Bifunctional Tellurium Nanodots for Photo-Induced ... - ACS Publications

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Bifunctional Tellurium Nanodots for PhotoInduced Synergistic Cancer Therapy Tao Yang,†,# Hengte Ke,†,# Qiaoli Wang,†,# Yong’an Tang,§ Yibin Deng,*,† Hong Yang,† Xiangliang Yang,§ Peng Yang,∥ Daishun Ling,⊥ Chunying Chen,∇ Yuliang Zhao,∇ Hong Wu,*,◆ and Huabing Chen*,†,‡ †

Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, College of Pharmaceutical Sciences and 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 § National Engineering Research Center for Nanomedicine and College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China ∥ School of Materials Science and Engineering, Yunnan University, Kunming 650071, China ⊥ Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, 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 Pharmacy, Air Force Military Medical University, Xi’an 710032, China

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‡

S Supporting Information *

ABSTRACT: Elemental tellurium (Te) nanoparticles are increasingly important in a variety of applications such as thermoelectricity, photoconductivity, and piezoelectricity. However, they have been explored with limited success in their biomedical use, and thus a tremendous challenge still exists in the exploration of Te nanoparticles that can treat tumors as an effective anticancer agent. Here, we introduce bifunctional Te nanodots with well-defined nanostructure as an effective anticancer agent for photo-induced synergistic cancer therapy with tumor ablation, which is accomplished using hollow albumin nanocages as a nanoreactor. Under near-infrared light irradiation, Te nanodots can produce effective photothermal conversion, as well as highly reactive oxygen species such as •O2− and dismutated •OH via a type-I mechanism through direct electron transfer, thereby triggering the potent in vivo hyperthermia and simultaneous intracellular reactive oxygen species at tumors. Moreover, Te nanodots possess perfect resistance to photobleaching, effective cytoplasmic translocation, preferable tumor accumulation, as well as in vivo renal elimination, promoting severe photo-induced cell damage and subsequent synergy between photothermal and photodynamic treatments for tumor ablation. These findings provide the insight of elemental Te nanodots for biomedical research. KEYWORDS: tellurium nanodot, albumin nanocage, photodynamic therapy, photothermal therapy, tumor ablation

T

unlike other chalcogen elements such as O, S, and Se, elemental Te has been explored with limited success in the exploration of its potential biological application, although nanoscaled Te compounds such as CdTe quantum dots have been explored for bioimaging, and nanowire-like Te nanoparticles synthesized through reduction of tellurite in microorganism have also been found to possess antibacterial activities.5,7−9 Hence, a tremendous challenge exists in the exploration of Te nano-

ellurium (Te) is a trace metalloid element at 0.005 ppm in earth’s crust. As a member of chalcogen family, Te generally has four oxidation states including TeO42−, TeO32−, elemental Te0, and metal telluride (Te2−).1,2 Interestingly, elemental Te holds an anisotropic growth tendency to form one-dimensional structures with band gap energy of 0.35 eV such as nanorods, nanoneedles, and nanowires through van der Waals interaction in their hexagonal lattice. These nanostructures generally act as p-type semiconductors to provide versatile characteristics including photoconductivity, catalytic activity, and thermoelectricity for electronic and photoelectronic applications.3−6 Unfortunately, © 2017 American Chemical Society

Received: June 16, 2017 Accepted: September 25, 2017 Published: September 25, 2017 10012

DOI: 10.1021/acsnano.7b04230 ACS Nano 2017, 11, 10012−10024

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Cite This: ACS Nano 2017, 11, 10012-10024

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Scheme 1. Schematic Illustration of (A) Te-NDs Preparation through Albumin Nanoreactor Approach, (B) Photoconversion Mechanism for Te-NDs To Generate Simultaneous Photothermal Effect and ROS under Single Wavelength NIR Light Irradiation, and (C) Their Intracellular Synergistic Photothermal and Photodynamic Treatments

homogeneous suspension of Te-NDs synthesized using HSA as a template (Figure S1), indicating that HSA plays a key role in the synthesis of Te-NDs. To demonstrate the reaction kinetics under the reduction of NaBH4, the absorption spectra of TeNDs were monitored at various reaction times. Te-NDs exhibited obvious broad-band absorbance even in NIR region when reaction time was more than 30 min (Figure 1A). The absorbances of Te-NDs at 785 nm were distinctly improved during the initial 60 min, followed by a slight increase after 60 min (Figure 1B). The reduction and growth of Te-NDs are primarily accomplished within the albumin nanocages during the initial 60 min. The first derivative of their absorbances at 785 nm further indicates that Te-NDs possess relatively high reaction rates during the initial 60 min (Figure 1B), owing to abundant precursors under potent reduction, while the excessive depletion of precursor might account for the reduced reaction rates after 60 min. To demonstrate the controlled synthesis of Te-NDs within albumin nanocages, we allowed the growth of Te-NDs within albumin nanocages for various reaction times, followed by transmission electron microscopy (TEM) imaging. Te-NDs possessed 3.4 ± 0.6 nm, 4.6 ± 0.6 nm, and 5.9 ± 0.5 nm Te nanocrystals in diameter at 1, 30, and 240 min (Figure 1C−E), respectively. Distinctly, the diameters of Te-NDs are effectively tuned through reaction time according to the first derivative of their absorbances at 785 nm, and Te-NDs also possess sizedependent absorbances (Figure 1B and Figure 1C−E). Hence, the size control of Te-NDs plays a key role in maximizing their NIR absorbances. Subsequently, the influence of several other factors such as temperature, pH, and Te/NaBH4 ratio on synthesis of Te-NDs were also evaluated in addition to reaction time.24 Te-NDs that were synthesized at various temperature, pH, and Te/NaBH4 ratio, respectively, displayed different absorbances at 785 nm (Figure S2), but they had similar average diameters ranging from 5.7 to 6.3 nm (Figure S3). Distinctly, the reaction with excessive reductant at higher temperature in basic solution preferably produces a higher amount of Te-NDs, instead of Te-NDs with a larger size. Thus, these factors are able to maximize the synthetic yields of TeNDs owing to their various reaction rates at different

particles with preferable characteristics for their biomedical applications such as cancer therapy. For cancer therapy, inorganic nanoparticles are generally demanded to possess an ability to effectively damage cancer cells, accompanied with some essential characteristics such as cancer targeting ability, ultrasmall particle size (e.g., < 10 nm) for potential renal excretion, as well as low adverse side effect.10−19 Although several synthetic approaches have been explored to fabricate one-dimensional Te nanoparticles with various morphologies including solvothermal method, chemical vapor deposition, and physical evaporation,20−22 very few synthetic approaches are exploited to synthesize Te nanoparticles that potentially possess therapeutic characteristics to satisfy fundamental demands for cancer therapy.23 Here, we report bifunctional Te nanodots (Te-NDs) for photo-induced synergistic cancer therapy using clinically accepted human serum albumin (HSA) as a nanoreactor (Scheme 1).24−30 TeNDs are capable of generating effective photothermal conversion and reactive oxygen species (ROS) under singlewave near-infrared (NIR) irradiation (Scheme 1), leading to potent in vivo hyperthermia and intracellular ROS in the tumor site. Moreover, Te-NDs exhibit ultrasmall size, enhanced NIR absorbance, ideal resistance to photobleaching, effective cytoplasmic translocation, preferable tumor accumulation, as well as renal excretion, thereby facilitating synergistic anticancer efficacy between photothermal therapy (PTT) and photodynamic therapy (PDT) for total tumor ablation.

RESULTS AND DISCUSSION Controlled Synthesis and Characterization of Te-NDs. An HSA nanoreactor was used to synthesize Te-NDs in hollow protein nanocages (Scheme 1A).24 Briefly, HSA in water was mixed with Na2TeO3 under vigorous vortexing, and pH was adjusted to be 12 for unfolding HSA.31,32 Then, NaBH4 as a strong reductant was further added to trigger the nucleation and growth of TeO32− into elemental Te nanocrystals through the reduction reaction of Na2TeO3 + NaBH4 + H2O = Te↓+ 2H2↑+ NaBO2 + 2NaOH in the expansive nanocages of nanoreactors.33 In the absence of HSA, the Te reduction reaction caused a muddy suspension, as compared to the 10013


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Figure 1. (A) Absorption spectra of Te-NDs synthesized at various time (inset: a magnified spectrum from 700 to 800 nm). (B) Normalized absorbance and its first derivative of Te-NDs at 785 nm. TEM images of Te-NDs synthesized at various reaction times including 1 min (C), 30 min (D), and 240 min (E), respectively. The inserts show the corresponding images at a higher magnification. (F) HR-TEM image of 5.9 nm Te-NDs.

3% phosphomolybdic acid. Te-NDs displayed the whole size of 8.6 ± 0.9 nm, 10.1 ± 1.5 nm, and 12.2 ± 1.9 nm, respectively (Figure S4), thereby indicating that Te-NDs hold the microstructure of Te nanocrystals as the core and protein nanocage as the shell with a thin thickness in the range of 2.5− 3.1 nm. Dynamic light scattering further indicates that these Te-NDs had the hydrodynamic diameters of 18.2, 21.4, and 24.8 nm owing to the presence of the hydration layer on TeNDs (Figure S5), indicating a potential passive targeting capability through enhanced permeation and retention (EPR) effect. Moreover, 5.9 nm Te-NDs exhibited a similar circular dichroism (CD) spectrum to that of HSA, indicating that the synthesis process has no significant influence on the secondary structure of albumin (Figure 2A). X-ray diffraction (XRD) pattern also shows that Te-NDs exhibited the characteristic Bragg’s peaks at (011) and (102) facets (Figure 2B). Thus, TeNDs had a hexagonal phase according to the characteristic peaks of standard Te phase, which is in agreement with their lattice fringe as shown in Figure 1F. Next, X-ray photoelectron spectroscopy (XPS) also suggested a composition of elemental

conditions. Then, Te-NDs with diameter of 5.9 nm, which were synthesized under pH 12 at 55 °C in the presence of Te/ NaBH4 ratio of 1:2 for 4 h reaction, were used for subsequent studies.34 The high-resolution transmission electron microscopy (HRTEM) image shows that Te-NDs with 5.9 nm in diameter exhibited a lattice fringe of d011 = 3.30 ± 0.02 Å (Figure 1F), which is in agreement with that of hexagonal element Te phase. The HSA nanoreactor allows the growth of Te nanocrystals with ultrasmall size within protein nanocages in the presence of potent reductant.27,28 In particular, the spherical morphology of Te-NDs is significantly different from reported one-dimensional Te nanostructures with relatively large size (Figure 1E),20−22 suggesting that this nanoreactor acts as an effective template for the synthesis of symmetrical NDs, although elemental Te has an anisotropic growth tendency. Additionally, ultrasmall size (650 nm). In addition, the molar extinction coefficient of Te-NDs was evaluated to be ∼109 M−1 cm−1 within the wavelength range of 650−900 nm. In particular, Te-NDs showed the molar extinction coefficient of 2.14 × 109 M−1 cm−1 at 785 nm (Figure S8), which is comparable to some typical photoabsorbing nanomaterials such as Au nanorods (109−1010 M−1 cm−1) at 808 nm.40−42 To demonstrate the photothermal effect of Te-NDs, we investigated their thermal behavior under 785 nm irradiation at 1.5 W cm−2. Te-NDs had a temperature elevation (ΔT) of 12.5 °C in 300 s at the concentration of 0.2 mM Te (Figure 3B), indicating a potential ability to induce hyperthermia, while PBS showed no significant temperature elevation. Moreover, TeNDs also had the concentration-dependent temperature increase, thereby displaying a strong ability to generate potent hyperthermia (>45 °C).24,43 Next, we further evaluated their photothermal conversion efficiency. Te-NDs had the photothermal conversion efficiency of 40.0%, which is preferable to

Te0 (Figure 2C). In addition, the energy dispersive X-ray spectroscopy (EDX) analysis of Te-NDs further exhibited the characteristic Te peaks at 3.77 and 4.05 keV (Figure 2D), indicating a composition of elemental Te without any other elements as well. Subsequently, 5.9 nm Te-NDs were found to possess a drug loading level of 2.0% with good encapsulation. Afterward, the chemical stability of Te-NDs was further evaluated in various aqueous solutions. Te-NDs were found to have a good chemical stability in water, serum, and PBS at pH 7.4 (Figure S6). Consequently, the albumin nanoreactor allows the effective growth of ultrasmall Te nanocrystals with stable nanostructure in albumin nanocages through the potent reduction. Absorption, Photothermal Effect, ROS Generation, and Photostability of Te-NDs. We evaluated their absorption spectrum. Figure 3A shows that Te-NDs had the absorbance peak at 278 nm with a widened shoulder at higher wavelength. To identify the optical response of Te nanostructure within Te-NDs, we performed the nonlinear Gauss fitting of this experimental absorption spectrum for peak-fit processing, which displays two peaks around 278 nm (Fit Peak 1) and 288 nm (Fit Peak 2), respectively. According to the absorption spectrum of HSA (Figure S7), it is reasonable to attribute the peak around 278 nm and shoulder around 288 nm to the optical absorption of HSA and essential optical response of Te nanostructure in the experimental spectrum, respectively. To further confirm the absorption of Te-NDs with 5.9 nm diameter, their absorption spectrum was further simulated using the discrete dipole approximation (DDA) method, which is powerful to simulate the optical response of nanoparticles with 10015


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Figure 3. (A) Nonlinear fitting of the experimental absorption spectra of Te-NDs and their absorption spectrum from DDA simulation. (B) Concentration-dependent temperature elevation of Te-NDs under 785 nm irradiation at 1.5 W cm−2. (C) Photothermal conversion efficiency of Te-NDs. (D) Concentration-dependent ROS generation from Te-NDs under 785 nm irradiation at 1.5 W cm−2 using DPBF as a probe. (E) ESR spectra of Te-NDs with or without oxygen using BMPO and DMPO as spin-trapping agents under 785 nm irradiation (1.5 W cm−2, 5 min), respectively. (F) Temperature elevation of Te-NDs under five irradiation/cooling cycles. (G) ROS generation of Te-NDs under 0, 10, and 20 min irradiation (785 nm, 1.5 W cm−2) using DPBF as a probe, respectively. (H) Cell viability of Te-NDs against 4T1 cells in the presence or absence of 10 mM Vc under 3 min irradiation (785 nm, 1.5 W cm−2) at 37 or 4 °C (student’s t-test, *p < 0.05, **p < 0.01).

relaxation, and thus their energy is emitted as the heat to achieve their photothermal conversion (Scheme 1B). To explore whether Te-NDs have the ability to produce ROS, we trapped ROS from Te-NDs in aqueous solution using 1,3-diphenyliso-benzofuran (DPBF).46 Te-NDs at 0.05 mM produced a detectable amount of ROS after a few minutes of irradiation (Figure 3D) and also exhibited a concentrationdependent ROS generation, suggesting a good capacity to

most of the extensively studied photothermal agents such as Au nanorods and organic dyes (Figure 3C).10,44,45 Although TeNDs have a relatively low extinction coefficient, their high photothermal conversion efficiency ensures their robust photothermal effect. Reasonably, Te-NDs as a semiconductor agents are effectively excited from valence band to conduction band under NIR irradiation, in which the excitons with electron−hole (e−−h+) pairs undergo the nonradiative 10016


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ACS Nano produce ROS under irradiation.47 Moreover, we further evaluated the quantum yield (ΦΔ) of Te-NDs to generate ROS. Te-NDs had the quantum yield of 0.12 (Figure S9), indicating a comparable ability to some typical NIR organic dyes such as indocyanine green (ΦΔ = 0.14),48 although TeNDs had a lower quantum yield as compared to some conventional photosensitizers such as ZnPc (ΦΔ = 0.56 at 670 nm in dimethylformamide). Hence, Te-NDs are able to effectively generate ROS that often induce photodynamic damage on tumor cells. To distinguish the type of ROS, the electron spin resonance (ESR) technique was employed to monitor ROS signals from Te-NDs in the presence or absence of molecular oxygen upon 785 nm irradiation using 2,2,6,6tetramethylpiperide (TEMP), 5-tert-butoxycarbonyl-5-methyl1-pyrroline N-oxide (BMPO), and 5,5-dimethyl-1-pyrroline-Noxide (DMPO) as the spin-trapping agents of singlet oxygen (1O2), superoxide radicals (•O2−), and hydroxyl radicals (•OH), respectively. The ESR spectra of Te-NDs in aqueous solutions showed no obvious signal of spin trapped adducts of 1 O2, while the characteristic 1:1:1:1:1:1 multiplicity was observed from BMPO-•O2− adduct under irradiation, and the 1:2:2:1 multiplicity was also detected from DMPO-•OH adduct. Distinctly, photoexcited Te-NDs cause the presence of •O2−/•OH (Figures 3E and S10).49,50 Moreover, the ESR signal of 1:1:1:1:1:1 multiplicity disappeared in O2-free solution, further confirming the generation of •O2− from oxygen in the presence of Te-NDs upon irradiation. Interestingly, the 1:2:2:1 multiplicity from DMPO-•OH adduct was also not observed without O2, implying that •OH might be produced from the rapid dismutation of •O2− instead of direct generation from H2O (Scheme 1B).51,52 In addition, the band energy levels of Te-NDs were also evaluated through the cyclic voltammetry (Figure S11). Distinctly, the photoexcited electrons in conduction band were directly transferred to oxygen molecules to produce reactive superoxide radicals, further confirming the generation of •O2− under irradiation and their subsequent dismutation into •OH. As a result, TeNDs undergo a type-I mechanism through their electron transfer to produce highly reactive ROS such as •O2− and dismutated •OH upon NIR light irradiation.47 To evaluate the photostability of Te-NDs, we further monitored their absorption spectra under NIR irradiation. They showed no distinct change in the absorbance at 785 nm during 15 min of irradiation (Figure S12), indicating an ideal resistance to photobleaching. To further confirm the photostability, we evaluated the ability of Te-NDs to maintain temperature elevation under irradiation. The solutions of TeNDs suffered from 5 min irradiation at 1.5 W cm−2 and then were cooled to room temperature in the absence of irradiation for 5 min, followed by another 4 cycles of irradiation/cooling. As shown in Figure 3F, Te-NDs had the temperature elevation of 27.0 °C after first irradiation/cooling cycle and exhibited no significant change in their temperature elevation and morphology after 5 cycles (Figure S13), suggesting that TeNDs have a good resistance to photobleaching. Moreover, the ability of Te-NDs to generate ROS under irradiation was also evaluated using DPBF as a probe. Figure 3G shows that DPBF trapping ROS from Te-NDs only exhibited a negligible change in its absorbance at 415 nm during 20 min irradiation, indicating that Te-NDs are able to maintain their ability to generate ROS even during 20 min irradiation. Obviously, TeNDs possess a preferable photostability as compared to the widely used organic dyes and inorganic nanomaterials with

surface plasmon resonance,10,44,45 thereby enabling the generation of both photothermal effect and ROS. As a result, photostable Te-NDs are able to cause a distinct photothermal effect through nonradiative relaxation and also trigger ROS generation through the reaction between exciton and water/ oxygen.53−55 Cellular Uptake, Endocytosis Pathway, Intracellular Distribution, Cytotoxicity, and Apoptotic Mechanism of Te-NDs. To demonstrate the ability of Te-NDs to be internalized by tumor cells, we evaluated their cellular uptakes by 4T1 murine breast cancer cells. Te-NDs exhibited timedependent cellular uptake of Te inside the cells (Figure S14), which are highly advantageous to the concentration-dependent hyperthermia and ROS generation in the cells. Next, the endocytic pathway of Te-NDs was also investigated using various pathway inhibitors (Figure S15A). Chlorpromazine caused the decrease of 50% in the cellular uptake of Te, suggesting that Te-NDs are primarily internalized through clathrin-mediated endocytosis.24 Subsequently, confocal laser scanning microscopy (CLSM) was also employed to directly confirm this clathrin-mediated endocytosis (Figure S15B). We also observed the intracellular distribution of Cy5.5-labeled TeNDs in 4T1 cells stained with LysoTracker Green DND-26 and Hoechst 33342 in the presence or absence of irradiation using CLSM. It indicates that Te-NDs are endocytosed into the lysosomes owing to their high co-localization of 92.0% with the lysosomes, while a poor co-localization of 48% was observed for those cells treated with Te-NDs after irradiation (Figure S16). Distinctly, Te-NDs trigger the disruption of lysosomal membranes. To further confirm the influence of Te-NDs on the lysosomes under irradiation, we employed acridine orange (AO) staining to evaluate the integrity of the lysosomes. In Figure S17, the cells incubated with PBS exhibited overlapped yellow fluorescence between red and green fluorescence under irradiation or not, indicating that PBS showed no influence on the integrity of the lysosomes regardless of irradiation. However, the cells treated with Te-NDs at the doses of more than 0.1 mM Te had a significant decrease of red fluorescence under irradiation as compared to that in the absence of irradiation, suggesting that the lysosomes were disrupted in the presence of Te-NDs under irradiation. The lysosomal disruption is highly advantageous to the translocation of TeNDs from the lysosomes into the cytoplasm, which might maximize the accessibility of ROS to target organelles such as mitochondria and nucleus for enhanced photodynamic cytotoxicity.56,57 Generally, this lysosomal disruption results from the photochemical internalization effect in the cells that is able to disrupt the lysosomal membranes through intracellular ROS (Figure S18).58 To evaluate the photo-induced cytotoxicity of Te-NDs against tumor cells, 4T1 cells were treated with Te-NDs for 24 h, followed by 785 nm irradiation at 1.5 W cm−2 for 3 min. TeNDs exhibited a severe cell injury (0.17 mM IC50) under irradiation (Figure 3H), while they only had a dark cytotoxicity of 6.9 mM IC50 in the absence of irradiation (Figure S19). To distinguish the role of photodynamic damage or hyperthermia in the cytotoxicity of Te-NDs, ROS-scavenger vitamin C was used to scavenge intracellular ROS from Te-NDs under irradiation to inhibit photodynamic activity due to its good ability to scavenge ROS (Figure S20), while we temporarily incubated 4T1 cells at ∼4 °C during irradiation to avoid photothermal damage. In the presence of 10 mM Vc, the 10017


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Figure 4. (A) Plasma concentration of Te from the mice treated with Te-NDs at the dose of 50 μmol kg−1 Te at various times. (B) Distribution of Te in various major tissues of the mice treated with Te-NDs at the dose of 50.0 μmol kg−1 Te at various times. (C) NIR fluorescence imaging of 4T1 tumor-bearing mice treated with Cy7.5-labeled Te-NDs at the dose of 50.0 μmol kg−1 Te during 72 h postinjection. (D) Long-term distribution of Te in various tissues of the mice treated with Te-NDs at the dose of 50.0 μmol kg−1 Te at various times. (E) Infrared thermography and (F) temperature elevation at the tumor of tumor-bearing mice treated with Te-NDs at various doses under 785 nm irradiation at 1.5 W cm−2. (G) DHE staining of tumor sections from the mice treated with Te-NDs at the dose of 50.0 μmol kg−1 Te in the presence or absence of ROS-scavenger Vc at 24 h post-injection upon irradiation. Scale bar, 200 μm.

In addition, to distinguish the mechanism of cell damage caused by Te-NDs, their apoptotic level in the absence or presence of 2.0 mM Vc was examined using AnnexinV/PI staining. Distinctly TeNDs resulted in both early and late apoptosis under PDT/PTT treatments, and PTT treatment alone resulted in the decrease of 50% in the late apoptosis (Figure S21). Reasonably, the photodynamic damage of TeNDs primarily contributes to half of the late apoptosis through superoxide radicals (•O2−), while their photothermal damage

relative cell viability of Te-NDs was increased (0.38 mM IC50) under irradiation, indicating that their cytotoxicity from PTT alone was distinctly decreased in the absence of ROS (Figure 3H). However, at ∼4 °C incubation, Te-NDs only possessed a IC50 of 0.69 mM, indicating a much lower cytotoxicity caused by PDT alone (Figure 3H). Thus, Te-NDs were found to have a synergistic index of 0.69, suggesting that Te-NDs have a significant synergistic effect between PDT and PTT efficiency. Reasonably, the lysosomal disruption might play an important role in their synergistic anticancer efficiency (Scheme 1C). 10018


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Figure 5. (A) Tumor growth profile of mice treated with Te-NDs at various doses in the presence or absence of Vc under 5 min irradiation (785 nm, 1.5 W cm−2) or not (** p < 0.01). (B) Photograph of tumors extracted from mice at 30 d post-irradiation. (C) H&E staining of tumor sections harvested from the mice treated with Te-NDs at the dose of 50.0 μmol kg−1 Te at 6 h post-irradiation. Scale bar, 100 μm. (D) Blood levels of ALP, ALT, AST. (E) Urea from the mice treated with Te-NDs at a single dose of 50.0 μmol kg−1 Te at various times.

accounts for both early apoptosis and the other half of late apoptosis through the hyperthermia. Pharmacokinetics, Biodistribution, Long-Term Clearance, Photo-Induced Hyperthermia, and ROS Generation in Vivo. To demonstrate the pharmacokinetic behavior, Te-NDs were intravenously injected into the mice at the dose of 50 μmol kg−1. As shown in Figure 4A, Te-NDs exhibited the elimination half-life time (t1/2β) of 1.4 h and area under the curve (AUC) of 111.6 μg mL−1 h (Table S1). To examine the in vivo biodistribution, Te-NDs were injected into the mice bearing 4T1 tumors via intravenous administration at the dose of 50 μmol kg−1 Te, and the distributions of Te at various tissues were evaluated at 12, 24, 48, and 72 h post-injection, respectively. Te-NDs were mainly distributed into the tumor and liver, indicating their preferable retention at the tumor owing to the EPR effect as well as tumor accumulation capability of albumin59,60 (Figure 4B). Clearly, the preferable tumor accumulation is able to facilitate concentration-dependent photothermal and photodynamic effects to maximize the anticancer efficacy. To further observe the retention behavior of Te-NDs at the tumor, we performed in vivo NIR fluorescence imaging of Cy7.5-labeled Te-NDs (Figures 4C and S22). TeNDs exhibited the maximal fluorescence intensity at the tumor

at 24 h post-injection and also maintained a good retention of fluorescence signals during 72 h,24 while Cy7.5 as a control showed a negligible fluorescence at the tumor. Thus, Te-NDs have a relatively long-term retention at the tumor, which is highly advantageous to achieve flexible light irradiation for PTT or PDT treatment. To assess the in vivo elimination of Te-NDs, we evaluated their long-term distributions at various tissues. The distributions of Te at various tissues were gradually decreased including heart, liver, spleen, lung, and kidney during 28 days (Figure 4D), suggesting an effective elimination from normal tissues through renal excretion due to their ultrasmall size. The detailed clearance pathway and in vivo fate of Te-NDs remain to be clarified in the future. To demonstrate the in vivo hyperthermia at the tumor, TeNDs were injected into the mice at the various doses, followed by infrared thermograph under 5 min irradiation (785 nm, 1.5 W cm−2) at 24 h post-injection. Te-NDs at the dose of 6.25 μmol kg−1 Te caused the temperature increase of ∼9.0 °C at the tumor under irradiation (Figure 4E,F), indicating a mild hyperthermia.43 Particularly, Te-NDs resulted in the temperature elevations of 13.0, 14.5, and 20.0 °C at the tumors at the doses of 12.5, 25.0, and 50.0 μmol kg−1 Te under irradiation, respectively (Figure 4E,F), indicating a potent dose-dependent 10019


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translocation, preferable tumor accumulation, ultrasmall size for effective renal excretion, and use of clinically acceptable albumin. Consequently, these synergistic characteristics of bifunctional Te-NDs account for their potential for effective photoactive cancer therapy. To validate the anticancer efficacy, hematoxylin and eosin (H&E) staining was employed to examine the tumor damage from Te-NDs at the dose of 50.0 μmol kg−1 Te under irradiation. Te-NDs resulted in destructive cell necrosis at the tumor at 6 h post-irradiation (Figure 5C).24 In contrast, PBS showed no detectable tumor damage regardless of irradiation (Figure 5C), and Te-NDs also exhibited no significant influence on the normal tissues such as heart, liver, spleen, lung, and kidney (Figure S25). Finally, the serum biochemistry assay was applied to evaluate the potential toxicity of Te-NDs. The levels of liver and kidney function markers including ALP, ALT, AST, and urea during 28 days post-injection had no distinct change as compared to their original levels (Figure 5D,E), confirming that Te-NDs have no detectable damage on the normal tissues, probably owing to their renal excretion ability.

hyperthermia behavior owing to both good photothermal conversion efficiency and enhanced tumor accumulation.24 In contrast, PBS only triggered a negligible temperature elevation at the tumor under irradiation. Furthermore, we also confirmed the generation of ROS at the tumor under irradiation using dihydroethidium (DHE) staining. Figure 4G shows that abundant ROS were observed from Te-NDs at the tumor under irradiation, while ROS were significantly scavenged at tumor in the presence of ROS-scavenger Vc. Clearly, Te-NDs have a sufficient capacity to generate in vivo ROS at the tumor under irradiation, which play a prerequisite role in PDT efficacy. Antitumor Efficacy, Histological Staining, and Serum Biochemistry Assay. To demonstrate the in vivo anticancer efficacy, Te-NDs were injected into the mice bearing 4T1 tumor at various doses through single-dose intravenous administration, followed by 5 min irradiation (785 nm, 1.5 W cm−2) of the tumors at 24 h post-injection. Then, the tumor volumes were monitored during the subsequent 30 days (Figure 5A,B). Te-NDs led to effective tumor ablation at the dose of 6.25 μmol kg−1 Te under irradiation, but exhibited significant tumor regrowth at 7 days post-irradiation, while the higher doses of 12.5, 25.0, or 50.0 μmol kg−1 Te caused the total tumor ablation without any regrowth (Figure 5A,B). Thus, the tumor temperature elevation of ∼13.0 °C from 12.5 μmol kg−1 Te-NDs can be considered as the threshold for total tumor ablation (Figure 4F).43 In contrast, PBS exhibited more than ∼30-fold increase of tumor volumes regardless of irradiation, and Te-NDs in the absence of irradiation also showed a similar tumor growth to those of PBS. To distinguish the role of ROS in the anticancer efficacy, ROS-scavenger Vc was administrated into the tumors through intratumor injection for scavenging ROS from Te-NDs at the tumor. Distinctly, Te-NDs at the dose of 12.5 μmol kg−1 exhibited significant tumor regrowth in the presence of Vc at 9 days post-irradiation (Figure 5A), indicating that only the hyperthermia is unable to ablate all the tumors in the absence of ROS. Thus, the presence of ROS at the tumor contributes to the total tumor ablation in addition to hyperthermia (Figure 4G).54 Moreover, the synergy of both PTT and PDT damages from Te-NDs might further reduce tissue oxygen depletion as compared to PDT alone and thus is potentially able to avoid possible hypoxia at the tumor under irradiation. Furthermore, we assessed the in vivo anticancer efficacy of Te-NDs at a low irradiation density of 0.35 W cm−2. Te-NDs still displayed a distinct tumor ablation at the dose of 50.0 μmol kg−1 Te (Figure S23), although this low irradiation density caused a lower hyperthermia of ∼15 °C (Figure S24). Distinctly, Te-NDs are able to effectively achieve potent synergistic efficacy even at a relatively low irradiation density. To date, although there are several existing nanoplatforms that integrate both photosensitizer and photothermal agents for achieving their synergistic treatments,53,61−67 some concerns are still encountered such as poor photostability, inconsistency of pharmacokinetics and excitation wavelengths of two encapsulated agents, absence of synergistic mechanism between PDT/PTT, and use of clinically unapproved ingredients. TeNDs display several characteristics that account for their effective tumor ablation including bifunctional characteristics of photothermal effects and highly toxic ROS through type-I mechanism under NIR irradiation, perfect resistance to photobleaching, distinct synergistic effect between PDT and PTT through lysosomal disruption-mediated cytoplasmic

CONCLUSIONS In summary, bifunctional Te-NDs with well-defined nanostructure are synthesized within albumin nanocages in a controllable manner through potent reduction. Electrons of Te-NDs can be excited from valence band to conduction band under NIR irradiation, causing both potent photothermal effect through nonradiative relaxation and abundant ROS such as •O2− and dismutated •OH through electron transfer. Moreover, Te-NDs with 5.9 nm in diameter as an inorganic nanomaterial exhibit the collective characteristics for efficient cancer therapy including NIR absorbance with good tissue transparence, ultrasmall size, ideal photostability, synergistic effect through effective cytoplasmic translocation, preferable tumor accumulation, and easy renal excretion, thereby facilitating total tumor ablation. These collective characteristics allow bifunctional TeNDs to afford preferable PTT/PDT efficacy as compared to conventional photosensitizers or PTT agents, owing to their ability to overcome several limitations such as complex compositions, frequently encountered wavelength mismatch between photosensitizer and PTT agent, severe photobleaching, as well as insufficient cell damage.54 To date, although several Te-containing polymer systems have been explored for drug delivery, there are very few reports on the nanostructure of elemental Te for cancer therapy.68−71 This system can serve as a valuable paradigm for synergistic photo-induced cancer therapy. EXPERIMENTAL SECTION Synthesis. Tellurium nanodots (Te-NDs) were synthesized using human serum albumin (HSA) as a nanoreactor. 10.0 mL of 25.0 mg mL−1 HSA was dropwise added to 2.0 mL of 20.0 mM Na2TeO3 under stirring. Next, 2.0 M NaOH was added to adjust the solution to pH 12. Next, 0.8 mL of 100.0 mM NaBH4 as the reductant was further added into the mixture at the Te/NaBH4 ratio of 1:2. Then, the reduction reaction was performed in the mixture at 55 °C for 4 h under vigorous stirring, followed by the formation of Te-NDs. Subsequently, Te-NDs were centrifuged through the ultrafiltration (100 kDa, Millipore) and purified through the dialysis against distilled water. The synthesis of Te-NDs at various temperatures (25 and 37 °C), pH (pH 8 and pH 10), and Te/NaBH4 ratio (1:1 and 1:1.5) was also performed according to the above procedures. For the synthesis of Cy5.5-labeled or Cy7.5-labeled Te-NDs, 1.0 mL of Cy5.5-NHS or Cy7.5-NHS (0.5 10020


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ACS Nano mg mL−1) was mixed with 4.0 mL Te-NDs and stirred in the dark overnight, followed by the purification through the dialysis against distilled water. Characterization. The morphology and energy-dispersive X-ray spectroscopy (EDX) of Te-NDs were characterized using transmission electron microscopy (TEM) imaging (Hitachi H-600). The microstructure of Te-NDs was evaluated using high-resolution TEM imaging (FEI Technai G2 S-Twin) at 200 kV. The secondary structures of HSA and protein in Te-NDs were monitored through their CD spectra using a spectrometer (JASCD J-815). The hydrodynamic diameter and size distribution were measured using dynamic light scattering (Zetasizer NanoZS90). X-ray diffraction (XRD) was carried out using an X-ray diffractometer (Bruker D8 Advance) with Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was evaluated using X-ray photoelectron spectrometer (Thermo Scientific Escalab 250Xi). The absorbance spectra were characterized using UV−vis spectrophotometer (UV2600, Shimadzu). The concentration of Te was determined using inductively coupled plasma optical emission spectrometer (ICP-OES, VARIAN, 710-ES). To evaluate the chemical stability of Te-NDs in various solutions including PBS, water, and cell culture medium containing 10% serum, their absorbances in these solutions were monitored at various times. To assess the molar extinction coefficient of Te-NDs, the absorbances of Te-NDs with various concentrations (1, 3, 5, 7, 9, and 10 μg mL−1) at various wavelengths were measured, and then their absorbances were plotted against the concentrations. The slope was calculated as the extinction coefficient by the linear fitting. Photothermal Effect and Photothermal Conversion Efficiency. To evaluate the photothermal effect of Te-NDs, the solutions of Te-NDs (0.5 mL) were irradiated for 5 min at various concentrations ranging from 0.2 mM to 1.0 mM (785 nm, 1.5 W cm−2), and the temperature was measured every 30 s using a digital thermometer. To evaluate the photothermal conversion efficiency, 0.5 mL of Te-NDs (1.0 mM) was exposed to 785 nm irradiation at 1.5 W cm−2 for 10 min, and then the laser was removed to cool down to room temperature. The temperature of the solution was recorded at an interval of 30 s during this process. The photothermal conversion efficiency (η) was calculated according to the following equation:

ηT =

785 nm irradiation using 2,2,6,6-tetramethylpiperide (TEMP), 5-tertbutoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO), and 5,5dimethyl-1-pyrroline-N-oxide (DMPO) as the spin-trapping agents of singlet oxygen (1O2), superoxide radicals (•O2−), and hydroxyl radicals (•OH), respectively. Spectra of spin trapped •O2− were acquired by mixing 5 μL BMPO (500 mM) with 100 μL Te-NDs. Then the samples were irradiated under 785 nm at 1.5 W cm−2 for 5 min, followed by ESR analysis. The ESR spectrum of Te-NDs in the presence of BMPO without irradiation was collected as a control. Spectra of spin trapped 1O2 and •OH from Te-NDs were also monitored using TEMP (5 μL, 500 mM) and DMPO (5 μL, 500 mM) according to the above procedures. In addition, the cyclic voltammetry (CV) was applied to determine the band energy levels of Te-NDs upon irradiation. Platinum wire was used as the counter electrode, a saturated calomel electrode (SCE) was used as the reference electrode, and Te-NDs films sprayed on ITO glass served as the working electrode. The photoelectrochemical experiment results were recorded with an electrochemical system (RST5000, China). Photostability. To evaluate the photostability for temperature elevation, 1.0 mM Te-NDs suffered from 785 nm irradiation at 1.5 W cm−2 for 5 min and then were cooled down to room temperature. Afterward, additional four irradiation/cooling cycles were repeated. During these cycles, the temperature was monitored, and their morphology was also observed after 5 cycles using TEM imaging. To evaluate the photostability for ROS generation, the solutions of TeNDs at same Te concentration were irradiated at 1.5 W cm−2 for 0 min, 10 min, 20 min, respectively, followed by ROS measurement. Cell Uptake. The cells (1.0 × 105 cells per well) were incubated with Te-NDs at the concentration of 0.1 mM Te for 2, 6, 12, and 24 h, respectively, followed by the cell collection and subsequent cell counting. Then, the cells were digested by nitric acid, and the concentration of Te was measured using ICP-OES. To confirm the cellular uptake, Cy5.5-labeled Te-NDs were also incubated with the cells for 2, 6, 12, and 24 h, respectively, followed by confocal laser scanning microscopy (CLSM) observation. Endocytic Pathway. 4T1 cells (1.0 × 105 cells per well) were treated with various inhibitors inlcuding chlorpromazine (10.0 μg mL−1), filipin (5.0 μg mL−1), and amiloride (100.0 μg mL−1) at 37 and 4 °C for 1 h incubation. Then, Te-NDs or Cy5.5-labeled Te-NDs were added into the cells at the dose of 0.1 mM Te. After 2 h incubation, the cells were washed 3 times using PBS, followed by the analysis of Te or CLSM observation. Intracellular Distribution. The cells (5.0 × 104 cells/well) in 20 mm glass-bottom dishes were incubated with Cy5.5-labeled Te-NDs at the concentration of 0.1 mM Te for 24 h at 37 °C, followed by 785 nm irradiation (3 min, 1.5 W cm−2) or not. Afterward, 0.3 mL Hoechst 33342 was added into the cells for 10 min incubation, and then 0.3 mL Lysotraker Green DND-26 (100 nM) was further added for 3 min incubation at 37 °C. Finally, the cells were observed using CLSM (Zeiss LSM710). Cytotoxicity. To distinguish the role of photodynamic damage and hyperthermia in the cytotoxicity of Te-NDs, ROS-scavenger Vitamin C (Vc, 10.0 mM) was used to scavenge intracellular ROS from TeNDs under irradiation for inhibiting their photodynamic activity, while we incubated 4T1 cells at ∼4 °C during irradiation to avoid photothermal cell damage. The cells were treated with Te-NDs at various doses of Te including 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mM for 24 h in the presence or absence of 10.0 mM Vc and then suffered from 785 nm irradiation (3 min, 1.5 W cm−2) at 37 or 4 °C. Then, the cell viability was measured using MTT assay. The synergistic index was calculated according to the equation of CI = DA/IC50(A) + DB/IC50(B), in which IC50(A) and IC50(B) are the respective IC50 values of drugs A and B in the presence of drug A or B alone, as well as DA and DB are drug concentrations of drugs A and B to reach IC50 in the presence of both drugs, respectively, and CI < 0.8 is considered as a significant synergy. To investigate the dark cytotoxicity, 4T1 cells were treated with Te-NDs at the doses of 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 8.0, and 10.0 mM for 24 h, respectively, and then their viability was evaluated using MTT.

hA(Tmax − Tamb) − Q 0 I(1 − 10−Aλ)

where h is the heat transfer coefficient, A is the surface area of the container, Tmax is the maximum system temperature, Tamb is the ambient surrounding temperature, I is the laser power, and Aλ is the absorbance at an excitation wavelength of 785 nm. Q0 is the rate of heat input (in units of mW) due to light absorption by the solvent. Reactive Oxygen Species (ROS), ROS Quantum Yield, and Electron Spin Resonance (ESR). The ROS generation of Te-NDs under 785 nm irradiation was measured using 1,3-diphenylbenzofuran (DPBF) as a probe. 2.97 mL of Te-NDs at different concentrations in quartz cuvettes were mixed with 30.0 μL of 3 mM DPBF in ethanol under continuous stirring. Under 785 nm irradiation at 1.5 W cm−2, the absorbances of DPBF at 415 nm were recorded at various times, followed by deducting the absorbance of Te-NDs themselves. To confirm the ability of vitamin C (Vc) to scavenge ROS from Te-NDs (0.2 mM), DPBF was also used to monitor the ROS generation of TeNDs in the presence of Vc at various concentrations of 0, 1.0, and 2.0 mM, respectively. For ROS quantum yield (ΦΔ) measurement, DPBF was used as a probe, and indocyanine green (ICG) was used as a reference compound (ΦΔICG = 0.14). The solutions of Te-NDs (0.1 mM) and ICG (0.6 μM) containing DPBF (30 μM) were irradiated at 785 nm (1.5 W cm−2) for 300 s. The absorbance of DPBF at 415 nm was measured every 30 s. ΦΔ was calculated using the equation ΦΔ = (ΦΔICG × W × IICG)/(WICG × I), where W and WICG are the DPBF photobleaching rates in the presence of Te-NDs and ICG, respectively. I and IICG are the rates of light absorption by Te-NDs and ICG, respectively. To distinguish the type of ROS, the electron spin resonance (ESR) technique was employed to monitor ROS signals from Te-NDs in the presence or absence of molecular oxygen upon 10021


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ACS Nano Lysosomal Disruption. Acridine orange (AO) as an intracellular indicator can emit red fluorescence (yellow after overlap) in intact lysosomes and generate green fluorescence in nuclei and cytoplasm. 4T1 cells were seeded on the glass slides in 35 mm dishes and treated with PBS and Te-NDs for 6 h, respectively. Then, the cells suffered from 785 nm irradiation at 1.5 W cm−2 for 3 min. After 1 h, the cells were washed using PBS and further incubated with 1.0 mL AO (10 μM) for 30 min. Finally, the cells were observed using CLSM (Zeiss LSM710). Observation of Intracellular ROS. Dihydroethidium (DHE) was used as the probe of ROS. 4T1 cells (1.0 × 105 cells per well) were treated with Te-NDs at the doses of 0.05, 0.1, 0.2, and 0.5 mM for 6 h, respectively. Then, the cells suffered from 3 min irradiation (785 nm, 1.5 W cm−2) or not. Next, 1.0 mL DHE (10 μM) was added for 30 min incubation, followed by CLSM (Zeiss LSM710). Apoptosis Assay. 4T1 cells (1.0 × 105 cells per well) were incubated with Te-NDs (0.2 mM) for 24 h with or without coincubation with 2.0 mM Vc. Then, the cells suffered from 785 nm irradiation (3 min, 1.5 W cm−2). After 24 h, the cells were collected and stained with an Annexin V-FITC/PI Apoptosis Detection Kit, followed by the flow cytometry analysis. Pharmacokinetics. Te-NDs were intravenously injected into healthy Balb/c mice at the dose of 50.0 μmol kg−1 (n = 3). Blood samples were collected at 5 min, 10 min, 30 min, 1, 2, 4, 8, 12, 24, and 48 h post-injection, respectively. Then, the samples were treated with nitric acid, followed by the analysis of Te using ICP-MS. Biodistribution and in Vivo Elimination. The female Balb/c mice bearing 4T1 tumor were constructed through the subcutaneous injection of 4T1 cells (2 × 106 cells/mouse). Te-NDs were intravenously injected at the dose of 50.0 μmol kg−1 (n = 3). Then, various tissues including heart, liver, spleen, lung, kidney, and tumor were extracted at 12, 24, 48, and 72 h post-injection, respectively. The tissues were treated with nitric acid, followed by the analysis of Te using ICP-OES. To investigate the long-term distribution, Te-NDs were intravenously injected into healthy Balb/c mice at a single dose of 50.0 μmol kg−1 (n = 3). Various tissues including heart, liver, spleen, lung, kidney, intestine, and urine were extracted at 3, 7, 14, 21, and 28 days, respectively. Finally, the samples were treated with nitric acid, followed by the analysis of Te using ICP-MS. In Vivo NIR Fluorescence Imaging. For NIR fluorescence imaging, Cy7.5-labeled Te-NDs were intravenously injected at the dose of 50.0 μmol kg−1 Te, and free Cy7.5 was used as a control at the dose of 0.5 mg kg−1. Then, the in vivo fluorescence imaging was performed at 0, 8, 12, 24, 48, and 72 h post-injection using IVIS Lumina II imaging. Finally, the average fluorescent intensity at tumor region was calculated. In Vivo Infrared Thermography. To monitor the in vivo photothermal effect, Te-NDs were injected into the tumor-bearing mice at the doses of 6.25, 12.5, 25.0, and 50.0 μmol kg−1, respectively, and then the tumors suffered from 1.5 W cm−2 and 0.35 mW cm−2 irradiation at 24 h post-injection, respectively. Meanwhile, the temperature at the tumor was monitored using an infrared camera (FLIR E50). Observation of in Vivo ROS at the Tumor. To validate the generation of ROS at the tumor, Te-NDs at a dose of 50.0 μmol kg−1 were injected into the tumor-bearing Balb/c mice. Then, Vc was injected into the tumor at the dose of 25.0 μmol kg−1 via intratumoral injection at 24 h post-injection. Meanwhile, 20.0 μmol kg−1 DHE was also injected into the tumor via intratumoral injection. After 0.5 h, the tumors were irradiated at 1.5 W cm−2 for 3 min or not. Afterward, the tumors were frozen to prepare the sections with the thickness of 10 μm. The tumor sections were observed using CLSM (Zeiss LSM710). In Vivo Anticancer Efficacy. Te-NDs were intravenously injected into the Balb/c mice bearing 4T1 tumor with the tumor size of 80− 100 mm3 at the doses of 6.25, 12.5, 25.0, and 50.0 μmol kg−1 (n = 5), respectively. The mice treated with Te-NDs at 12.5 μmol kg−1 were further injected with Vc at the dose of 25.0 μmol kg−1 via intratumor administration at 24 h post-injection. Then, the tumors suffered from 785 nm irradiation (5 min, 1.5 W cm−2) or not. Subsequently, the tumor volume (V) was calculated as follows: V = L × W2/2, where L

and W are the longest and widest dimensions, respectively. Finally, the tumors were extracted and imaged after 30 days. To verify the anticancer efficacy of Te-NDs at a low irradiation density, Te-NDs were injected into the mice at the doses of 6.25, 12.5, 25.0, and 50.0 μmol kg−1 (n = 5). After 24 h post-injection, the tumors suffered from 0.35 mW cm−2 irradiation for 20 min, followed by tumor volume measurements. Histological Staining. Te-NDs were intravenously injected into the Balb/c mice bearing 4T1 tumors at a single dose of 50.0 μmol kg−1. Then, the tumors suffered from 5 min of irradiation (785 nm, 1.5 W cm−2) at 24 h post-injection. The tumor, heart, liver, spleen, lung, and kidney were dissected from the mice at 6 h post-irradiation and fixed in a 4% formaldehyde solution for 24 h at room temperature. The various tissues were frozen, and the sections with the thickness of 10 μm were made on a cryostat. The H&E staining (BBC Biochemical, Mount Vernon, WA) was performed, and the sections were finally observed using an IX73 bright-field microscopy. Serum Biochemistry Assay. Te-NDs were intravenously injected into healthy Balb/c mice at a single dose of 50.0 μmol kg−1 (n = 3). 0.5 mL of blood from each mouse was collected at 0, 7, 14, 21, and 28 days, respectively. Then, the blood samples were used to perform the blood biochemistry examination.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b04230. Additional results (Figure S1−25, and Table S1) (PDF)

AUTHOR INFORMATION Corresponding Authors

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

Chunying Chen: 0000-0002-6027-0315 Yuliang Zhao: 0000-0002-9586-9360 Huabing Chen: 0000-0003-1637-2872 Author Contributions #

These authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (31422021, 51473109, 11304238, and 81473166), National Basic Research Program (2014CB931900), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases, and Project 16NBI02 from CAS Key laboratory of Nano-Bio Interface of Suzhou Institute of NanoTech and Nano-Bionics, CAS. We thank Dr. Hervé Portalès for assistance with the discussion about absorbance. REFERENCES (1) Srivastava, P.; Nikhil, E. V. R.; Bragança, J. M.; Kowshik, M. AntiBacterial TeNPs Biosynthesized by Haloarcheaon Halococcus Salifodinae BK3. Extremophiles 2015, 19, 875−884. (2) Baesman, S. M.; Bullen, T. D.; Dewald, J.; Zhang, D.; Curran, S.; Islam, F. S.; Beveridge, T. J.; Oremland, R. S. Formation of Tellurium Nanocrystals during Anaerobic Growth of Bacteria that Use Te Oxyanions as Respiratory Electron Acceptors. Appl. Environ. Microbiol. 2007, 73, 2135−2143. 10022


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