Albumin-Bioinspired Gd:CuS NanotheranosticAgent for In Vivo Photoacoustic/MagneticResonance Imaging-Guided Tumor-TargetedPhotothermal TherapyWeitao 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 andDetection-Treatment Technology, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin University, Tianjin300072, China‡Institute of Photomedicine, Shanghai Skin Disease Hospital, The Institute for Biomedical Engineering & Nano Science, TongjiUniversity 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 increasinginterest and becoming more widely used for skin cancer therapy in theclinic, as a result of its noninvasiveness and low systemic adverseeffects. However, there is an urgent need to develop biocompatiblePTT agents, which enable accurate imaging, monitoring, anddiagnosis. Herein, a biocompatible Gd-integrated CuS nanotheranos-tic agent (Gd:CuS@BSA) was synthesized via a facile and environ-mentally friendly biomimetic strategy, using bovine serum albumin(BSA) as a biotemplate at physiological temperature. The as-preparedGd:CuS@BSA nanoparticles (NPs) with ultrasmall sizes (ca. 9 nm)exhibited high photothermal conversion efficiency and good photo-stability under near-infrared (NIR) laser irradiation. With doped Gdspecies and strong tunable NIR absorbance, Gd:CuS@BSA NPsdemonstrate prominent tumor-contrasted imaging performance bothon the photoacoustic and magnetic resonance imaging modalities. The subsequent Gd:CuS@BSA-mediated PTT resultshows high therapy efficacy as a result of their potent NIR absorption and high photothermal conversion efficiency. Theimmune response triggered by Gd:CuS@BSA-mediated PTT is preliminarily explored. In addition, toxicity studies in vitroand in vivo verify that Gd:CuS@BSA NPs qualify as biocompatible agents. A biodistribution study demonstrated that theNPs can undergo hepatic clearance from the body. This study highlights the practicality and versatility of albumin-mediatedbiomimetic mineralization of a nanotheranostic agent and also suggests that bioinspired Gd:CuS@BSA NPs possesspromising imaging guidance and effective tumor ablation properties, with high spatial resolution and deep tissuepenetration.

KEYWORDS: biomimetic mineralization, CuS, photothermal therapy, photoacoustic, MR imaging

Cancer has been recognized as one of the leading causesof mortality worldwide for decades.1,2 Among variousstrategies for tumor treatment, photothermal therapy

(PTT) has attracted particular attention.3 Agent-mediated PTTemploys photothermal agents to effectively thermally ablatecancer cells and tissues by converting absorbed laser energyinto heat with minimal invasiveness and tumor-specificlocalization.4 To further improve the therapeutic efficiency of

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

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

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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 foraccurate tumor diagnosis.9−11 Photoacoustic (PA) imaging, asa recent and promising imaging modality, integrates theadvantages of optical imaging and ultrasonic imaging and hassome distinguishing superiorities, including nonionization, highsensitivity, and background-free detection.12−15 Consequently,MR/PA dual-modal imaging is expected to be a promisingapproach for accurate cancer diagnosis with good imagingsensitivity and spatial resolution.16−18

High-performing, biocompatible contrast agents are urgentlyneeded for imaging guidance during cancer PTT. Copperchalcogenides and, in particular, CuS nanoparticles (CuS NPs),with strong near-infrared (NIR) absorbance, photostability, andlow toxicity, are ideal candidates for in vivo PA imaging andPTT.2,19−23 To date, CuS NPs have been widely synthesized inorganic or aqueous phases. Although CuS NPs produced by anorganic synthetic strategy have a regular morphology and highcrystallinity, harsh reaction conditions are usually required, suchas increased temperatures (150−180 °C), an oxygen-freeatmosphere, and toxic organic solvents.21,24−27 An additionalphase transfer process is required prior to biomedicalapplications. In contrast, an aqueous synthetic strategy cansimplify the synthesis but mostly involves a relatively high refluxtemperature (∼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 NP-based nanotheranostic agents for in vivo imaging-guided PTT.Recently, albumin-mediated biomimetically mineralized

inorganic nanoparticles have attracted considerable interestdue to a multitude of advantages including much milderreaction conditions (near room temperature, in aqueoussolutions), “green” processing, good reproducibility, biocom-patibility, and robust stability.32 Bovine serum albumin (BSA),as a commercially available protein, has been frequentlyreported to assist in the preparation of various inorganicnanoparticles (e.g., fluorescent Au nanoclusters,29,33 Cu nano-clusters,34 CdSe,35 and HgS36 quantum dots (QDs)) for in vivo

bioapplications. Our group has reported a bioinspired strategyto synthesize gadolinium-based hybrid nanoparticles as apositive blood pool contrast agent with high relaxivity and aprolonged imaging time window.37 On this basis, Gd2O3/Aunanoclusters and Ag2S NIR QDs were developed by Yan’sgroup for in vivo imaging.38,39 Very recently, Wang et al.prepared cypate-grafted gadolinium oxide nanocrystals (Cy-GdNCs) for PA/MR/near-infrared imaging-guided pH-respon-sive PTT and illustrated the process of albumin biomineraliza-tion.40 Herein, we develop a bioinspired and straightforwardstrategy for biomimetic mineralization of Gd:CuS@BSAnanotheranostic hybrid NPs (Gd:CuS@BSA NPs) using analbumin-mediated strategy. The obtained Gd:CuS@BSAexhibits a pronounced increase of temperature under NIRlaser irradiation with intense PA signals and acceptablelongitudinal relaxivity (r1 = 16.032 mM−1·s−1), favoring PA/MR bimodal imaging and subsequent PTT treatment. Inaddition, the potential immunogenic response induced byGd:CuS@BSA is also discussed.

RESULTS AND DISCUSSION

Characterization of Size, Morphology, Structure, andComposition. In this work, multifunctional Gd:CuS@BSANPs as theranostic agents were synthesized via a bioinspiredsynthetic route (Scheme 1), where BSA acted as the stabilizerand reaction scaffold. Due to the great affinity of carboxylgroups to ions, Cu and Gd species were intensely anchored byBSA during the reaction. At the initial stage, the yellowish greenturbid solution was observed and implied the formation of theGd3+−BSA−Cu2+ complex. This mixed solution rapidly becametransparent and a purple-blue color upon adjustment toapproximately pH 12, which may be ascribed to theconformational transformation of BSA from a three-dimen-sional (3D) folding structure into an unfolded configurationunder alkaline conditions. Upon the injection of Na2S·9H2Osolution, the solution turned brown immediately, suggesting thenucleation of NPs (Figure S1 in the Supporting Information).Dark green cotton-like powder was collected by lyophilizationand can be redispersed well in deionized water (Figure 1A).

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

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Notably, the size distribution and the UV−vis absorptionspectrum demonstrated no noticeable changes when the NPpowder was redispersed in an aqueous phase (Figure S2 in theSupporting Information). On the basis of the TEM observation,energy-dispersive spectrometry (EDS) analysis, and the X-rayphotoelectron spectroscopy (XPS) spectrum (Figure 1B,C andFigure S3 in the Supporting Information), the Gd:CuS@BSANPs are monodisperse with an average size of ∼9 nm andcomposition of O, Gd, Cu, and S elements. In addition,elemental mapping images (Figure 1D) and line scanning dataof a Gd:CuS@BSA bulk further indicate the existence and thehomogeneous distribution of S, Cu, Gd, and O elements(Figure S4 in the Supporting Information). Additionally, bymeans of XPS Peak 4.1 software, the XPS spectra of Gd (4d)and O (1s) are analyzed. It can be claimed that peaks at143.737 and 141.286 eV in the Gd 4d spectrum are allocated tothe Gd2O3 and Gd(OH)3, respectively. Peaks at 532.096 and530.909 eV are the characteristics of oxygen in Gd(OH)3 andGd2O3, while the peak at 532.6 eV is attributed to the oxygen in−COOH and −OH present in BSA (Figure S5 in theSupporting Information).37 Circular dichroism (CD) andFourier transform infrared spectroscopy (FTIR) character-izations were adopted to examine the conformation of BSA andGd:CuS@BSA. As shown in Figure S6 in the SupportingInformation, the characteristic bands of Gd:CuS@BSA NPswere very consistent with those of pure BSA, suggesting thepresence of BSA on the surface of Gd:CuS NPs. As displayed inFigure 1E, compared to the CD spectra of pure BSA, the peakat 210 nm in the CD spectra of Gd:CuS@BSA NPs showed aslight blue shift, and the peak at 221 nm remarkably vanished,suggesting an increase in random coil structures. It is believedthat this change in secondary structure is beneficial incontrolling the synthesis of NPs.38 With respect to the TEM

diameter, a slight increase in hydrodynamic diameter (HD) ofthe obtained Gd:CuS@BSA NPs (Figure 1F) indicates thepresence of individual nanoparticles in the solution. Due to theadequate carboxyl groups in BSA, the obtained Gd:CuS@BSANPs were granted a negatively charged surface (−22 mV,Figure S7 in the Supporting Information). Investigation of theoptical properties (Figure 1G) revealed that the Gd:CuS@BSANPs showed pronounced absorbance in a wide NIR range(650−1000 nm). In particular, it is demonstrated that the 980nm laser has a better penetration ability than that at 808 nm,and the NPs showed more intense absorbance at 980 nm thanat 808 nm (Figure 1G). This absorption property favors betterPA imaging and PTT in vivo.To optimize the reaction conditions, the effect of the Cu/S

molar ratio, reaction time, and ion concentrations on theresulting NPs were investigated. As presented in Figure 2A,more intense absorption at 980 nm was achieved whenGd:CuS@BSA NPs were synthesized at higher feeding ratio ofCu/S, and 1/8 was selected as the optimized ratio of Cu/S fortypical synthesis. With a fixed Cu/S feeding ratio, Gd:CuS@BSA NPs with a maximum absorption were obtained afterreacting 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 increaseand higher absorbance in the NIR range (600−1000 nm) withthe increasing Cu ion concentration. Moreover, the good linearfit (0.994) further reveals a significant positive correlationbetween the Cu2+ concentration and absorbance at 980 nm(Figure 2C, inset). The strong absorption in the NIR rangedemonstrates that Gd:CuS@BSA can act as a potentialtheranostic agent for NIR laser-induced photoacousticimaging-guided PTT.

Figure 1. Characterization of the main physicochemical properties of the nanoparticles. (A) Digital photos of lyophilized powder andredispersed solution. (B) TEM and HRTEM (inset) images. (C) EDS of the nanoparticles. (D) FESEM image and the correspondingelemental 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.

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Photothermal Effect of Gd:CuS@BSA. Encouraged bythe 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

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−visabsorption 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) Temperatureincrease 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 afunction of time (0−300 s). (E) Plot of the temperature increase (ΔT) over a period of 300 s versus Cu2+ concentration. (F) Infrared thermalimages 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 sat varied power densities of 0.3 and 0.6 W/cm2, respectively. (G) Photothermal effect of a Gd:CuS@BSA NP aqueous solution irradiated witha 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 = 212.2s) 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 variousconcentrations of Gd:CuS@BSA NPs embedded in agar gel cylinders.

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experiment. As shown in Figure 2D, the temperature of theGd:CuS@BSA droplet samples increased rapidly undercontinuous NIR irradiation. After 5 min irradiation (0.6 W/cm2), the NP droplet containing 0.96 mM of Cu ions presenteda dramatic temperature increase of 21.9 °C (Figure 2E), whileno significant temperature increase was detected on the DIwater. In addition, infrared thermal images were acquired tomonitor the photothermal effect of the Gd:CuS@BSA NPs,indicating that the temperature of Gd:CuS@BSA can reach upto 45.8 °C (laser power = 0.3 W/cm2) and 50.2 °C (laserpower = 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 temper-ature change curve was recorded as a function of continuousirradiation time until the solution reached a steady-statetemperature, and then the laser was shut off. The time constantfor heat transfer of this system can be obtained by applyinglinear time data versus ln θ from this cooling stage. According tothe as-obtained data (Figure 2G,H), the photothermalconversion efficiency of Gd:CuS@BSA was calculated as32.3%, which is comparable to that in previous reports.42,43

Thus, the obtained BSA-bioinspired Gd:CuS@BSA NPs holdgreat potentials as promising candidates for PTT.

In Vitro MR/PA Dual-Modal Imaging Performance. Toevaluate the capacity of Gd:CuS@BSA NPs as effective T1-weighted MR imaging contrast agents, their longitudinal (T1)and transverse (T2) relaxation times were measured using a1.41 T NMR analyzer. As shown in Figure 3A, Gd:CuS@BSAexhibits 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@BSAmay be due to the confined tumbling of Gd3+ in abiomacromolecule, resulting in a longer rotational correlationtime.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 = 3.843mM−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 desiredT1-weighted contrast effect.46 T1-weighted MR images (Figure3C) and their corresponding signal intensity (Figure 3D)further confirm that the Gd:CuS@BSA NPs exhibit strongerenhanced T1 signals (800%, compared with H2O) comparedwith those of Magnevist (400%, compared with H2O) at thesame Gd3+ concentration. This demonstrates that Gd:CuS@BSA NPs can act as highly efficient T1-weighted MR imagingcontrast agents for biomedical imaging.

Figure 4. Stability assessment. (A−E) Colloidal stability of Gd:CuS@BSA NPs measured using changes in UV−vis absorption. Gd:CuS@BSAwas dispersed in different solutions and incubated over a period of 7 days, including (A) H2O, (B) PBS (1× , pH 7.4), (C) borate buffer (50mM, 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) Variationof size distribution of Gd:CuS@BSA NPs (inset: digital photographs). (F−I) Photostability of Gd:CuS@BSA: (F) variation in UV−visabsorption spectrum (inset: corresponding absorption at 980 nm), (G) size distribution (inset: digital photographs before and after laserirradiation), (H) photothermal conversion curves for four cycles, and (I) relaxation time stability.

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To assess their potential for in vitro PA imaging, Gd:CuS@BSA NPs at various mass concentrations were embedded inagar gel cylinders to produce photoacoustic imaging phantomson a multispectral optical tomography (MSOT) imagingsystem. As presented in Figure 3E,F, the Gd:CuS@BSA NPsexhibit excitation light-wavelength- and mass-concentration-dependent PA signals. A more intense signal was produced at alonger wavelength. A quantitative analysis demonstrated alinear correlation between the PA signal intensity and theGd:CuS@BSA NP concentrations ranging from 0 to 5 mg/mL,as presented in the inset of Figure 3F. These results verify thatGd: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 withvarious solutions (DI water, phosphate-buffered saline (PBS),borate buffer, and Dulbecco’s modified Eagle medium(DMEM)) over 7 days. UV−vis absorption and sizedistribution were monitored as the key criteria to assesscolloidal stability. As presented in Figure 4A−E, the UV−visabsorption curves of Gd:CuS@BSA remain almost unchanged,and the HDs were stable at approximately 15 nm. Digitalphotographs in the inset of Figure 4E show that nomacroscopic aggregates can be observed even after storagefor 7 days. Taken together, these results suggest the goodcolloidal stability of Gd:CuS@BSA NPs in physiologicalcircumstances, which should benefit from the BSA coatingshell. In addition, since photostability of agents used for

photothermal therapy is of great importance, the photostabilityof the Gd:CuS@BSA NPs was investigated by comparing theirUV−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 afterlaser irradiation, the UV−vis absoption is almost consistentwith that of nonirradiated samples (Figure 4F), and the size ofthe Gd:CuS@BSA NPs remains approximately 15 nm withoutaggregations (Figure 4G). As shown in Figure 4H, Gd:CuS@BSA NPs retain a rather robust photothermal conversion afterfour cycles of NIR laser irradiation. Moreover, the relaxationtimes (T1 and T2) of Gd:CuS@BSA NPs before and afterexposure to laser irradiation were monitored. As summarized inFigure 4I, no significant change in their relaxivity was observed,suggesting the good structural stability of Gd:CuS@BSA NPsagainst laser exposure. Taken together, these results demon-strate the outstanding photostability of Gd:CuS@BSA NPs,which is attributed to infrared absorption derived from energyband−band transitions rather than surface plasmons.47 Thepromising stability of Gd:CuS@BSA NPs suggests theirsignificant potential for further in vitro and in vivo applications.

Cytotoxicity and Photothermal Ablation Study inTumor Cells. Prior to in vivo applications, standard CCK-8 andhemocompatibility assays were performed to evaluate thebiocompatibility of the as-prepared Gd:CuS@BSA NPs. Asshown in Figure 5A, no obvious cell toxicity was observed after24 and 48 h incubations. The relative viability was maintainedup to 84% even after incubation for 48 h at the concentration of

Figure 5. In vitro biocompatibility, PTT effect, and in vivo PA/MR imaging, blood circulation profile, and biodistribution. (A) Cytotoxicity ofthe 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 atvarious 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 (0−250mg/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 cellscostained with calcein AM (live cells, green) and PI (dead cells, red) after different treatments: control, Gd:CuS@BSA only, laser only, andGd: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 beforeand after intravenous injection of Gd:CuS@BSA at different time points. The corresponding (F) PA and (G) MR signal intensities at thetumor area. (H) Blood circulation profile of Gd:CuS@BSA NPs. (I) Biodistribution (heart, liver, spleen, lung, kidney, and tumor) ofGd:CuS@BSA NPs in the SK-OV-3 tumor-bearing mice at different postinjection time points (2, 24, and 48 h).

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250 μg/mL. Likewise, the calculated hemolysis ratio shown inFigure 5B is less than 2% at the maximum experimentalconcentration (250 μg/mL), suggesting that the Gd:CuS@BSANPs are hemocompatible and could be administered intra-venously for in vivo cancer treatment. This good biocompat-ibility profited from the biocompatible synthesis route, excellentwater 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 evaluatedvia a standard CCK-8 assay after treatment. As displayed inFigure 5C, the relative viabilities of the SK-OV-3 cells decreasedramatically alone with an increase of Gd:CuS@BSA NPs afterbeing exposed to laser irradiation for 5 min, whereas littlecytotoxicity was observed in the absence of laser irradiation.Ultimately, about 90% of the SK-OV-3 cells were killed by theGd:CuS@BSA-induced thermal effect (concentration = 250μg/mL). For deeper insight of the photothermal effect, cellcostaining with calcein AM (live cells, green) and PI (deadcells, red) was conducted after parallel treatments. As shown inthe fluorescence microscope images (Figure 5D), cells in theexperimental group (Gd:CuS@BSA plus laser) show severeapoptosis caused by the induced hyperpyrexia, compared withthe other three control groups, demonstrating the significantphotothermal ablation effect of Gd:CuS@BSA on tumor cells invitro.Gd:CuS@BSA for In Vivo Tumor-Targeted PA/MR

Imaging. PA imaging as a hybrid imaging modality integratesthe advantages of ultrasound imaging and optical imaging based

on the PA effect and thus holds great potentials for in vivodiagnosis and therapy visualizations with deep tissue pene-tration and fine sensitivity.48,49 In this study, each SK-OV-3tumor-bearing mouse was intravenously administrated with 150μL of Gd:CuS@BSA NPs in PBS (Cu2+ concentration = 13mM), and cross-sectional PA images were acquired at differentpostinjection (p.i.) time points. As presented in Figure 5E, theaverage PA intensity derived from the tumor site increasedcontinuously until 24 h p.i. Quantitative analysis in Figure 5Fshows the PA signal intensity at 24 h p.i. was enhanced by 9-fold compared with the signal preinjection, suggesting that thetumor homing of Gd:CuS@BSA benefited from the enhancedpermeability and retention (EPR) effect during bloodcirculation.50−52 The subsequent PA signal decrease after 24h 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 ofGd:CuS@BSA (dosage: 0.08 mmol Gd/kg mice), T1-weightedMR images were obtained at 2, 24, and 48 h p.i. As shown inFigure 5E, the MR enhancement at the tumor site keptincreasing until 24 h compared with the preinjection image.Such a significant increase in signal is attributed to the selectiveaccumulation of Gd:CuS@BSA in the tumor area via the EPReffect. Quantitative measurement shows that the T1 signalintensity increased by 1.4-fold, 2.25-fold, and 1.5-fold at 2, 24,and 48 h, respectively (Figure 5G). These results demonstratethe high MR contrast effect of Gd:CuS@BSA, which could have

Figure 6. In vivo thermal imaging and PTT. (A) Thermal imaging of SK-OV-3 tumor-bearing mice before and after intravenous injection withPBS 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 tumor-bearing mice after PTT and (F) hematoxylin and eosin (H&E)-stained slices of tumor tissues collected from different groups after treatment.

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a synergistic effect along with PA imaging for accurate tumordiagnosis.In order to investigate the blood circulation behavior of the

Gd:CuS@BSA NPs, blood samples were collected intermit-tently at indicated time points from the mice administratedwith Gd:CuS@BSA NPs. Cu2+ levels in the blood samples weremeasured 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 second-phase blood circulation time of 0.34 and 4.68 h, respectively.The mice were sacrificed after administration of Gd:CuS@BSANPs at 2, 24, and 48 h. As shown in the distribution diagram(Figure 5I), a larger quantity of Gd:CuS@BSA NPsaccumulated in organs of the reticuloendothelial system, suchas the liver and spleen. At 24 h p.i., the accumulation efficiencyof the Gd:CuS@BSA NPs at the tumor site was calculated to be∼8% ID/g, which is in agreement with the PA/MR imagingresults.Gd:CuS@BSA for In Vivo Photothermal Therapy. SK-

OV-3 tumor-bearing mice injected with Gd:CuS@BSA andPBS were irradiated using a 980 nm NIR laser for 5 min. Fromthe real-time thermal images recorded using an IR thermalcamera (Figure 6A), the temperature of the tumor regions wasincreased by ca. 21 °C (ca. 30−51 °C) in the presence of

Gd:CuS@BSA upon laser irradiation, which is much higherthan the ca. 6 °C temperature increase observed in the controlgroup (PBS + NIR laser; Figure 6B). This is attributed to thestrong absorption of Gd:CuS@BSA NPs at 980 nm and theirhigh photothermal conversion efficiency.Changes in tumor volume represent a direct index to

evaluate therapeutic effects. As shown in Figure 6C, the tumorsof group 4 (Gd:CuS@BSA + NIR laser) exhibited a remarkableregression 2 days after the PTT treatment and were completelyeliminated on day 6 p.i., while the tumors of the control groupstreated with PBS (group 1), Gd:CuS@BSA (group 2) only, andNIR 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 tumorablation, and the NIR laser or Gd:CuS@BSA NPs alone haveno inhibitory effects on tumor growth. Moreover, the bodyweight of the mice remained stable throughout the experiment(Figure 6D), suggesting no systemic side effects during PTT.Representative photographs of dissected tumors after 15 daystreatment are shown in Figure 6E, which demonstrated thatGd:CuS@BSA NPs impose significant photothermal damageon the tumors upon NIR laser irradiation, which quickly causescomplete tumor ablation without any regrowth during the 15

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&E-stained images of tissues (heart, liver, spleen, lung, kidney, and intestine) of the mice harvested from the control and 15 days after intravenousinjection of Gd:CuS@BSA NPs.

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days following treatment, compared with the other threegroups (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. Asdisplayed in Figure 6F, no obvious necrosis or karyolysis couldbe found in the control group samples (1−3). On the contrary,as expected, the tumor tissue structures of mice treated withGd:CuS@BSA NPs and NIR laser irradiation (group 4) werealmost completely destroyed. There was a significant increase inthe number of severe necrotic cells present in the group 4samples, indicating the thoroughness and high efficiency ofGd:CuS@BSA NPs for PTT. Major organs (heart, liver, spleen,lung, and kidney) of the mice were collected after PTT andsliced for H&E staining examinations. As shown in Figure S8(Supporting Information), the H&E staining images indicatedno noticeable signs of toxic side effects of Gd:CuS@BSA-mediated 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 relatedcytokines, such as IFN-γ, TNF-α, and IL-2 were measured afterPTT treatment. As presented in Figure S9 in the SupportingInformation, it was found that these changes were insignificantat the investigated time points (2, 24, and 48 h). In addition, animmunohistochemical analysis on the CD8+ T cell expressionat 3 days post-therapy was conducted. As reported in a previousstudy, naive CD8+ cytotoxic T cells in humans and mice areconsidered as the dominant cells mediating tumor regression.53

As shown in Figure S10 (Supporting Information), CD8+ Tcell infiltration demonstrated a slight increasing trend in thetumors of the experimental group (Gd:CuS@BSA plus NIRlaser) compared with the other three groups treated with PBSonly, Gd:CuS@BSA only, and laser only. Quantitative analysisof CD8+ T cells in the tumors of mice after the differenttreatments was carried out using flow cytometry. As seen inFigure S11 in the Supporting Information, the percentage ofCD8+ T cells in tumors treated with Gd:CuS@BSA plus laseralso 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 notthat strong. This finding is consistent with the previousreport.54 The triggered immunogenic response largely dependson the composition, morphology, size, and surface conditionsof the agents and whether they carry immunologic adjuvant ornot.55,56 Some unique nanomaterials, for instance, single-wallednanotubes,57 and polypyrrole composite nanoparticles withspecial morphology can arouse obvious immunogenic re-sponse.58 Nevertheless, as investigated by Liu’s group, forthose types of non-adjuvant-like photothermal agents includinggraphene oxide, gold nanorods, and ICG, there is relativelyweak capability of activating immune response.57 Ongoingefforts are encouraged on screening novel immunologicadjuvants to further intensify the triggered immune response.Such work in this nanotheranostic system is under consid-eration, and it can be believed that this protein-bioinspiredsynthetic route favors maintaining the biological activity of theinvolved adjuvants. The photothermal and immunologiccombination therapy will be effective for cancer treatment bydestroying the primary tumor and inhibiting cancer metastasisat distant sites.59

In Vivo Toxicology Analysis. Toxicology analysis ofGd:CuS@BSA NPs was investigated via in vivo bloodbiochemistry test, blood routine analysis, and H&E staining

examination. Mice were treated with Gd:CuS@BSA NPs at adosage of 20 mg/kg. For the blood biochemistry test, wefocused on the six important hepatic and kidney functionindicators, such as aspartate alkaline phosphatase (ALP),aminotransferase (AST), alanine albumin (ALB), amino-transferase (ALT), albumin/globulin (A/G) ratio, and bloodurea nitrogen (BUN). As displayed in Figure 7A−D, nosignificant difference on the levels of these markers between thetreatment and control groups was observed, indicating the goodhepatic and kidney safety profile of Gd:CuS@BSA. Con-currently, 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 meancorpuscular hemoglobin concentration (MCHC), were meas-ured. As expected, all of these eight markers are still in thenormal range, and there is no observable difference from thosemice in the control group, suggesting good hemocompatibilityof Gd:CuS@BSA (Figure 7E−L). To further evaluate the invivo toxicity, especially the potential tissue damage, inflamma-tion, or lesions that Gd:CuS@BSA may cause, H&E stainingexamination was conducted. As shown in Figure 7M, comparedwith the control group, the tissue structure of major organsfrom mice administered with Gd:CuS@BSA NPs are almostintact. No obvious cell necrosis or inflammatory infiltrate areobserved in the major organs after 15 days. It can be inferredthat the bioinspired Gd:CuS@BSA NPs are biocompatible inliving mice, which is crucial for in vivo biomedical applications.

CONCLUSIONSIn summary, a kind of bioinspired Gd:CuS@BSA nano-theranostic agent was synthesized through an albumin-mediated biomimetic mineralization strategy. This one-potsynthesis was found to be effective, environmentally benign,and straightforward. The as-prepared Gd:CuS@BSA NPsexhibit pronounced T1-weighted MR and PA dual-modalimaging signals as well as a strong NIR photothermalconversion capability with good stability. In vivo experimentalresults reveal the significant tumor-targeted PA/MR imagingperformance of the Gd:CuS@BSA NPs and their facilitation ofimaging-guided PTT for potent tumor ablation with highresolution and sensitivity. A preliminary study on immunologicfactors suggests that Gd:CuS@BSA NPs should be integratedwith additional adjuvants to intensify the immune responseassociated with Gd:CuS@BSA-mediated PTT. As a likelyconsequence of the biocompatible synthetic route, noobservable toxicity or side effects were found either in vitroor in vivo. Gd:CuS@BSA NPs present great potential as atheranostic agent for bimodal imaging-guided PTT.

EXPERIMENTAL SECTIONMaterials. Copper(II) chloride dihydrate (CuCl2·2H2O),

gadolinium(III) chloride hexahydrate (GdCl3·6H2O), and sodiumsulfide 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 ReagentCo., Ltd. (Shanghai, China). Live/dead cell viability/cytotoxicity assaykit (calcein AM and PI) was obtained from KeyGEN bioTECH(Nanjing, China). Cell counting lit-8 (CCK-8) was ordered fromDojindo Laboratory. All chemicals were used without furtherpurification. Deionized water (18.2 MΩ·cm resistivity at 25 °C) wasused throughout the entire experiments.

Synthesis of Gd:CuS@BSA NPs. Gd:CuS@BSA NPs wereprepared according to a biomineralization strategy in aqueous solution

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at physiological temperature (37 °C). Typically, an aqueous CuCl2·2H2O solution (0.05 mmol, 5 mL, 37 °C), an aqueous GdCl3·6H2Osolution (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 magneticstirring in a water bath (37 °C). Upon being mixed, a light greenturbidity appeared. Subsequently (3 min later), a NaOH solution (1M, 500 μL) was introduced to adjust the pH of the system to ∼12, andthe mixture became a transparent deep blue. Subsequently, 400 μL ofNa2S·9H2O (242.16 mg/mL) was quickly injected into the abovesystem, and the solution turned deep brown. After 4 h, the reactionwas 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 wascollected and redissolved in 3 mL of PBS (1×, pH 7.4) for further use.The exact concentrations of Gd3+ and Cu2+ were measured using ICP-MS.Material Characterization. The TEM images of the Gd:CuS@

BSA NPs were obtained using a Tecnai G2 F20 instrument operated atan acceleration voltage of 200 kV. Element mapping images and EDSline scanning results were obtained using a field-emission scanningelectron microscope (FESEM, Ultra55, Zeiss, Germany). Dynamiclight scattering (Nano ZS, Malvern) was used to record thehydrodynamic size distribution and ζ-potential of the Gd:CuS@BSANPs. The UV−visible absorption spectrum and OD980 were measuredusing a Cary 50 spectrophotometer (Varian). The XPS measurementswere performed using a PHI-5000 CESCA system (PerkinElmer) withradiation from an Al Kα (1486.6 eV) X-ray source. The CD spectra ofpure BSA and Gd:CuS@BSA were collected using a spectropolarim-eter system (BioLogic, MOS-450).Measurement of Aqueous Gd:CuS@BSA NP Photothermal

Effect. A NIR laser (980 nm, BWT Beijing Ltd.) was used during thephotothermal effect measurements and the following in vitro/vivo PTTexperiments. An aqueous Gd:CuS@BSA solution (1 mL) was placedin 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 adigital thermometer (with an accuracy of 0.1 °C) was inserted into thesolution to measure the real-time temperature, and the change oftemperature was recorded every 50 s.Studies on Colloidal Stability, Relaxivity Stability, and

Photostability. The as-prepared Gd:CuS@BSA NPs were mixedwith DI water, PBS (0.1 M, pH 7.4), borate buffer (10 mM, pH 8.2),or DMEM containing 1% penicillin−streptomycin and 10% fetalbovine serum. Subsequently, the temporal evolution profiles of theUV−vis absorption and HD were carefully monitored throughout astorage period of up to 7 days. Moreover, photostability wasinvestigated by recording the changes in size distribution, UV−visspectra, and relaxation times (T1 and T2) of the Gd:CuS@BSA NPsbefore and after NIR laser irradiation (2 h, 24 h, and 7 days).Phototemperature cycling tests were also conducted repeatedly toobserve increases in temperature induced by the laser.Relaxivity Characterization and MR Imaging in Vitro. The

longitudinal (T1) and transverse (T2) relaxation times of theGd:CuS@BSA NPs were measured using a 1.41 T minispec mq 60NMR analyzer (Bruker, Germany) at 37 °C. The in vitro MR phantomimages were acquired using a MicroMR-25 mini MR system (NiumagCorporation, Shanghai, China). The measurement parameters were asfollows: 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 conductedusing an ovarian carcinoma cell line (SK-OV-3) to evaluate the in vitrocytotoxicity 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 thenthe cells were incubated in the culture medium for 24 h at 37 °C under5% CO2 atmosphere. The culture medium was then removed, and thecells 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 (groups1, 2, and 3) and 48 h (group 4). Subsequently, cells of group 3 wereexposed to a 980 nm laser with a power density of 0.6 W/cm2 for 5min. The CCK-8 agentia (10 μL, 5 mg/mL) was added into the fourplates replacing the culture medium, and cells were incubated for afurther 4 h. Finally, the OD450 value (abs.) of each well was measuredusing a multifunction microplate reader (Infinite M200 Pro,Switzerland).

The cell live/dead assays were carried out to evaluate the efficiencyof 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 1−4were treated with PBS, laser only (0.6 W/cm2, 5 min), Gd:CuS@BSAonly (250 mg/L), and Gd:CuS@BSA (250 mg/L) with laserirradiation (0.6 W/cm2, 5 min), respectively. A mixed solutioncontaining 2 μM of calcein AM and 8 μM of PI was then added to thewells. After being stained for 40 min, cells were washed with PBS andexamined using a fluorescence microscope to observe their live/deadstatus.

Hemolysis Assay. Blood samples obtained from volunteers (1mL) were diluted with 2 mL of PBS, and then RBCs were separatedfrom the serum using centrifugation at 2000 rpm for 10 min. Afterbeing washed at least four times, the RBCs were then diluted with 10mL of PBS. Subsequently, 200 μL of the diluted RBC suspension wasmixed with 1 mL of PBS (negative control), deionized water (positivecontrol), or Gd:CuS@BSA NPs at different concentrations (10−250mg/L). After incubation for 2 h at 37 °C, the mixtures werecentrifuged at 12 000 rpm for 10 min. All of the obtained supernatantswere added to a 96-well plate, and their absorbance at 570 nm wasmeasured using a multifunction microplate reader (Infinite M200 Pro,Switzerland). Finally, the percentage hemolysis of the RBCs wascalculated 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 animalexperimental procedures were performed in adherence with a standardprotocol approved by the Institutional Animal Care and UseCommittee of Tongji University. Tumor models were established byinjecting SK-OV-3 cells (3 × 106), suspended in 70 μL of PBS,subcutaneously into the right thigh of each female Balb/c mouse (5weeks old, body weight ca. 22 g).

For in vivo PA imaging, tumor-bearing mice, anaesthetized usingchloral hydrate (5%, 8 μL/g), were administered Gd:CuS@BSA NPs(Cu2+ concentration = 13 mM) dispersed in 100 μL of PBS via the tailvein. PA signals at varying time points (preinjection, 2, 24, and 48 h)were acquired using a MSOT imaging system (iTheramedical, invision128, Germany). The excitation wavelength was set from 700 to 950nm 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 MRimaging system (MesoMR, 21.3 MHz, Shanghai Niumag Corporation,China). The images were acquired before and after intravenousinjection at a given time (dosage = 0.05 mmol Gd/kg mice) using afat-saturated 3D gradient echo imaging sequence. The detailed MRimaging parameters were set as follows: FOV read = 90 mm, FOVphase = 90 mm, TR/TE = 300 ms/13.5 ms, slices = 6, slice width = 3.5mm, slice gap = 0.5 mm, flip angle = 90°.

In Vivo Photothermal Imaging and PTT. When the tumorvolume reached ∼100 mm3, SK-OV-3 tumor-bearing mice wererandomly 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 (980nm, 0.8 W/cm2, 5 min). During the NIR irradiation, an infraredthermal camera (InfReC, Thermal Gear, G100EX/G120EX) was usedto monitor the temperature changes of the tumor sites. Tumor sizeand body weight of the mice before and after treatment were measuredusing a caliper and an electronic balance, respectively. The tumorvolume can be calculated according to the normal equation (volume =width2 × length/2) commonly used in previous reports.60,61

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In Vivo Blood Circulation Behavior and BiodistributionAnalysis. For the blood circulation study, the SK-OV-3 tumor-bearingmice (n = 3) were intravenously injected with 200 μL of Gd:CuS@BSA (Cu2+ content = 0.2 mg/mL). Then, the blood samples werecollected from the eye socket at the indicated time points (15 min, 30min, 1, 2, 4, 12, and 24 h), followed by weighing and dissolution indigestive chloroazotic acid (HCl/HNO3 = 1:3) to measure the amountof Cu in the blood using ICP-MS. For the biodistribution study, SK-OV-3 tumor-bearing mice (n = 3) were intravenously injected with200 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) andthe tumor were collected and digested using aqua regia (6 mL, VHCl/VHNO3

= 3/1) overnight. The solutions were placed on a heating plateto volatilize the acid components and then made up to 10 mL usingdeionized water. The Cu2+ content of the samples was quantified usingICP-MS.Immunohistochemistry Assay. A total of 12 female melanoma-

tumor-bearing mice with full immunity (C57BL/6, 6−8 weeks) wereused in this study (n = 3), and treatment was performed in accordancewith the aforementioned protocols. Tumors located in the center werecollected after 3 days of treatment, paraffin-embedded, and cutsectionally (4 μm). Then, three slides from each tumor wererehydrated and treated with citrate buffer (0.01 M, pH 6.0). Theywere then stained using anti-mouse CD8 (primary antibody, 1:100dilution) for 1 h at 37 °C, followed by incubation with biotin-conjugated goat anti-mouse IgG (secondary antibody) and detectedusing 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. Allhistological 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 wereconsidered 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 carriedout 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 20mg/kg. At 15 days p.i., the mice were anaesthetized, and the eyeballswere removed, followed by collection of blood samples for bloodchemistry tests and routine blood analysis. The mice treated with PBSwere used as the blank control. Subsequently, the main organs of themice (heart, liver, spleen, lung, kidney, and intestine) were harvestedand fixed using 4% paraformaldehyde. Tissue samples were thenembedded in paraffin, sliced (4 μm), and stained using H&E. All of theobtained biopsy samples were imaged using an optical microscope(Leica, 20× magnification).

ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsnano.6b05760.

Detailed experimental section, digital photos of reactionprocess, stability of Gd:CuS@BSA before and afterlyophilization photothermal, element line scanningFESEM images, ζ-potential spectra, FTIR spectra, XPSanalysis, H&E-stained images of major organs after PTT,and immune-related data (PDF)

AUTHOR INFORMATION

Corresponding Authors*E-mail: jinchang@tju.edu.cn.*E-mail: bingbozhang@tongji.edu.cn.

Author Contributions⊥W.Y. and W.G. contributed equally.NotesThe authors declare no competing financial interest.

ACKNOWLEDGMENTSThis work was supported by the National Natural ScienceFoundation of China (81571742, 81371618, 51373117,51573128, 81601603), Shanghai Innovation Program(14ZZ039), Key Project of Tianjin Natural Science Foundation(13JCZDJC33200), National High Technology Program ofChina (2012AA022603), the Doctoral Base Foundation ofEducational Ministry of China (20120032110027), Program forOutstanding Young Teachers in Tongji University, and theFundamental Research Funds for the Central Universities.

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