Size-Dependent Ag2S Nanodots for Second Near-Infrared

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Size-Dependent Ag2S Nanodots for Second Near-Infrared Fluorescence/Photoacoustics Imaging and Simultaneous Photothermal Therapy Tao Yang,†,# Yong’an Tang,‡,# Ling Liu,† Xiaoyan Lv,† Qiaoli Wang,† Hengte Ke,† Yibin Deng,† Hong Yang,† Xiangliang Yang,‡ Gang Liu,§ Yuliang Zhao,∥ and Huabing Chen*,†,⊥ †

Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China ‡ National Engineering Research Center for Nanomedicine and College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China § State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen 361102, China ∥ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Center of Excellence for Nanosciences, National Center for Nanoscience and Technology of China, Beijing 100190, China ⊥ School of Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, and School of Radiation Medicine and Protection, Soochow University, Suzhou 215123, China S Supporting Information *

ABSTRACT: Ag2S nanoparticles are increasingly important in biomedicine, such as in cancer imaging. However, there has been only limited success in the exploration of theranostic Ag2S nanoparticles for photoinduced cancer imaging and simultaneous therapy. Here we report size-dependent Ag2S nanodots (NDs) with well-defined nanostructure as a theranostic agent for multimodal imaging and simultaneous photothermal therapy. The NDs are precisely synthesized through carefully controlled growth of Ag2S in hollow human serum albumin nanocages. These NDs produce effective fluorescence in second near-infrared (NIRII) region, distinct photoacoustic intensity, and good photothermal conversion in a size-dependent manner under light irradiation, thereby generating sufficient in vivo fluorescence and photoacoustic signals as well as potent hyperthermia at tumors. Moreover, Ag2S NDs possess ideal resistance to photobleaching, effective cellular uptake, preferable tumor accumulation, and in vivo elimination, thus facilitating NIR-II fluorescence/photoacoustics imaging with both ultrasensitivity and microscopic spatial resolution and simultaneous photothermal tumor ablation. These findings provide insight into the clinical potential of Ag2S nanodots for cancer theranostics. KEYWORDS: Ag2S nanodot, albumin nanocage, second near-infrared fluorescence, photothermal therapy, tumor ablation

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radiative transitions as well as reactive oxygen species through singlet-to-triplet transitions or a photothermal effect through surface plasmon resonance/nonradiative transitions.10−15 These photoconversion characteristics effectively cause fluorescence or photoacoustic (PA) imaging and simultaneously generate photothermal therapy (PTT) or photodynamic therapy (PDT), thereby leading to significant advantages of

hotoinduced diagnostic and therapeutic nanoparticles have been recognized as theranostic breakthrough toward personalized nanomedicine.1−4 Theranostic nanoparticles are generally fabricated through integration of both imaging and therapeutic agents for fluorescence imaging and simultaneous photoinduced cancer therapy.1,3,5,6 Recently, single-component theranostic agents have been explored as an emerging frontier, including gold nanoparticles, single-walled carbon nanotubes, and organic fluorophore dyes (e.g., cyanine and chlorin e6).6−10 This type of agent is demanded to provide fundamental photoconversion into fluorescence through © 2017 American Chemical Society

Received: November 22, 2016 Accepted: January 24, 2017 Published: January 24, 2017 1848

DOI: 10.1021/acsnano.6b07866 ACS Nano 2017, 11, 1848−1857

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Scheme 1. Schematic Illustration of Theranostic Ag2S Nanodots with Clinical Potential Synthesized through Precisely Controlled Growth in Albumin Nanocages for in Vivo NIR-II Fluorescence/Photoacoustic Imaging and Photothermal Therapy

theranostic demands such as theranostic characteristics, ultrasmall size, ideal resistance to photobleaching, preferable tumor accumulation, and potential in vivo clearance.28,29 Very recently, some proteins such as bovine serum albumin and ferritin as nanocages have been found to allow the nucleation and growth of metal ions into metal clusters or metal oxide/sulfide nanoparticles through electrostatic interactions and subsequent reduction/precipitation reactions.6,30−32 These proteins might act as effective templates for the synthesis of inorganic nanoparticles within their hollow nanocages, exhibiting multiple advantages such as facile and reproducible methodology, biocompatibility, and good stability. Herein we demonstrate multifunctional Ag2S nanodots (Ag2SNDs) as a theranostic agent. These NDs are synthesized through precisely controlled growth of Ag2S within clinically acceptable human serum albumin (HSA) nanocages (Scheme 1). These nanodots produce remarkable fluorescence in the NIR-II region, distinct PA signals, and a good photothermal effect in a size-dependent manner under irradiation with singlewavelength NIR light, thereby generating sufficient in vivo fluorescence and PA signals as well as potent hyperthermia at tumors. Moreover, Ag2S-NDs possess ideal resistance to photobleaching, effective cellular uptake, prolonged blood circulation, preferable tumor accumulation, and in vivo clearance, thus facilitating fluorescence/PA imaging with both ultrasensitivity and microscopic spatial resolution and simultaneous photothermal tumor ablation.

simple composition, single-wavelength irradiation, and excellent in vivo theranostic performance without compromising either imaging or therapy.16,17 Very recently, stoichiometric semiconductor metal sulfide nanoparticles such as CuS and Ag2S nanocrystals have been extensively explored for potent PTT or fluorescence imaging because of their good near-infrared (NIR) absorbance, enhanced resistance to photobleaching, effective photoconversion efficiency, ultrasmall size, and ideal inertness (e.g., the solubility product constant is Ksp = 6.3 × 10−50 for Ag2S).18−21 Unfortunately, unlike single-component theranostic agents, this class of semiconductor nanocrystals only possess either fluorescence or a photothermal effect instead of theranostic characteristics.18,22 For instance, Ag2S nanocrystals as a narrowband-gap semiconductor (∼1.5 eV) have been developed with limited success in the exploration of theranostic characteristics because of their poor photoconversion into a thermal effect or reactive oxygen species, although they have been synthesized with various nanostructures for fluorescence imaging in the NIR and second NIR (NIR-II) regions.22−27 Hence, a critical challenge remains in the rational design of stoichiometric nanocrystals such as Ag2S nanostructures with clinical potential for cancer theranostics. Despite such strategies to synthesize various Ag2S nanocrystals, including thermal decomposition, microemulsion method, biomimetic approach, sol−gel method, hydrothermal method, and microwave method, advances in the development of Ag2S nanostructures with theranostic characteristics have been hampered by the scarcity of available synthetic methods. In particular, it is highly desired to explore a facile synthetic approach that is accomplished under mild conditions using clinically acceptable ingredients, thereby satisfying fundamental

RESULTS AND DISCUSSION Controlled Synthesis and Growth Mechanism of Ag2S-NDs. HSA was used to synthesize Ag2S-NDs within hollow albumin nanocages through biomineralization (Scheme 1849

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Figure 1. (A) Absorption spectra of Ag2S-NDs synthesized with the Ag:S ratio of 1:2 at different reaction times. (B) Their absorbance at 785 nm at different reaction times during 720 min and the corresponding first derivative curve. (C−E) TEM images of Ag2S-NDs synthesized with the Ag:S ratio of 1:2 at various reaction times: (C) 5 min; (D) 60 min; (E) and 240 min.

Figure 2. (A) Absorption spectra of Ag2S-NDs synthesized with Ag:S ratios of 1:0.5, 1:1, and 1:2 at 240 min, respectively. (B, C) TEM images of Ag2S-NDs synthesized with Ag:S ratios of (B) 1:0.5 and (C) 1:1 at 240 min, respectively, HR-TEM images of Ag2S-NDs synthesized with Ag:S ratios of (D) 1:0.5, (E) 1:1, and (F) 1:2 at 240 min.

1).32 Briefly, HSA in water was mixed with AgNO3 for electrostatic interactions between Ag+ ions and carboxyl groups in HSA, and then NaOH was added to adjust the solution to pH 12.0 to cause expansive unfolded conformation.33,34 Afterward, Na2S was further added to trigger the nucleation and growth of Ag+ into Ag2S nanocrystals through the reaction 2Ag+ + S2− = Ag2S(s) in expansive albumin nanocages.22,35 The absorption spectrum of Ag2S-NDs was measured at various reaction times (Figure 1A). Ag2S-NDs had remarkable broadband absorbances even in the NIR region during the reaction time, suggesting that Ag2S-NDs might have a good

potential to achieve photoconversion under irradiation with NIR light.32 To understand the reaction kinetics, the absorbance at 785 nm was further monitored at various reaction times (Figure 1B). The nucleation and growth of Ag2S-NDs were primarily accomplished during initial 240 min, followed by slow growth during the subsequent 480 min (Figure 1B). The first derivative of the absorbance at 785 nm further suggests that Ag2S-NDs had a controllable growth rate during the initial 240 min in the presence of excess precursors, followed by distinctly reduced growth rates owing to excess depletion of the Ag+ precursor (Figure 1B). To validate the 1850

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Figure 3. (A) Fluorescence spectra of Ag2S-NDs with diameters of 4.1, 7.9, and 9.8 nm. (B) Temperature elevations of Ag2S-NDs with diameters of 4.1, 7.9, and 9.8 nm at a concentration of 1.0 mM Ag under 5 min irradiation (785 nm, 1.5 W cm−2). (C) Photothermal conversion efficiency of 9.8 nm Ag2S-NDs, which was calculated to be 35.0%. (D) XRD pattern of Ag2S-NDs. (E) XPS analysis of Ag2S-NDs. (F) Size distribution of Ag2S-NDs using DLS.

Å or d112 = 2.83 ± 0.02 Å (Figure 2D−F), in accord with those of standard monoclinic Ag2S phase. Distinctly, albumin nanocages allow the growth of stoichiometric Ag2S nanocrystals through the diffusion-controlled precipitation reaction. Size-Dependent Fluorescence in the NIR-II Region and Photothermal Effect of Ag2S-NDs. We further evaluated the fluorescence spectra of Ag2S-NDs with diameters of 4.1, 7.9, and 9.8 nm. Figure 3A shows that all of these Ag2SNDs displayed a narrow full width at half-maximum of ∼8.0 nm in their fluorescence spectra, reasonably resulting from their monodisperse size distribution. Moreover, larger Ag2S-NDs caused stronger fluorescence intensity in the NIR-II region, indicating that their fluorescence intensity strongly relies on the particle size (Figure 3A), while the emission peak of Ag2S-NDs is not dependent on the particle size. Supposedly, Ag2S-NDs might be out of strong quantum confinement regime because of their larger particle size compared with the Bohr radius.37 Hence, Ag2S-NDs might still retain a fluorescence emission peak at 1060 nm even though their size varies from 4.1 to 9.8 nm. Moreover, the emission peak of Ag2S-NDs at 1060 nm might also be correlated with the coordination of albumin with Ag2S on the surface that is able to cause a slight change in the surface properties,33,34,38 and a full understanding of this emission peak is still to be clarified in future. Subsequently, the fluorescence quantum yields of these Ag2S-NDs with various diameters were measured to be 1.26−1.32% using IR 26 (0.1% in dichloroethane) as a reference (Figure S4),25 which are preferable to those of existing Ag2S quantum dots synthesized in aqueous solutions.23,35,39 Presumably, the excitons in the conduction band of Ag2S-NDs radiatively recombine under NIR light irradiation, leading to the emission of photons as fluorescence on their way back to the valence band. Therefore, Ag2S-NDs possess a good potential to act as a fluorescent agent in the NIR-II region. To demonstrate the photothermal effect, we evaluated the temperature elevations of Ag2S-NDs with various diameters under 785 nm irradiation. As shown in Figure 3B, Ag2S-NDs with a diameter of 9.8 nm caused a rapid temperature increase

controlled synthesis, we allowed the growth of Ag2S-NDs within nanocages for various reaction times. The Ag2S-NDs had diameters of 4.6 ± 0.9, 7.1 ± 0.8, and 9.8 ± 0.4 nm after 5, 60, and 240 min of growth, respectively (Figure 1C−E). The controllable growth rate of Ag2S-NDs allows the formation of Ag2S-NDs with tailor-made particle sizes within albumin nanocages through control of the reaction time. Meanwhile, the Ag2S-NDs also exhibited size-dependent absorbance (Figure 1B−E), suggesting that size control may be advantageous to maximize their absorbance in the NIR region. To demonstrate the growth mechanism of Ag2S-NDs in the nanocages through precipitation reaction, we monitored the absorbances of Ag2S-NDs synthesized at various initial Ag:S ratios including 1:0.5, 1:1, and 1:2 during 720 min. Distinctly, lower initial Ag:S ratios led to enhanced absorbances of Ag2SNDs (Figures 2A and S1) as a result of higher growth rates caused by excess precursors. Particularly, we found that the Ag:S ratios of 1:0.5, 1:1, and 1:2 resulted in the formation of Ag2S-NDs with diameters of 4.1 ± 0.5, 7.9 ± 0.2, and 9.8 ± 0.4 nm, respectively (Figures 2B,C and 1E), suggesting that the particle size is highly dependent on the Ag:S ratio. Thus, the growth of Ag2S-NDs is considered to proceed by a diffusioncontrolled growth mechanism, since the particle size distinctly depends on the initial ratio of reactants.36 To further confirm the growth mechanism of Ag2S-NDs, we evaluated the influence of the HSA concentration and reaction temperature on the growth of Ag2S-NDs. All of these Ag2S-NDs had similar diameters in the range of 9.1−9.8 nm, indicating that both factors have no distinct influence on the final particle size (Figures S2 and S3), although they had slightly different growth rates. Thus, Ag2S-NDs undergo diffusion-controlled growth rather than thermodynamically controlled growth. Consequently, we are able to achieve controlled growth of Ag2SNDs through control of the initial reactant ratio in addition to the reaction time. Subsequently, high-resolution transmission electron microscopy (HR-TEM) showed that these Ag2S-NDs with diameters of 4.1, 7.9, and 9.8 nm had lattice fringes of d121 = 2.60 ± 0.01 1851

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Figure 4. (A) Fluorescence mapping of Ag2S-NDs at various concentrations in the NIR-II region. (B) PA mapping of Ag2S-NDs at various concentrations. (C) Temperature elevation of Ag2S-NDs under five irradiation/cooling cycles (785 nm, 1.5 W cm−2). (D) Intracellular distribution of Cy5.5-labeled Ag2S-NDs in 4T1 cells stained with Hoechst 33342 and Lysotracker DND-26 Green. (E) Viability of Ag2S-NDs against 4T1 cells under 3 min irradiation (785 nm, 1.5 W cm−2) or in the dark. (F) Plasma concentrations of Ag2S-NDs with various diameters at a dose of 50.0 μmol kg−1 Ag at different times after intravenous administration. (G) Biodistribution of Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag at 24 h postinjection. (H) NIR fluorescence imaging of 4T1-tumor-bearing mice treated with Cy7.5-labeled Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag at 72 h postinjection. (I) Long-term distributions of Ag2S-NDs in various major tissues at a dose of 50.0 μmol kg−1 Ag during 30 days postinjection.

of ∼32 °C in 300 s at a concentration of 1.0 mM Ag, while smaller Ag2S-NDs with diameters of 4.1 and 7.9 nm exhibited temperature elevations of 11.8 and 19.5 °C, respectively. Distinctly, Ag2S-NDs also exhibited size-dependent temperature elevations, primarily resulting from their size-dependent molar extinction coefficients in the NIR region (1.0, 2.7, and 3.4 L g−1 cm−1 for 4.1, 7.9, and 9.8 nm Ag2S-NDs, respectively; Figure S5), since they had similar photothermal conversion efficiencies of 33.7−35.0% (Figures 3C and S6). These photothermal conversion efficiencies are also comparable to those of most of the existing photothermal nanoparticles, such as Au nanorods (∼22.8%) and indocyanine green-loaded micelles (∼25.2%) (Figure S7).2,40 Possibly, nonradiative relaxation from the excitons in the conduction band might account for the effective photothermal conversion ability of Ag2S-NDs.32 In addition, Ag2S-NDs also exhibited concentration-dependent temperature elevations (Figure S8). Therefore, the temperature elevation due to Ag2S-NDs could potentially be maximized through control of the particle size and concentration. Reasonably, the albumin nanocages provide an effective approach to synthesize Ag2S-NDs with both fluorescence and a photothermal effect. As a result, Ag2S-NDs with a diameter of 9.8 nm were selected as typical nanodots for subsequent studies. Characterization of Ag2S-NDs. The X-ray diffraction (XRD) pattern shows that Ag2S-NDs displayed several

characteristic Bragg peaks for the [111], [112], [103], and [121] facets (Figure 3D). Distinctly, Ag2S-NDs had a monoclinic phase according to the characteristic peaks of standard Ag2S phases (JCPDS no. 14-0072),13 in accord with the lattice fringes shown in Figure 2F. X-ray photoelectron spectroscopy (XPS) analysis of Ag2S-NDs was further performed to validate their chemical composition. There were two peaks at 373.4 and 367.4 eV (Figure 3E), confirming a stoichiometric composition of Ag2S. Moreover, the secondary structure of albumin within the Ag2S-NDs was also studied using circular dichroism (CD) spectroscopy (Figure S9). Ag2SNDs exhibited two peaks at 210 and 222 nm, which are similar to those of free HSA from its α-helix structure.41 Clearly, Ag2SNDs have a corona of HSA without an obvious change in its secondary structure. Dynamic light scattering (DLS) shows that Ag2S-NDs had a hydrodynamic diameter of 32.8 nm due to the presence of the protein corona on the Ag2S core (Figure 3F), suggesting a potential passive targeting ability through the enhanced permeation and retention (EPR) effect. Subsequently, the chemical stability of Ag2S-NDs was further investigated. Ag2S-NDs had good chemical stability in various solutions, including water and serum as well as phosphatebuffered saline (PBS) at pH 6.2 and 7.4, over 72 h (Figure S10). As a consequence, the albumin nanocages provide an effective template for achieving the controlled synthesis of 1852

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Figure 5. (A) In vivo NIR-II fluorescence imaging and (B) their calculated intensity in 4T1-tumor-bearing mice treated with Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag during 48 h postinjection. (C) In vivo PA imaging and (D) their calculated intensity in the 4T1-tumor-bearing mice treated with Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag during 24 h postinjection. (E) Infrared thermography and (F) temperature elevations in the tumors of tumor-bearing mice treated with Ag2S-NDs at various doses during 5 min irradiation (785 nm, 1.5 W cm−2). (G) Tumor growth profiles of 4T1-tumor-bearing mice treated with Ag2S-NDs with or without 5 min irradiation (785 nm, 1.5 W cm−2) and (H) photograph of tumors extracted from the mice at 30 days postirradiation. (I) Blood levels of ALP, ALT, AST, and urea from mice treated with Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag at various times.

irradiation, suggesting a perfect resistance to photobleaching. Next, we further investigated the capacity of Ag2S-NDs to maintain their temperature elevation under irradiation. The solution of Ag2S-NDs was irradiated at 1.5 W cm−2 for 5 min, and then the irradiation was removed for 5 min of cooling, followed by another four irradiation/cooling cycles. Ag2S-NDs had no obvious change in temperature elevation during the five cycles (Figure 4C). Thus, Ag2S-NDs possess a preferable resistance to photobleaching compared with extensively used organic dyes and inorganic nanomaterials with surface plasmon resonance. Cellular Uptake, Endocytic Pathway, Intracellular Distribution, and Cytotoxicity of Ag2S-NDs. The cell internalization of Ag2S-NDs was investigated on 4T1 murine breast tumor cells. Ag2S-NDs exhibited time-dependent cellular uptakes (Figure S13), which is highly important to cause enhanced theranostic performance. Moreover, we studied the endocytic pathway of Ag2S-NDs using various pathway inhibitors (Figure S14). Chlorpromazine caused a decrease of ∼55.0% in the cellular uptake of Ag2S-NDs, indicating clathrinmediated endocytosis. In addition, the intracellular distribution of Cy5.5-labeled Ag2S-NDs was also observed in 4T1 cells that were stained with Hoechst 33342 and Lysotracker DND-26 Green (Figure 4D). Red-fluorescent Ag2S-NDs exhibited a colocalization percentage of 93.8% with the lysosomes after 2 h of incubation, suggesting that Ag2S-NDs were preferably endocytosed into the lysosomes through clathrin-mediated

stoichiometric Ag2S-NDs with stable nanostructure through precipitation reaction. Fluorescence Mapping, Photoacoustic Mapping, and Photostability of Ag2S-NDs. To characterize the ability of Ag2S-NDs to generate fluorescence, we first evaluated their fluorescence mapping using an NIR-II fluorescence imaging system. Ag2S-NDs exhibited concentration-dependent fluorescence signals in aqueous solutions under 785 nm irradiation due to their emission in the NIR-II region (Figure 4A). Ag2SNDs might act as an imaging agent for potential NIR-II fluorescence imaging with deep light penetration, low autofluorescence background, and weak tissue scattering.19,38 To evaluate the capacity to produce PA signals, we monitored the photoacoustic spectrum of Ag2S-NDs. Ag2S-NDs exhibited a gradual decrease in the PA signal in the range of 720−980 nm (Figure S11). Reasonably, Ag2S-NDs are able to possess a distinct photoacoustic signal under excitation at 785 nm. Furthermore, the PA mapping of Ag2S-NDs at various concentrations was also observed. Ag2 S-NDs exhibited concentration-dependent PA signals under excitation at 800 nm due to their thermoelastic expansion (Figure 4B).42 Thus, Ag2S-NDs possess a potential ability to generate both fluorescence and PA imaging modalities through their concentration-dependent NIR-II fluorescence and PA signals. The photostability of Ag2S-NDs under NIR irradiation was also evaluated (Figure S12). Ag2S-NDs exhibited no distinct change in their absorbance at 785 nm during 15 min of 1853

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PA signals can be activated upon thermoelastic expansion from hyperthermia.42 Thus, the ability of Ag2S-NDs to generate PA imaging was evaluated in 4T1-tumor-bearing mice (Figure 5C). Only a few PA signals were observed from endogenous oxyhemoglobin and deoxyhemoglobin in tumor vasculatures before injection. However, Ag2S-NDs resulted in a 2.4-fold increase in the PA signal in tumor at 24 h postinjection compared with that before injection (Figure 5C,D) due to their good photothermal effect and tumor accumulation. Particularly, Ag2S-NDs were uniformly distributed into the tumor from tumor vasculatures at 24 h postinjection (Figure 5C), indicating that Ag2S-NDs are able to deeply penetrate into tumor cells. Clearly, Ag2S-NDs possess a microscopic PA imaging ability with spatial resolution and provide a complementary imaging modality compared with their fluorescence imaging in the NIRII region. Thus, the combination of NIR-II fluorescence and PA imaging modalities is able to provide both microscopic spatial resolution and macroscopic ultrasensitivity for tumor imaging, treatment guidance, and monitoring. In Vivo Hyperthermia, Anticancer Efficacy, H&E Staining, and Serum Biochemistry Assay. To evaluate the in vivo hyperthermia in tumor, Ag2S-NDs were injected into mice at various doses, followed by infrared thermography during 5 min irradiation (785 nm, 1.5 W cm−2) at 24 h postinjection (Figure 5E). PBS as a control exhibited a minor temperature elevation in tumor under irradiation, indicating that light irradiation alone is unable to generate hyperthermia in tumor. Ag2S-NDs resulted in temperature elevations of ∼7.7 and ∼12.5 °C in tumor at doses of 10.0 and 30.0 μmol kg−1 Ag under irradiation, respectively (Figure 5F), suggesting mild hyperthermia at these doses.47 Importantly, Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag resulted in a temperature elevation of ∼19.0 °C in tumor under irradiation (Figure 5F), indicating potent hyperthermia from Ag2S-NDs. Moreover, the steady hyperthermia in tumor during 5 min irradiation also implies that Ag2S-NDs possess good in vivo stability, presumably resulting from their inertness, good chemical stability, and photostability. Possibly, both the good photothermal effect and preferable tumor accumulation are responsible for their potent hyperthermia in tumor, which plays an essential role in generating ideal PTT efficacy. To demonstrate the in vivo anticancer efficacy, we injected Ag2S-NDs into the 4T1-tumor-bearing mice at various doses through single-dose intravenous administration. Then the tumors were irradiated for 5 min (785 nm, 1.5 W cm−2) at 24 h postinjection, followed by measurements of tumor size during the subsequent 30 days (Figure 5G,H). PBS as a control had an approximately 25-fold increase of tumor volume in the absence or presence of irradiation, indicating that no anticancer efficacy is triggered by the irradiation itself. In the absence of irradiation, Ag2S-NDs resulted in tumor growth similar to that of PBS, suggesting an inactive behavior. Interestingly, Ag2SNDs resulted in effective tumor ablation at doses of both 10.0 and 30.0 μmol kg−1 Ag under irradiation but still displayed remarkable tumor regrowth at 8 and 14 days postirradiation, respectively (Figure 5G,H). Remarkably, the mild hyperthermia with a temperature elevation of ∼12.6 °C is unable to achieve total tumor ablation because of the presence of residual tumor cells surviving the photothermal cell damage, even though significant tumor inhibition was still achieved at the dose of 30.0 μmol kg−1 Ag. Importantly, Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag resulted in total tumor ablation without any regrowth (Figure 5G,H). Obviously, the potent hyperthermia

endocytosis. To evaluate the photothermal damage of Ag2SNDs against tumor cells, 4T1 cells were irradiated for 3 min (785 nm, 1.5 W cm−2) after 24 h of incubation with Ag2S-NDs. Ag2S-NDs exhibited remarkable cell damage, with an IC50 value of 0.17 mM under irradiation (Figure 4E), while they had no significant cytotoxicity in the absence of irradiation. Distinctly, both the enhanced cellular uptake and potent photothermal effect of Ag2S-NDs account for their severe cell damage under irradiation.32 Pharmacokinetics, Biodistribution, Tumor Retention, and in Vivo Elimination. We evaluated the pharmacokinetic behavior of Ag2S-NDs in Balb/c mice. As shown in Figure 4F, Ag2S-NDs with a diameter of 9.8 nm displayed an enhanced elimination half-life (t1/2β) of 5.7 h compared with those of 7.9 and 4.1 nm Ag2S-NDs as controls (t1/2β = 3.1 and 2.3 h, respectively).43 Ag2S-NDs also exhibited a distinct increase of the area under the curve (AUC0−∞) compared with Ag2S-NDs with smaller diameters (Table S1). Distinctly, both the prolonged circulation time and higher AUC0−∞ of Ag2S-NDs might be advantageous to facilitate their subsequent tumor accumulation. To evaluate the in vivo biodistribution, Ag2S-NDs were administered to mice bearing 4T1 tumors via intravenous injection at a dose of 50.0 μmol kg−1 Ag, followed by measurements of Ag in various tissues at 24 h postinjection. Ag2S-NDs were found to be mainly distributed into tumor, liver, and kidney, suggesting their preferable accumulation in tumor, and the tumor uptake efficiency was calculated to be 10.6% ID/g (Figure 4G).43 Obviously, Ag2S-NDs exhibit good tumor accumulation due to their enhanced circulation and EPR effect.44 To demonstrate the retention ability of Ag2S-NDs in tumor, we conducted in vivo NIR fluorescence imaging of Cy7.5labeled Ag2S-NDs at a dose of 50.0 μmol kg−1 Ag (Figures 4H and S15). Ag2S-NDs displayed distinct fluorescence signals in tumor at 48 h postinjection, while Cy7.5 as a control exhibited negligible fluorescence in tumor because of its quick elimination. Thus, Ag2S-NDs show relatively long-term retention in tumor, which is highly advantageous for flexible imaging or PTT treatment. To further demonstrate the in vivo elimination of Ag2S-NDs, we monitored their long-term distribution in various major tissues at a dose of 50.0 μmol kg−1 Ag. As depicted in Figure 4I, Ag2S-NDs exhibited timedependent distributions in heart, liver, spleen, lung, and kidney. In particular, most of the Ag2S-NDs were gradually eliminated from these major tissues over 30 days through renal clearance as a result of their ultrasmall size, thereby avoiding potential toxicity concerns.43,45,46 Fluorescence Imaging in the NIR-II Region and PA Imaging. Fluorescence imaging in the NIR-II region has aroused great attention because of its minimal autofluorescence and negligible tissue scattering as well as enhanced light penetration depth. We intravenously injected Ag2S-NDs into 4T1-tumor-bearing mice at a dose of 50.0 μmol kg−1 Ag, followed by subsequent NIR-II fluorescence imaging at different times. The fluorescence signals from Ag2S-NDs appeared in the tumor at 4 h postinjection and gradually increased during 48 h postinjection (Figure 5A). Ag2S-NDs displayed the strongest fluorescence intensity at 24 h postinjection (Figure 5B), which is in accordance with their tumor retention behavior shown in Figure 4H. Thus, Ag2S-NDs are able to provide ultrasensitive NIR-II fluorescence imaging for tumor visualization because of their good fluorescence quantum yield and preferable tumor accumulation. 1854

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ACS Nano with the temperature elevation of ∼19.0 °C causes the destructive cell necrosis for total tumor ablation. Therefore, Ag2S-NDs possess a good ability to achieve photothermal tumor ablation, possibly due to their multiple properties including ideal photostability, good photothermal effect, effective cellular uptake, and preferable tumor accumulation. To confirm the PTT efficacy of Ag2S-NDs, we evaluated their photothermal damage to tumor using hematoxylin and eosin (H&E) staining. Ag2S-NDs led to destructive cell necrosis and severe hemorrhagic inflammation in the tumor at 6 h postirradiation (Figure S16). In contrast, Ag2S-NDs exhibited no remarkable damage to the tumor in the absence of irradiation. In addition, Ag2S-NDs also resulted in no significant damage to the normal major tissues including heart, liver, spleen, lung, and kidney (Figure S17). To demonstrate the potential toxicity of Ag2S-NDs, a serum biochemistry assay was performed during 30 days. The levels of liver function markers (ALP, ALT, and AST) and kidney function marker (urea) exhibited no distinct changes compared to their original levels (Figure 5I), suggesting that Ag2S-NDs have no obvious hepatic and renal toxicity at a dose of 50.0 μmol kg−1 Ag, reasonably due to their ideal inertness and in vivo elimination through renal excretion.

Characterization. The morphology was observed using transmission electron microscopy (TEM) imaging (Hitachi HT7700). The microstructure was obtained using high-resolution TEM (FEI Technai G2 S-Twin, 200 kV). UV−vis and fluorescence spectra were detected using a UV−vis spectrophotometer (UV2600, Shimadzu) and a fluorescence spectrophotometer (Fluoromax-4, HORIBA), respectively. The hydrodynamic diameter was measured using dynamic light scattering (DLS) (Zetasizer ZS90, Malvern). Wide-angle X-ray diffraction (XRD) measurements (Bruker D8 Advance) were utilized to characterize Ag2S-NDs in the range of 2θ from 20° to 80°. X-ray photoelectron spectroscopy (XPS) was performed using an XPS spectrometer (Thermo Scientific Escalab 250Xi). Circular dichroism spectra of HSA and Ag2S-NDs were measured at room temperature using a CD spectrometer (JASCO J-815). The concentration of Ag was measured using inductively coupled plasma optical emission spectrometry (ICP-OES) (Varian 710-ES) and inductively coupled plasma mass spectrometry (ICP-MS) (Thermo ELEMENT 2). Photothermal Effect. To evaluate the photothermal effect, 0.3 mL of Ag2S-NDs at concentrations of 0.05, 0.1, 0.2, and 0.5 mM were subjected to 785 nm irradiation at 1.5 W cm−2 for 5 min. A thermometer was applied to monitor the temperature at different irradiation times. To further compare the photothermal effects of Ag2S-NDs of various sizes, various Ag2S-NDs (1.0 mM) were exposed to 5 min irradiation (785 nm, 1.5 W cm−2), followed by temperature monitoring. Photostability, Photothermal Conversion Efficiency, and Chemical Stability. To investigate the photostability of Ag2S-NDs, Ag2S-NDs (0.5 mM) were irradiated at 785 nm (1.5 W cm−2, 5 min; LASER ON), followed by cooling to room temperature without irradiation for 5 min (LASER OFF). Afterward, we further repeated four LASER ON/OFF cycles for temperature monitoring. To measure the photothermal conversion efficiency, irradiation was applied to the solutions (0.3 mL) of Ag2S-NDs at a concentration of 0.5 mM Ag in quartz cuvettes. When the temperature remained steady, the irradiation was removed for cooling to room temperature. The temperature was monitored at an interval of 30 s during this experiment. Finally, the photothermal conversion efficiency (η) was calculated. To evaluate the chemical stability, the absorbances of Ag2SNDs at different times were measured in various solvents including deionized water, phosphate buffers at pH 6.2 and pH 7.4, and RPMI 1640 cell medium containing 10% fetal bovine serum during 72 h. Fluorescence Quantum Yield. The quantum yield of Ag2S-NDs was measured using IR 26 in dichloroethane (DCE) as a reference (QY ≈ 0.1%) because of its maximal emission around 1100 nm in the NIR-II window. A series solutions of IR 26 in DCE and Ag2S-NDs with various absorbances of ∼0.05, ∼0.10, ∼0.15, ∼0.20, and ∼0.25 at 785 nm were prepared, and then the absorption and emission spectra of these samples were monitored. The integrated fluorescence intensity of the fluorescence spectrum was plotted against the absorbance at 785 nm. Fluorescence Mapping in the NIR-II Region and Photoacoustic Mapping. For NIR-II fluorescence imaging, Ag2S-ND solutions with various concentrations (0.37, 0.75, 1.5, 3.0, and 6.0 mM) were prepared and further imaged using an NIR-II fluorescence imaging system, and then the fluorescence intensity was acquired. Ag2S-ND solutions with various concentrations (2.0, 3.0, 4.0, and 5.0 mM) in the tubes were scanned using an Endra Nexus 128 system at an excitation wavelength of 800 nm. Then the average PA intensity was calculated. Cellular Uptake, Endocytic Pathway, and in Vitro Photothermal Cytotoxicity. Ag2S-NDs (0.1 mM Ag) were incubated with 4T1 cells (1.0 × 106 4T1 cells/well). After 6 or 24 h of incubation, the cells were collected for cell counting. Afterward, Ag was extracted from the counted cells under ultrasonication and further analyzed using ICP-OES. To distinguish the endocytic pathway, 4T1 cells (1.0 × 106 cells/well) were exposed to various inhibitors including 5 μg mL−1 filipin, 10 μg mL−1 chlorpromazine, and 100 μg mL−1 amiloride for 1 h of incubation at 37 or 4 °C in serum-free RPMI 1640 medium. Then Ag2S-NDs (0.1 mM Ag) were added to the medium for 2 h of incubation, followed by the extraction of Ag from the collected cells

CONCLUSIONS We have reported size-dependent Ag2S nanodots as a theranostic agent for multimodal imaging and simultaneous photothermal therapy. Ag2S-NDs with well-defined nanostructure were synthesized through precisely controlled growth of Ag2S in albumin nanocages. We precisely regulated the particle size of Ag2S-NDs through control of the reaction time and initial ratio of reactants. In particular, Ag2S-NDs possess sizedependent NIR-II fluorescence intensity, photothermal effect, and blood circulation. Moreover, Ag2S-NDs exhibit the collective characteristics of ideal resistance to photobleaching, good cellular uptake, preferable tumor accumulation, and in vivo elimination, thereby enabling Ag2S-NDs to generate ultrasensitive fluorescence imaging in the NIR-II region and PA imaging with microscopic spatial resolution as well as potent photothermal tumor ablation. As a nanoplatform, these ultrasmall nanodots coated with clinically approved HSA can also easily be functionalized with various imaging or therapeutic agents for versatile theranostic applications. This study provides insight on clinically potential Ag2S nanodots for cancer theranostics. EXPERIMENTAL SECTION Synthesis. For the synthesis of Ag2S-NDs, 250.0 mg of HSA was dissolved in 9.0 mL of deionized water, and then 1.0 mL of AgNO3 solution (20.0 mM) was slowly added under vigorous stirring. Next, NaOH (2.0 M) was used to adjust the solution to pH 12, and Na2S (0.1 M) was further added to the mixture at the Ag:S ratios of 1:0.5, 1:1, and 1:2, respectively. Then the mixture was allowed to react at 55 °C for 4 h under vigorous stirring, followed by dialysis for 24 h. To study the influence of temperature and HSA concentration on the growth of Ag2S-NDs, HSA at various concentrations (5.0, 10.0, and 25.0 mg mL−1) and different temperatures (25, 37, and 55 °C) were applied to the synthesis of Ag2S-NDs. For the conjugation of Ag2SNDs with Cy7.5-NHS or Cy5.5-NHS, 4.0 mL of Ag2S-NDs (5.0 mM) coated with HSA were mixed with 1.0 mL of Cy7.5-NHS or Cy5.5NHS (0.5 mg mL−1) under stirring overnight, followed by dialysis against distilled water. Finally, Ag2S-NDs were obtained after centrifuge ultrafiltration (100 kDa MW, 5000 rpm, 20 min each) in a solution of PBS (pH 7.4, 10.0 mM). 1855

DOI: 10.1021/acsnano.6b07866 ACS Nano 2017, 11, 1848−1857

Article

ACS Nano

with a thickness of 10 μm were made. H&E staining was performed, followed by the observation using an IX73 bright-field microscope (Olympus). Serum Biochemistry Assay and in Vivo Elimination. Ag2SNDs were injected intravenously into healthy Balb/c mice at a single dose of 50.0 μmol kg−1 (n = 3). Blood (0.5 mL) from each mouse was collected for blood biochemistry examination. Meanwhile, various tissues, including heart, liver, spleen, lung, and kidney, were extracted and digested using a mixture of aqua regia and perchloric acid at 280 °C. Finally, the Ag concentrations were determined using ICP-OES.

under ultrasonication. Finally, the concentration of Ag was measured using ICP-OES. For photothermal cytotoxicity, 4T1 cells were incubated with Ag2S-NDs at 0.02, 0.05, 0.1, 0.2, and 0.5 mM for 24 h, and then the cells were subjected to 785 nm irradiation at 1.5 W cm−2 for 3 min. After 24 h, the cell viability was measured using the MTT assay. Intracellular Distribution. 4T1 cells (5.0 × 104 cells/well) were seeded in a glass-bottom dish for 24 h and then incubated with Cy5.5labeled Ag2S-NDs (0.1 mM Ag) for 2 h at 37 °C. After washing, 0.2 mL of Lysotracker Green DND-26 (100 nM) was used to stain the lysosomes for 5 min at 37 °C, followed by the addition of Hoechst 33342 (1.0 mL, 1.0 μg mL−1) for another 10 min of incubation. Finally, confocal laser scanning microscopy (Zeiss LSM710) was employed to observe the cells. Pharmacokinetics and Biodistribution. Balb/c mice and nude mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. and treated in accordance with the guidelines from Soochow University Laboratory Animal Center. To investigate the pharmacokinetic behavior, Ag2S-NDs with various diameters were administrated into healthy Balb/c mice at a dose of 50.0 μmol kg−1 Ag via tail vein, and blood samples were collected at 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. Then the blood samples were extracted and digested using a mixture of aqua regia and perchloric acid at 280 °C. Afterward, the concentrations of Ag were obtained using ICP-MS. Afterward, t1/2α, t1/2β, and AUC data were fitted using the 3P97 software. To demonstrate the biodistribution, Ag2S-NDs (50.0 μmol kg−1) were intravenously administered into Balb/c mice bearing 4T1 tumors, which were sacrificed at 24 h postinjection for extraction of heart, liver, spleen, lung, kidney, and tumor. Next, various tissues were digested using a mixture of aqua regia and perchloric acid at 280 °C. Finally, the Ag concentrations were determined using ICP-MS for calculation of the biodistribution. In Vivo Retention Behavior, Fluorescence Imaging in the NIR-II Region, and PA Imaging. For the in vivo retention behavior of Ag2S-NDs, Cy7.5-labeled Ag2S-NDs or free Cy7.5 (0.5 mg kg−1 Cy7.5, equal to 50.0 μmol kg−1 Ag) was injected intravenously into 4T1-tumor-bearing mice. Under isoflurane anesthesia, in vivo NIR fluorescence imaging was performed using an IVIS Lumina II imaging system at 0, 6, 12, 24, 48, and 72 h postinjection, respectively. Then the fluorescence intensity at the tumor site was calculated. For in vivo fluorescence imaging of Ag2S-NDs in the NIR-II region, an NIR-II fluorescence imaging system was applied. The excitation light was provided by a 785 nm diode laser with an 880 nm long-pass filter. After a single dose of 50.0 μmol kg−1 Ag2S-NDs was injected into the 4T1-tumor-bearing mice, NIR-II fluorescence imaging was performed. For in vivo PA imaging, Ag2S-NDs (50.0 μmol kg−1) were injected into the 4T1-tumor-bearing mice via intravenous injection. Then the mice were observed using the Endra Nexus 128 system at an excitation wavelength of 800 nm at 2, 4, 8, 12, and 24 h postinjection. Finally, the average PA intensity at the tumor was calculated. In Vivo Infrared Thermography, in Vivo Anticancer Efficacy, and Histological Staining. For in vivo infrared thermography, Ag2SNDs were injected into 4T1-tumor-bearing mice at doses of 10.0, 30.0, and 50.0 μmol kg−1. The tumors were irradiated for 5 min (785 nm, 1.5 W cm−2) at 24 h postinjection, and the imaging was performed using a Fotric 225 infrared camera during the irradiation. To examine the anticancer efficacy, Ag2S-NDs were injected into mice bearing 4T1 tumors (approximately 60 mm3) via intravenous administration at doses of 10.0, 30.0, and 50.0 μmol kg−1, respectively, followed by 5 min irradiation of the tumors (785 nm, 1.5 W cm−2) at 24 h postinjection. Then the tumor volume (V) was measured according to the equation of V = L × W2/2, where W and L are the widest and longest tumor dimensions, respectively. Finally, the mice were sacrificed by cervical dislocation under anesthetic status after the experiments. To confirm the in vivo photothermal damage against tumors, Ag2S-NDs were injected into mice bearing 4T1 tumors (∼60 mm3) via intravenous administration at a dose of 50.0 μmol kg−1, followed by 5 min irradiation of the tumors (785 nm, 1.5 W cm−2) at 24 h postinjection. The tumors were obtained at 6 h postirradiation and further fixed in a 4% formaldehyde solution. Then tumor sections

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07866. Additional results (Figures S1−S17 and Table S1) (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Gang Liu: 0000-0003-2613-7286 Yuliang Zhao: 0000-0002-9586-9360 Huabing Chen: 0000-0003-1637-2872 Author Contributions #

T.Y. and Y.T. contributed equally.

Notes

The authors declare no competing financial interest.

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