shown in Figure 3A, Gd:CuS@BSA exhibits a high r1 value of 16.032 mM−1·s−1 in aqueous solution, which is 5 times higher than that of the commercial Magnevist (Gd-DTPA, r1 = 3.217 mM−1·s−1) under the same conditions. The appealing MR enhancement ability of the Gd:CuS@BSA may be due to the confined tumbling of Gd3+ in a biomacromolecule, resulting in a longer rotational correlation time.44,45 In addition to the improved longitudinal relaxivity (r1), the transverse relaxivity (r2) was also significantly higher (r2 = 29.477 mM−1·s−1) than that of Magnevist (r2 = 3.843 mM−1·s−1; Figure 3B). The relatively low r2/r1 ratio (r2/r1 = 1.8 < 3) of Gd:CuS@BSA NPs contributed to producing a desired T1-weighted contrast effect.46 T1-weighted MR images (Figure 3C) and their corresponding signal intensity (Figure 3D) further confirm that the Gd:CuS@BSA NPs exhibit stronger enhanced T1 signals (800%, compared with H2O) compared with those of Magnevist (400%, compared with H2O) at the same Gd3+ concentration. This demonstrates that Gd:CuS@ BSA NPs can act as highly efficient T1-weighted MR imaging contrast agents for biomedical imaging.

experiment. As shown in Figure 2D, the temperature of the Gd:CuS@BSA droplet samples increased rapidly under continuous NIR irradiation. After 5 min irradiation (0.6 W/ cm2), the NP droplet containing 0.96 mM of Cu ions presented a dramatic temperature increase of 21.9 °C (Figure 2E), while no significant temperature increase was detected on the DI water. In addition, infrared thermal images were acquired to monitor the photothermal effect of the Gd:CuS@BSA NPs, indicating that the temperature of Gd:CuS@BSA can reach up to 45.8 °C (laser power = 0.3 W/cm2) and 50.2 °C (laser power = 0.6 W/cm2) within 60 s (Figure 2F). Therefore, Gd:CuS@BSA NPs demonstrate concentration/laser power/ irradiation time-dependent photothermal behaviors. The photothermal conversion efficiency was further evaluated according to a previous report.41 A sample temperature change curve was recorded as a function of continuous irradiation time until the solution reached a steady-state temperature, and then the laser was shut off. The time constant for heat transfer of this system can be obtained by applying linear time data versus ln θ from this cooling stage. According to the as-obtained data (Figure 2G,H), the photothermal conversion efficiency of Gd:CuS@BSA was calculated as 32.3%, which is comparable to that in previous reports.42,43 Thus, the obtained BSA-bioinspired Gd:CuS@BSA NPs hold great potentials as promising candidates for PTT. 10249

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Figure 5. In vitro biocompatibility, PTT effect, and in vivo PA/MR imaging, blood circulation profile, and biodistribution. (A) Cytotoxicity of the Gd:CuS@BSA in SK-OV-3 cells after 24 and 48 h incubation. (B) Hemolysis of Gd:CuS@BSA NPs after incubation with red bloo cells at various concentrations (0−250 μg/mL) for 2 h, using PBS and deionized water as a negative and positive control, respectively. Inset: Hemolysis photo after centrifugation. (C) Viability of SK-OV-3 cells incubated with Gd:CuS@BSA NPs at varying concentrations (0−250 mg/L) before and after irradiation for 5 min using a 980 nm laser at a power density of 0.6 W/cm2. (D) Fluorescence images of SK-OV-3 cells costained with calcein AM (live cells, green) and PI (dead cells, red) after different treatments: control, Gd:CuS@BSA only, laser only, and Gd:CuS@BSA plus laser. Scale bars: 100 μm. (E) Time-dependent in vivo PA/MR dual-modal imaging in SK-OV-3 tumor-bearing mice before and after intravenous injection of Gd:CuS@BSA at different time points. The corresponding (F) PA and (G) MR signal intensities at the tumor area. (H) Blood circulation profile of Gd:CuS@BSA NPs. (I) Biodistribution (heart, liver, spleen, lung, kidney, and tumor) of Gd:CuS@BSA NPs in the SK-OV-3 tumor-bearing mice at different postinjection time points (2, 24, and 48 h).

To assess their potential for in vitro PA imaging, Gd:CuS@ BSA NPs at various mass concentrations were embedded in agar gel cylinders to produce photoacoustic imaging phantoms on a multispectral optical tomography (MSOT) imaging system. As presented in Figure 3E,F, the Gd:CuS@BSA NPs exhibit excitation light-wavelength- and mass-concentrationdependent PA signals. A more intense signal was produced at a longer wavelength. A quantitative analysis demonstrated a linear correlation between the PA signal intensity and the Gd:CuS@BSA NP concentrations ranging from 0 to 5 mg/mL, as presented in the inset of Figure 3F. These results verify that Gd:CuS@BSA NPs can act as an ideal contrast agent for MR/ PA dual-modal imaging. Gd:CuS@BSA Stability Study. The colloidal stability of Gd:CuS@BSA was investigated through incubation with various solutions (DI water, phosphate-buffered saline (PBS), borate buffer, and Dulbecco’s modified Eagle medium (DMEM)) over 7 days. UV−vis absorption and size distribution were monitored as the key criteria to assess colloidal stability. As presented in Figure 4A−E, the UV−vis absorption curves of Gd:CuS@BSA remain almost unchanged, and the HDs were stable at approximately 15 nm. Digital photographs in the inset of Figure 4E show that no macroscopic aggregates can be observed even after storage for 7 days. Taken together, these results suggest the good colloidal stability of Gd:CuS@BSA NPs in physiological circumstances, which should benefit from the BSA coating shell. In addition, since photostability of agents used for

photothermal therapy is of great importance, the photostability of the Gd:CuS@BSA NPs was investigated by comparing their UV−vis absorption spectra, size distribution, relaxation time, and temperature variations before and after laser irradiation (Figure 4F−I). It was demonstrated that even at 7 days after laser irradiation, the UV−vis absoption is almost consistent with that of nonirradiated samples (Figure 4F), and the size of the Gd:CuS@BSA NPs remains approximately 15 nm without aggregations (Figure 4G). As shown in Figure 4H, Gd:CuS@ BSA NPs retain a rather robust photothermal conversion after four cycles of NIR laser irradiation. Moreover, the relaxation times (T1 and T2) of Gd:CuS@BSA NPs before and after exposure to laser irradiation were monitored. As summarized in Figure 4I, no significant change in their relaxivity was observed, suggesting the good structural stability of Gd:CuS@BSA NPs against laser exposure. Taken together, these results demonstrate the outstanding photostability of Gd:CuS@BSA NPs, which is attributed to infrared absorption derived from energy band−band transitions rather than surface plasmons.47 The promising stability of Gd:CuS@BSA NPs suggests their significant potential for further in vitro and in vivo applications. Cytotoxicity and Photothermal Ablation Study in Tumor Cells. Prior to in vivo applications, standard CCK-8 and hemocompatibility assays were performed to evaluate the biocompatibility of the as-prepared Gd:CuS@BSA NPs. As shown in Figure 5A, no obvious cell toxicity was observed after 24 and 48 h incubations. The relative viability was maintained up to 84% even after incubation for 48 h at the concentration of 10250

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Figure 6. In vivo thermal imaging and PTT. (A) Thermal imaging of SK-OV-3 tumor-bearing mice before and after intravenous injection with PBS or Gd:CuS@BSA followed by a 980 nm laser irradiation for 5 min. (B) Corresponding temperature change curves at tumor sites. (C) Relative tumor growth curves and (D) changes in body weight during PTT. (E) Representative dissected tumor pictures of SK-OV-3 tumorbearing mice after PTT and (F) hematoxylin and eosin (H&E)-stained slices of tumor tissues collected from different groups after treatment.

250 μg/mL. Likewise, the calculated hemolysis ratio shown in Figure 5B is less than 2% at the maximum experimental concentration (250 μg/mL), suggesting that the Gd:CuS@BSA NPs are hemocompatible and could be administered intravenously for in vivo cancer treatment. This good biocompatibility profited from the biocompatible synthesis route, excellent water solubility, and BSA encapsulation. The in vitro PTT effect was further assessed on SK-OV-3 cells. The cytotoxicity of the Gd:CuS@BSA was also evaluated via a standard CCK-8 assay after treatment. As displayed in Figure 5C, the relative viabilities of the SK-OV-3 cells decrease dramatically alone with an increase of Gd:CuS@BSA NPs after being exposed to laser irradiation for 5 min, whereas little cytotoxicity was observed in the absence of laser irradiation. Ultimately, about 90% of the SK-OV-3 cells were killed by the Gd:CuS@BSA-induced thermal effect (concentration = 250 μg/mL). For deeper insight of the photothermal effect, cell costaining with calcein AM (live cells, green) and PI (dead cells, red) was conducted after parallel treatments. As shown in the fluorescence microscope images (Figure 5D), cells in the experimental group (Gd:CuS@BSA plus laser) show severe apoptosis caused by the induced hyperpyrexia, compared with the other three control groups, demonstrating the significant photothermal ablation effect of Gd:CuS@BSA on tumor cells in vitro. Gd:CuS@BSA for In Vivo Tumor-Targeted PA/MR Imaging. PA imaging as a hybrid imaging modality integrates the advantages of ultrasound imaging and optical imaging based

on the PA effect and thus holds great potentials for in vivo diagnosis and therapy visualizations with deep tissue penetration and fine sensitivity.48,49 In this study, each SK-OV-3 tumor-bearing mouse was intravenously administrated with 150 μL of Gd:CuS@BSA NPs in PBS (Cu2+ concentration = 13 mM), and cross-sectional PA images were acquired at different postinjection (p.i.) time points. As presented in Figure 5E, the average PA intensity derived from the tumor site increased continuously until 24 h p.i. Quantitative analysis in Figure 5F shows the PA signal intensity at 24 h p.i. was enhanced by 9fold compared with the signal preinjection, suggesting that the tumor homing of Gd:CuS@BSA benefited from the enhanced permeability and retention (EPR) effect during blood circulation.50−52 The subsequent PA signal decrease after 24 h p.i. is attributed to metabolism of a portion of the Gd:CuS@ BSA. In vivo MR imaging on SK-OV-3 tumor-bearing mice was subsequently conducted. After an intravenous injection of Gd:CuS@BSA (dosage: 0.08 mmol Gd/kg mice), T1-weighted MR images were obtained at 2, 24, and 48 h p.i. As shown in Figure 5E, the MR enhancement at the tumor site kept increasing until 24 h compared with the preinjection image. Such a significant increase in signal is attributed to the selective accumulation of Gd:CuS@BSA in the tumor area via the EPR effect. Quantitative measurement shows that the T1 signal intensity increased by 1.4-fold, 2.25-fold, and 1.5-fold at 2, 24, and 48 h, respectively (Figure 5G). These results demonstrate the high MR contrast effect of Gd:CuS@BSA, which could have 10251

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Figure 7. In vivo toxicology assessment of Gd:CuS@BSA NPs. (A−D) Blood biochemistry test: (A) AST, ALP, and ALT; (B) ALB; (C) A/G; (D) BUN. (E−L) Routine blood analysis: (E) WBC; (F) RBC; (G) HGB; (H) PLT; (I) HCT; (J) MCV; (K) MCH; (L) MCHC. (M) H&Estained images of tissues (heart, liver, spleen, lung, kidney, and intestine) of the mice harvested from the control and 15 days after intravenous injection of Gd:CuS@BSA NPs.

a synergistic effect along with PA imaging for accurate tumor diagnosis. In order to investigate the blood circulation behavior of the Gd:CuS@BSA NPs, blood samples were collected intermittently at indicated time points from the mice administrated with Gd:CuS@BSA NPs. Cu2+ levels in the blood samples were measured using inductively coupled plasma mass spectrometry (ICP-MS). As seen in the blood circulation curve (Figure 5H), a two-compartment model is presented with a first- and secondphase blood circulation time of 0.34 and 4.68 h, respectively. The mice were sacrificed after administration of Gd:CuS@BSA NPs at 2, 24, and 48 h. As shown in the distribution diagram (Figure 5I), a larger quantity of Gd:CuS@BSA NPs accumulated in organs of the reticuloendothelial system, such as the liver and spleen. At 24 h p.i., the accumulation efficiency of the Gd:CuS@BSA NPs at the tumor site was calculated to be ∼8% ID/g, which is in agreement with the PA/MR imaging results. Gd:CuS@BSA for In Vivo Photothermal Therapy. SKOV-3 tumor-bearing mice injected with Gd:CuS@BSA and PBS were irradiated using a 980 nm NIR laser for 5 min. From the real-time thermal images recorded using an IR thermal camera (Figure 6A), the temperature of the tumor regions was increased by ca. 21 °C (ca. 30−51 °C) in the presence of

Gd:CuS@BSA upon laser irradiation, which is much higher than the ca. 6 °C temperature increase observed in the control group (PBS + NIR laser; Figure 6B). This is attributed to the strong absorption of Gd:CuS@BSA NPs at 980 nm and their high photothermal conversion efficiency. Changes in tumor volume represent a direct index to evaluate therapeutic effects. As shown in Figure 6C, the tumors of group 4 (Gd:CuS@BSA + NIR laser) exhibited a remarkable regression 2 days after the PTT treatment and were completely eliminated on day 6 p.i., while the tumors of the control groups treated with PBS (group 1), Gd:CuS@BSA (group 2) only, and NIR laser only (group 3) demonstrate rapid tumor growth. This suggests that the heat (ca. 50 °C) generated by Gd:CuS@ BSA NPs upon NIR laser irradiation is sufficient for tumor ablation, and the NIR laser or Gd:CuS@BSA NPs alone have no inhibitory effects on tumor growth. Moreover, the body weight of the mice remained stable throughout the experiment (Figure 6D), suggesting no systemic side effects during PTT. Representative photographs of dissected tumors after 15 days treatment are shown in Figure 6E, which demonstrated that Gd:CuS@BSA NPs impose significant photothermal damage on the tumors upon NIR laser irradiation, which quickly causes complete tumor ablation without any regrowth during the 15 10252

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examination. Mice were treated with Gd:CuS@BSA NPs at a dosage of 20 mg/kg. For the blood biochemistry test, we focused on the six important hepatic and kidney function indicators, such as aspartate alkaline phosphatase (ALP), aminotransferase (AST), alanine albumin (ALB), aminotransferase (ALT), albumin/globulin (A/G) ratio, and blood urea nitrogen (BUN). As displayed in Figure 7A−D, no significant difference on the levels of these markers between the treatment and control groups was observed, indicating the good hepatic and kidney safety profile of Gd:CuS@BSA. Concurrently, standard blood parameters, including red blood cells (RBCs), white blood cell (WBCs), hemoglobin (HGB), platelets (PLT), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), were measured. As expected, all of these eight markers are still in the normal range, and there is no observable difference from those mice in the control group, suggesting good hemocompatibility of Gd:CuS@BSA (Figure 7E−L). To further evaluate the in vivo toxicity, especially the potential tissue damage, inflammation, or lesions that Gd:CuS@BSA may cause, H&E staining examination was conducted. As shown in Figure 7M, compared with the control group, the tissue structure of major organs from mice administered with Gd:CuS@BSA NPs are almost intact. No obvious cell necrosis or inflammatory infiltrate are observed in the major organs after 15 days. It can be inferred that the bioinspired Gd:CuS@BSA NPs are biocompatible in living mice, which is crucial for in vivo biomedical applications.

days following treatment, compared with the other three groups (PBS, laser only, and Gd:CuS@BSA NPs only). H&E staining analysis was used to further evaluate the therapeutic effect of the Gd:CuS@BSA NPs on the tumors. As displayed in Figure 6F, no obvious necrosis or karyolysis could be found in the control group samples (1−3). On the contrary, as expected, the tumor tissue structures of mice treated with Gd:CuS@BSA NPs and NIR laser irradiation (group 4) were almost completely destroyed. There was a significant increase in the number of severe necrotic cells present in the group 4 samples, indicating the thoroughness and high efficiency of Gd:CuS@BSA NPs for PTT. Major organs (heart, liver, spleen, lung, and kidney) of the mice were collected after PTT and sliced for H&E staining examinations. As shown in Figure S8 (Supporting Information), the H&E staining images indicated no noticeable signs of toxic side effects of Gd:CuS@BSAmediated PTT. Potential Immunogenic Response Study. We explored the potential immunogenic response induced by the Gd:CuS@ BSA-mediated PTT. The changes in serum levels of related cytokines, such as IFN-γ, TNF-α, and IL-2 were measured after PTT treatment. As presented in Figure S9 in the Supporting Information, it was found that these changes were insignificant at the investigated time points (2, 24, and 48 h). In addition, an immunohistochemical analysis on the CD8+ T cell expression at 3 days post-therapy was conducted. As reported in a previous study, naive CD8+ cytotoxic T cells in humans and mice are considered as the dominant cells mediating tumor regression.53 As shown in Figure S10 (Supporting Information), CD8+ T cell infiltration demonstrated a slight increasing trend in the tumors of the experimental group (Gd:CuS@BSA plus NIR laser) compared with the other three groups treated with PBS only, Gd:CuS@BSA only, and laser only. Quantitative analysis of CD8+ T cells in the tumors of mice after the different treatments was carried out using flow cytometry. As seen in Figure S11 in the Supporting Information, the percentage of CD8+ T cells in tumors treated with Gd:CuS@BSA plus laser also displays a slight increase from ∼12.3 to 15.6%. These results indicate that the immunogenic response induced by the photothermal effect of Gd:CuS@BSA was not that strong. This finding is consistent with the previous report.54 The triggered immunogenic response largely depends on the composition, morphology, size, and surface conditions of the agents and whether they carry immunologic adjuvant or not.55,56 Some unique nanomaterials, for instance, single-walled nanotubes,57 and polypyrrole composite nanoparticles with special morphology can arouse obvious immunogenic response.58 Nevertheless, as investigated by Liu’s group, for those types of non-adjuvant-like photothermal agents including graphene oxide, gold nanorods, and ICG, there is relatively weak capability of activating immune response.57 Ongoing efforts are encouraged on screening novel immunologic adjuvants to further intensify the triggered immune response. Such work in this nanotheranostic system is under consideration, and it can be believed that this protein-bioinspired synthetic route favors maintaining the biological activity of the involved adjuvants. The photothermal and immunologic combination therapy will be effective for cancer treatment by destroying the primary tumor and inhibiting cancer metastasis at distant sites.59 In Vivo Toxicology Analysis. Toxicology analysis of Gd:CuS@BSA NPs was investigated via in vivo blood biochemistry test, blood routine analysis, and H&E staining

CONCLUSIONS In summary, a kind of bioinspired Gd:CuS@BSA nanotheranostic agent was synthesized through an albuminmediated biomimetic mineralization strategy. This one-pot synthesis was found to be effective, environmentally benign, and straightforward. The as-prepared Gd:CuS@BSA NPs exhibit pronounced T1-weighted MR and PA dual-modal imaging signals as well as a strong NIR photothermal conversion capability with good stability. In vivo experimental results reveal the significant tumor-targeted PA/MR imaging performance of the Gd:CuS@BSA NPs and their facilitation of imaging-guided PTT for potent tumor ablation with high resolution and sensitivity. A preliminary study on immunologic factors suggests that Gd:CuS@BSA NPs should be integrated with additional adjuvants to intensify the immune response associated with Gd:CuS@BSA-mediated PTT. As a likely consequence of the biocompatible synthetic route, no observable toxicity or side effects were found either in vitro or in vivo. Gd:CuS@BSA NPs present great potential as a theranostic agent for bimodal imaging-guided PTT. EXPERIMENTAL SECTION Materials. Copper(II) chloride dihydrate (CuCl 2 ·2H 2 O), gadolinium(III) chloride hexahydrate (GdCl3·6H2O), and sodium sulfide nonahydrate (Na2S·9H2O) were obtained from Sigma-Aldrich. Albumin from BSA was purchased from Alfa Aesar China Co., Ltd. Sodium hydroxide was ordered from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Live/dead cell viability/cytotoxicity assay kit (calcein AM and PI) was obtained from KeyGEN bioTECH (Nanjing, China). Cell counting lit-8 (CCK-8) was ordered from Dojindo Laboratory. All chemicals were used without further purification. Deionized water (18.2 MΩ·cm resistivity at 25 °C) was used throughout the entire experiments. Synthesis of Gd:CuS@BSA NPs. Gd:CuS@BSA NPs were prepared according to a biomineralization strategy in aqueous solution 10253

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ACS Nano at physiological temperature (37 °C). Typically, an aqueous CuCl2· 2H2O solution (0.05 mmol, 5 mL, 37 °C), an aqueous GdCl3·6H2O solution (0.025 mmol, 1 mL, 37 °C), and a BSA solution (50 mg/mL, 5 mL, 37 °C) were mixed in a one-neck flask (25 mL) with magnetic stirring in a water bath (37 °C). Upon being mixed, a light green turbidity appeared. Subsequently (3 min later), a NaOH solution (1 M, 500 μL) was introduced to adjust the pH of the system to ∼12, and the mixture became a transparent deep blue. Subsequently, 400 μL of Na2S·9H2O (242.16 mg/mL) was quickly injected into the above system, and the solution turned deep brown. After 4 h, the reaction was completed, and the solution was dialyzed (MWCO = 8000− 14 000 Da) against deionized water for 24 h to remove excess Cu2+ and Gd3+. Upon lyophilization, a dark green cotton-like powder was collected and redissolved in 3 mL of PBS (1×, pH 7.4) for further use. The exact concentrations of Gd3+ and Cu2+ were measured using ICPMS. Material Characterization. The TEM images of the Gd:CuS@ BSA NPs were obtained using a Tecnai G2 F20 instrument operated at an acceleration voltage of 200 kV. Element mapping images and EDS line scanning results were obtained using a field-emission scanning electron microscope (FESEM, Ultra55, Zeiss, Germany). Dynamic light scattering (Nano ZS, Malvern) was used to record the hydrodynamic size distribution and ζ-potential of the Gd:CuS@BSA NPs. The UV−visible absorption spectrum and OD980 were measured using a Cary 50 spectrophotometer (Varian). The XPS measurements were performed using a PHI-5000 CESCA system (PerkinElmer) with radiation from an Al Kα (1486.6 eV) X-ray source. The CD spectra of pure BSA and Gd:CuS@BSA were collected using a spectropolarimeter system (BioLogic, MOS-450). Measurement of Aqueous Gd:CuS@BSA NP Photothermal Effect. A NIR laser (980 nm, BWT Beijing Ltd.) was used during the photothermal effect measurements and the following in vitro/vivo PTT experiments. An aqueous Gd:CuS@BSA solution (1 mL) was placed in a quartz cuvette at a series of concentrations (Cu2+ concentration = 0−0.96 mM) and was exposed to the NIR laser (980 nm, 0.6 W/cm2) for 5 min. Simultaneously, a thermocouple probe connected to a digital thermometer (with an accuracy of 0.1 °C) was inserted into the solution to measure the real-time temperature, and the change of temperature was recorded every 50 s. Studies on Colloidal Stability, Relaxivity Stability, and Photostability. The as-prepared Gd:CuS@BSA NPs were mixed with DI water, PBS (0.1 M, pH 7.4), borate buffer (10 mM, pH 8.2), or DMEM containing 1% penicillin−streptomycin and 10% fetal bovine serum. Subsequently, the temporal evolution profiles of the UV−vis absorption and HD were carefully monitored throughout a storage period of up to 7 days. Moreover, photostability was investigated by recording the changes in size distribution, UV−vis spectra, and relaxation times (T1 and T2) of the Gd:CuS@BSA NPs before and after NIR laser irradiation (2 h, 24 h, and 7 days). Phototemperature cycling tests were also conducted repeatedly to observe increases in temperature induced by the laser. Relaxivity Characterization and MR Imaging in Vitro. The longitudinal (T1) and transverse (T2) relaxation times of the Gd:CuS@BSA NPs were measured using a 1.41 T minispec mq 60 NMR analyzer (Bruker, Germany) at 37 °C. The in vitro MR phantom images were acquired using a MicroMR-25 mini MR system (Niumag Corporation, Shanghai, China). The measurement parameters were as follows: T1-weighted sequence, spin echo, TR/TE = 500/18.2 ms, matrix acquisition = 90 × 90, NS = 2, FOV = 80 mm × 80 mm, slices = 8, slice width = 5.0 mm, slice gap = 0.55 mm, 0.55 T, 32.0 °C. Relaxivity values (r1 and r2) were calculated by fitting the 1/T1 and 1/ T2 relaxation times (s−1) versus Gd3+ concentration (mM) curves. In Vitro Cytotoxicity and Photothermal Therapy Assays of the Gd:CuS@BSA NPs. A standard CCK-8 assay was conducted using an ovarian carcinoma cell line (SK-OV-3) to evaluate the in vitro cytotoxicity of Gd:CuS@BSA. Typically, SK-OV-3 cells (5 × 103/well) were seeded into four 96-well plates (groups 1, 2, 3, and 4), and then the cells were incubated in the culture medium for 24 h at 37 °C under 5% CO2 atmosphere. The culture medium was then removed, and the cells were incubated with fresh medium containing 100 μL of

Gd:CuS@BSA NPs at varied concentrations (0, 10, 25, 50, 100, 200, and 250 mg/L) at 37 °C under 5% CO2 for an additional 24 h (groups 1, 2, and 3) and 48 h (group 4). Subsequently, cells of group 3 were exposed to a 980 nm laser with a power density of 0.6 W/cm2 for 5 min. The CCK-8 agentia (10 μL, 5 mg/mL) was added into the four plates replacing the culture medium, and cells were incubated for a further 4 h. Finally, the OD450 value (abs.) of each well was measured using a multifunction microplate reader (Infinite M200 Pro, Switzerland). The cell live/dead assays were carried out to evaluate the efficiency of the PTT. Briefly, SK-OV-3 cells were divided into four groups (1− 4) and were seeded into a 24-well plate. Then, cells of groups 1−4 were treated with PBS, laser only (0.6 W/cm2, 5 min), Gd:CuS@BSA only (250 mg/L), and Gd:CuS@BSA (250 mg/L) with laser irradiation (0.6 W/cm2, 5 min), respectively. A mixed solution containing 2 μM of calcein AM and 8 μM of PI was then added to the wells. After being stained for 40 min, cells were washed with PBS and examined using a fluorescence microscope to observe their live/dead status. Hemolysis Assay. Blood samples obtained from volunteers (1 mL) were diluted with 2 mL of PBS, and then RBCs were separated from the serum using centrifugation at 2000 rpm for 10 min. After being washed at least four times, the RBCs were then diluted with 10 mL of PBS. Subsequently, 200 μL of the diluted RBC suspension was mixed with 1 mL of PBS (negative control), deionized water (positive control), or Gd:CuS@BSA NPs at different concentrations (10−250 mg/L). After incubation for 2 h at 37 °C, the mixtures were centrifuged at 12 000 rpm for 10 min. All of the obtained supernatants were added to a 96-well plate, and their absorbance at 570 nm was measured using a multifunction microplate reader (Infinite M200 Pro, Switzerland). Finally, the percentage hemolysis of the RBCs was calculated according to the following formula: hemolysis ratio (%) = (A(sample, 570 nm) − A(negative, 570 nm))/(mean value of A(positive, 570 nm) − A(negative, 570 nm)) × 100%. Animal Model and In Vivo PA/MR Imaging. All animal experimental procedures were performed in adherence with a standard protocol approved by the Institutional Animal Care and Use Committee of Tongji University. Tumor models were established by injecting SK-OV-3 cells (3 × 106), suspended in 70 μL of PBS, subcutaneously into the right thigh of each female Balb/c mouse (5 weeks old, body weight ca. 22 g). For in vivo PA imaging, tumor-bearing mice, anaesthetized using chloral hydrate (5%, 8 μL/g), were administered Gd:CuS@BSA NPs (Cu2+ concentration = 13 mM) dispersed in 100 μL of PBS via the tail vein. PA signals at varying time points (preinjection, 2, 24, and 48 h) were acquired using a MSOT imaging system (iTheramedical, invision 128, Germany). The excitation wavelength was set from 700 to 950 nm with a 10 nm interval, and regions of interest were fixed at 20 mm. The MR imaging study was conducted using a 0.5 ± 0.08 T MR imaging system (MesoMR, 21.3 MHz, Shanghai Niumag Corporation, China). The images were acquired before and after intravenous injection at a given time (dosage = 0.05 mmol Gd/kg mice) using a fat-saturated 3D gradient echo imaging sequence. The detailed MR imaging parameters were set as follows: FOV read = 90 mm, FOV phase = 90 mm, TR/TE = 300 ms/13.5 ms, slices = 6, slice width = 3.5 mm, slice gap = 0.5 mm, flip angle = 90°. In Vivo Photothermal Imaging and PTT. When the tumor volume reached ∼100 mm3, SK-OV-3 tumor-bearing mice were randomly divided into four groups (n = 3, in each group): namely, (1) PBS (150 μL); (2) Gd:CuS@BSA NPs only (dose = 10 mg/kg Cu2+, 150 μL); (3) NIR laser only (980 nm, 0.8 W/cm2, 5 min); (4) Gd:CuS@BSA (dose = 10 mg/kg Cu2+, 150 μL) plus NIR laser (980 nm, 0.8 W/cm2, 5 min). During the NIR irradiation, an infrared thermal camera (InfReC, Thermal Gear, G100EX/G120EX) was used to monitor the temperature changes of the tumor sites. Tumor size and body weight of the mice before and after treatment were measured using a caliper and an electronic balance, respectively. The tumor volume can be calculated according to the normal equation (volume = width2 × length/2) commonly used in previous reports.60,61 10254

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ACS Nano In Vivo Blood Circulation Behavior and Biodistribution Analysis. For the blood circulation study, the SK-OV-3 tumor-bearing mice (n = 3) were intravenously injected with 200 μL of Gd:CuS@ BSA (Cu2+ content = 0.2 mg/mL). Then, the blood samples were collected from the eye socket at the indicated time points (15 min, 30 min, 1, 2, 4, 12, and 24 h), followed by weighing and dissolution in digestive chloroazotic acid (HCl/HNO3 = 1:3) to measure the amount of Cu in the blood using ICP-MS. For the biodistribution study, SKOV-3 tumor-bearing mice (n = 3) were intravenously injected with 200 μL of Gd:CuS@BSA NPs at a dosage of 20 mg/kg. Subsequently, the mice were sacrificed at varying p.i. time points (2, 24, and 48 h), and then the major organs (heart, liver, spleen, lung, and kidney) and the tumor were collected and digested using aqua regia (6 mL, VHCl/ VHNO3 = 3/1) overnight. The solutions were placed on a heating plate to volatilize the acid components and then made up to 10 mL using deionized water. The Cu2+ content of the samples was quantified using ICP-MS. Immunohistochemistry Assay. A total of 12 female melanomatumor-bearing mice with full immunity (C57BL/6, 6−8 weeks) were used in this study (n = 3), and treatment was performed in accordance with the aforementioned protocols. Tumors located in the center were collected after 3 days of treatment, paraffin-embedded, and cut sectionally (4 μm). Then, three slides from each tumor were rehydrated and treated with citrate buffer (0.01 M, pH 6.0). They were then stained using anti-mouse CD8 (primary antibody, 1:100 dilution) for 1 h at 37 °C, followed by incubation with biotinconjugated goat anti-mouse IgG (secondary antibody) and detected using a SABC-POD kit (Wuhan Boster Bio-Engineering Ltd. Co., Wuhan, China). Tissue sections were stained using a 3,3′diaminobenzidine chromogen and a hematoxylin counterstain. All histological sections were observed using a microscope (400× objective lens), and 10 fields in each section were selected randomly. Cells with brown granules in the cell membrane or cytoplasm were considered as positively stained cells. Cytokine Detection. Serum samples were isolated from mice after different treatments and diluted for analysis. Cytokines, such as IFN-γ, TNF-α, and IL-2 were detected using ELISA kits (Dakewe Biotech) according to the vendors’ instructions. All measurements were carried out in triplicate. In Vivo Biosafety Analysis. Healthy female Balb/c mice (n = 4) were injected with 200 μL of Gd:CuS@BSA NPs at a dosage of 20 mg/kg. At 15 days p.i., the mice were anaesthetized, and the eyeballs were removed, followed by collection of blood samples for blood chemistry tests and routine blood analysis. The mice treated with PBS were used as the blank control. Subsequently, the main organs of the mice (heart, liver, spleen, lung, kidney, and intestine) were harvested and fixed using 4% paraformaldehyde. Tissue samples were then embedded in paraffin, sliced (4 μm), and stained using H&E. All of the obtained biopsy samples were imaged using an optical microscope (Leica, 20× magnification).

Author Contributions ⊥

W.Y. and W.G. contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (81571742, 81371618, 51373117, 51573128, 81601603), Shanghai Innovation Program (14ZZ039), Key Project of Tianjin Natural Science Foundation (13JCZDJC33200), National High Technology Program of China (2012AA022603), the Doctoral Base Foundation of Educational Ministry of China (20120032110027), Program for Outstanding Young Teachers in Tongji University, and the Fundamental Research Funds for the Central Universities. REFERENCES (1) Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat. Rev. Cancer 2005, 5, 161−171. (2) Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869−10939. (3) Yi, X.; Yang, K.; Liang, C.; Zhong, X.; Ning, P.; Song, G.; Wang, D.; Ge, C.; Chen, C.; Chai, Z.; et al. Imaging-Guided Combined Photothermal and Radiotherapy to Treat Subcutaneous and Metastatic Tumors Using Iodine-131-Doped Copper Sulfide Nanoparticles. Adv. Funct. Mater. 2015, 25, 4689−4699. (4) Cai, X.; Jia, X.; Gao, W.; Zhang, K.; Ma, M.; Wang, S.; Zheng, Y.; Shi, J.; Chen, H. A Versatile Nanotheranostic Agent for Efficient DualMode Imaging Guided Synergistic Chemo-Thermal Tumor Therapy. Adv. Funct. Mater. 2015, 25, 2520−2529. (5) Lee, N.; Cho, H. R.; Oh, M. H.; Lee, S. H.; Kim, K.; Kim, B. H.; Shin, K.; Ahn, T.-Y.; Choi, J. W.; Kim, Y.-W.; et al. Multifunctional Fe3O4/TaOx Core/Shell Nanoparticles for Simultaneous Magnetic Resonance Imaging and X-ray Computed Tomography. J. Am. Chem. Soc. 2012, 134, 10309−10312. (6) Guo, W.; Yang, W.; Wang, Y.; Sun, X.; Liu, Z.; Zhang, B.; Chang, J.; Chen, X. Color-Tunable Gd-Zn-Cu-In-S/ZnS Quantum Dots for Dual Modality Magnetic Resonance and Fluorescence Imaging. Nano Res. 2014, 7, 1581−1591. (7) Harisinghani, M. G.; Barentsz, J.; Hahn, P. F.; Deserno, W. M.; Tabatabaei, S.; van de Kaa, C. H.; de la Rosette, J.; Weissleder, R. Noninvasive Detection of Clinically Occult Lymph-Node Metastases in Prostate Cancer. N. Engl. J. Med. 2003, 348, 2491−2499. (8) Yang, W.; Guo, W.; Gong, X.; Zhang, B.; Wang, S.; Chen, N.; Yang, W.; Tu, Y.; Fang, X.; Chang, J. Facile Synthesis of Gd−Cu−In− S/ZnS Bimodal Quantum Dots with Optimized Properties for Tumor Targeted Fluorescence/MR In Vivo Imaging. ACS Appl. Mater. Interfaces 2015, 7, 18759−18768. (9) Weissleder, R.; Pittet, M. J. Imaging in the Era of Molecular Oncology. Nature 2008, 452, 580−589. (10) Judenhofer, M. S.; Wehrl, H. F.; Newport, D. F.; Catana, C.; Siegel, S. B.; Becker, M.; Thielscher, A.; Kneilling, M.; Lichy, M. P.; Eichner, M.; et al. Simultaneous PET-MRI: A New Approach for Functional and Morphological Imaging. Nat. Med. 2008, 14, 459−465. (11) Lee, S. Y.; Jeon, S. I.; Jung, S.; Chung, I. J.; Ahn, C.-H. Targeted Multimodal Imaging Modalities. Adv. Drug Delivery Rev. 2014, 76, 60− 78. (12) Sim, C.; Kim, H.; Moon, H.; Lee, H.; Chang, J. H.; Kim, H. Photoacoustic-Based Nanomedicine for Cancer Diagnosis and Therapy. J. Controlled Release 2015, 203, 118−125. (13) Wang, L. V.; Hu, S. Photoacoustic Tomography: In Vivo Imaging From Organelles to Organs. Science 2012, 335, 1458−1462. (14) Pu, K.; Shuhendler, A. J.; Jokerst, J. V.; Mei, J.; Gambhir, S. S.; Bao, Z.; Rao, J. Semiconducting Polymer Nanoparticles as Photoacoustic Molecular Imaging Probes in Living Mice. Nat. Nanotechnol. 2014, 9, 233−239.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b05760. Detailed experimental section, digital photos of reaction process, stability of Gd:CuS@BSA before and after lyophilization photothermal, element line scanning FESEM images, ζ-potential spectra, FTIR spectra, XPS analysis, H&E-stained images of major organs after PTT, and immune-related data (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 10255

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