Clearable Theranostic Platform with a pH-Independent

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Biological and Medical Applications of Materials and Interfaces

Clearable Theranostic Platform with pH-independent Chemodynamic Therapy Enhancement Strategy for Synergetic Photothermal Tumor Therapy Qian Chen, Yu Luo, Wenxian Du, Zhuang Liu, Shengjian Zhang, Jiahui Yang, Heliang Yao, Tianzhi Liu, Ming Ma, and Hangrong Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02905 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 3, 2019

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ACS Applied Materials & Interfaces

Clearable Theranostic Platform with pHindependent Chemodynamic Therapy Enhancement Strategy for Synergetic Photothermal Tumor Therapy Qian Chen 1, 2, Yu Luo 3, Wenxian Du 1, 2, Zhuang Liu 4, Shengjian Zhang 4, Jiahui Yang 5, Heliang Yao 1, Tianzhi Liu 1, 2, Ming Ma 1, Hangrong Chen1* 1State

Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai

Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, P. R. China; 2University of Chinese Academy of Sciences, Beijing, 100049, P. R. China; 3School of Chemical Science and Engineering, Tongji University, Shanghai 200092, P. R. China; 4Department of Radiology, Shanghai Cancer Hospital, Fudan University, Shanghai, 200032, P. R. China; 5Department of Bruker Bbio, Bruker (Shanghai) Scientific Technology Co. Ltd, Shanghai, 200233, P. R. China KEYWORDS: chemodynamic therapy; photothermal therapy; pH-independent; Fenton-like reaction; clearance; cancer theranostic

ABSTRACT: Chemodynamic therapy (CDT) is an emerging field, which utilizes intratumoral iron-mediated Fenton chemistry for cancer therapy. However, the slightly acidic tumor environment is improper for the classical Fenton reaction which is generally energetic at a

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narrow pH range (e.g., pH=3-4). Herein, a kind of ultrasmall bovine serum albumin(BSA)modified chalcopyrite nanoparticles (BSA-CuFeS2 NPs) was synthesized via a facile aqueous biomineralization strategy, which shows high dispersity and biocompatibility. Interestingly, the obtained BSA-CuFeS2 shows a pH-independent Fenton-like reaction, which could exert Fentonlike activity to efficiently generate •OH under weak acidic tumor environment. Combining with the extraordinarily high photothermal conversion (38.8%), BSA-CuFeS2 shows the synergistic function of high photothermal therapy and enhanced chemodynamic therapy, i.e, PTT/CDT. Importantly, such ultrasmall BSA-CuFeS2 NPs with around 4.9 nm can be quickly clearable out the body through kidneys and liver, thus effectively avoiding long-term toxicity and systemic toxicity. Moreover, BSA-CuFeS2 NPs can be acted as an efficient T2-weighted MRI contrast agent to guide tumor ablation in vivo. This work offers a universal approach to boost production •OH by a pH-independent Fenton-like reaction strategy and achieves MRI-guided synergistic enhanced photothermal-chemodynamic therapy for high efficient tumor treatment.

INTRODUCTION Reactive oxygen species (ROS), including hydroxyl radicals (·OH) and singlet oxygen (1O2), significantly suppress cancer cells by breaking biomolecules, thus inducing cellular apoptosis or necrosis.1-6 With the widespread attention of ROS-mediated cancer treatment strategies in recent years, chemodynamic therapy (CDT), as an emerging noninvasive therapeutic agent for cancer, employs overproduced endogenous hydrogen peroxide (H2O2) to convert less-reactive H2O2 into more cytotoxic · OH through an intratumoral iron-mediated Fenton chemistry.7-11 To date, several iron-based nanoparticles (NPs), such as amorphous iron nanoparticles (AFeNPs), Fe3O4 nanoparticles, Fe2O3 nanoparticles, and FePt nanoparticles have been designed for generating ·


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OH by endogenous H2O2-dependent Fenton reaction.12-17 The pH value of tumor microenvironment (around 6.5) is not beneficial for Fenton reaction, which is generally energetic at a narrow pH range (e.g., pH=3-4).18-20 So, a slightly acidic tumor environment usually decrease the · OH productivity and overall oxidation efficacy of the classical pH-dependent Fenton reaction.10,

11

Therefore, construction of a pH-independent Fenton-like reaction to

increase production · OH, especially in the weak acidic tumor environment is extremely expected. Photothermal therapy (PTT) with minimal invasiveness is effective therapeutic modality based on the near-infrared (NIR) laser, which utilizes photothermal conversion to generate a local heating effect for tumor ablation.21-25 The hyperthermia of the tumor area not only kills the cancer cell but also accelerates the hydroxyl radical production of CDT, resulting in a synergism of CDT/PTT.8 In recent years, PTT and CDT nanotherapeutic agents, such as Au nanorods,22, 26 semiconducting polymer NPs,27, 28 metal sulfide,29-31 amorphous iron oxide,32 and metal-organic framework (MOF)14 have been designed for cancer therapy in vivo. Additionally, it is vital that the nanotherapeutic agent can be rapidly cleared to decrease the accumulation of nanotherapeutic agent in the major organs, increasing long-term biosafety. It is reported that ultrasmall NPs ( ~ 6 nm) can be clearable through kidneys, eliminating from the body more efficiently.33-36 Herein, for the first time, we develop a novel kind of ultrasmall BSA-CuFeS2 NPs as a multifunctional theranostic platform via an eco-friendly bioinspired albumin-mediated strategy, which possesses distinctively pH-independent Fenton-like reaction property to efficiently generate·OH and excellent photothermal-conversion efficiency for synergetic enhancement of CDT/PTT therapy (Scheme 1). The obtained ultrasmall BSA-CuFeS2 NPs can efficiently work in


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a broad pH range to catalyze H2O2 to generate ROS and exhibit prominent photothermal performance. Importantly, the ultrasmall size endows BSA-CuFeS2 NPs with the ability to be rapidly excreted from the body through the kidneys and liver, thus effectively avoiding long-term toxicity and systemic toxicity. BSA-CuFeS2 NPs show no apparent toxicity in vivo. Furthermore, distinctively synergetic enhanced CDT/PTT efficiency has been proved in vivo. RESULTS AND DISCUSSION Design, Synthesis, and Characterization of BSA-CuFeS2 NPs. Ultrasmall BSA-CuFeS2 NPs were synthesized via an eco-friendly aqueous biomineralization strategy at physiological temperature (37 °C). BSA was used as a stabilizer to anchor Cu and Fe ions by the outstanding affinity of carboxyl groups and surfactant to enhance their biocompatibility.37-39 After injecting Na2S·9H2O, the solution changed into black rapidly, indicating the formation of CuFeS2. Transmission electron microscopy (TEM) images show BSA-CuFeS2 NPs are mono-dispersed with the size of 4.9 ± 0.9 nm (Figure 1a, 1b, and 1d), which could be readily cleared through the kidneys.40-42 High-resolution TEM images (HRTEM) obviously reveals the interplanar spacings of 0.305 nm, in agreement with the (111) lattice plane of CuFeS2 (Figure 1c). It is noting that X-ray diffraction (XRD) pattern of BSA-CuFeS2 displays no characteristic peaks of CuFeS2 which could be covered by a broad peak of BSA around 22 ° (Figure 1e).38,

43

Therefore, the XRD pattern of CuFeS2 without BSA modification was also conducted to confirm the existence of CuFeS2. The XRD pattern of obtained NPs is consistent with standard peaks (JCPDS 41-1404) of CuFeS2. The elements mapping of obtained NPs shows the homogeneous distribution of Cu, Fe, and S, confirming the chemical composition of CuFeS2 (Figure S1). The valence state information of BSA-CuFeS2 measured by X-ray photoelectron spectroscopy (XPS)


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accord with previous studies,44,

45

confirming the formation of CuFeS2 (Figure 1g, h, i). The

BSA modification endows NPs with excellent the aqueous solubility and stability, and the hydrodynamic diameter is about 42.5 nm (Figure 1f), which is much suitable for the application in vivo. It is noting that the hydrodynamic diameter is larger than the TEM size, likely because of the fact that the hydrodynamic size comprises the hydration shell, inorganic CuFeS2, and surface of BSA, thereinto, the size of BSA is 4.5 nm × 14.2 nm × 21.6 nm.46 Photothermal Property of BSA-CuFeS2 NPs. The optical absorption spectra of BSACuFeS2 NPs show strong absorption in the NIR region (Figure 2a). The extinction coefficients were calculated to be 18.1 Lg−1 cm−1 by Lambert-Beer law (A = εCL) (Figure 2b), which is significantly higher than that of CuFeSe2 NPs (5.8 Lg−1cm−1),47 carbon nanodots (0.35 Lg−1cm−1),48 black phosphorus quantum dots (14.8 Lg−1cm−1),49 and Cu2−xSe NCs (2.9 Lg−1cm−1),50 at 808 nm, etc., suggesting the strong NIR light absorption capability. BSA-CuFeS2 NPs solution with varying concentrations was irradiated by the 808 laser for 300 s to investigate their photothermal performance. Obviously, BSA-CuFeS2 NPs show concentration-dependent photothermal effect (Figure 2c), and the temperature of BSA-CuFeS2 NPs solution rapidly increases to the highest temperature of 63 °C (Figure 2d), demonstrating the ultrasmall BSACuFeS2 NPs can rapidly transform NIR light into thermal energy. Besides, BSA-CuFeS2 NPs exhibit remarkable photothermal stability during the recycling temperature variations (Figure 2e and Figure S2). Probably due to the unique band structure and high Néel temperature of 823 K,51, 52 the photothermal conversion efficiency (η) of BSA-CuFeS2 NPs reaches up to be 38.8% (Figure 2f), which is much higher than those of Cu9S5 nanocrystals (25.7%),53 Au nanorods (21%),54 and gold nanovesicles (37%).55 Such excellent photothermal-conversion efficiency of BSA-CuFeS2 NPs shows a significant potential as a photothermal agent.


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In Vitro catalytic performance of BSA-CuFeS2 NCs. Considering high chemical activity of hydroxyl radicals (•OH) damaging cancer cell, 3,3,5,5-tetramethylbenzidine (TMB) was used to investigate the potential of BSA-CuFeS2 NPs to generate •OH.56 BSA-CuFeS2 NPs with H2O2 can catalyze the reaction of TMB to cause a blue color reaction at varying pH conditions (Figure 3a, b, and Figure S3), with maximum absorbance at 652 nm. Remarkably different from amorphous Fe0 nanoparticles and Fe3O4, which show classical pH-dependent Fenton reaction (i.e., generally energetic at a narrow pH range (e.g., pH=3-4)), it is interesting to find that BSACuFeS2 NPs can produce a comparable amount of ·OH radicals in varying pH conditions (7.4, 6.5, 5.4, 4 and 3), indicating the BSA-CuFeS2 NPs display pH-independent Fenton-like reaction to generate·OH radicals for enhanced CDT. This pH-independent Fenton-like reaction of the BSA-CuFeS2 NPs, similarly as Cu-based Fenton-like reactions, could work over a wide pH range including neutral pH conditions,10,

11, 57

since the hydrolyzed complex generated in Cu-

based Fenton-like reaction is the aqueous solubility, which is favorable for overall oxidation efficiency.58, 59 To further explore the Fenton-like reaction mechanism of the BSA-CuFeS2 NPs, the Cu ion release at different pH values were measured. The dialysis bag loading BSA-CuFeS2 NPs solution (1 mL, 500 μg/mL) were put into 25 mL PBS solution with 2 mM of H2O2 at different pHs of 7.4, 6.5, and 5.4. Then 1 mL PBS solution was removed and analyzed by ICPAES at predesignated time intervals (5, 10, 20, 40, 60, 120 and 240 min) (Figure S4). No obvious Cu ions release could be detected at different pHs (7.4, 6.5, and 5.4), indicating BSACuFeS2 NPs could be served as heterogeneous Fenton-like catalyst to boost the production of •OH in the CuFeS2-based Fenton-like reaction.56, 60-62 Moreover, with increasing of temperatures (e.g., 25, 37, 45, 55 ℃ ), the absorbance significantly increase at the beginning of the reaction and barely noticeable differences after 30 minutes of reaction (Figure S5). These results suggest


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that the heat produced by BSA-CuFeS2 can improve •OH producing efficiency, which is synergistic in favor of effective suppression of tumor growth. Furthermore, the electron spin resonance (ESR) was used for exploring the production •OH by using DMPO as a spin trap. A higher ESR signal and a typical 1:2:2:1 multiple peak could be observed in the presence of H2O2, BSA-CuFeS2 NPs (Figure 3c). Furthermore, the concentration of •OH does not have obvious differences at different pH conditions (Figure 3d), in accordance with the aforementioned TMB coloration experiment. Additionally, fluorescence probe 2´,7´-dichlorofluorescein diacetate (DCFH-DA) was used to further confirm ROS production in the 4T1 cellular environment. DCFH-DA is easily oxidized by ROS, emitting green fluorescence. Much higher green fluorescence is found in the CLSM image of the co-incubation groups of BSA-CuFeS2 NPs and H2O2-treated cancer cells compared with the control group of cells (PBS, H2O2 only, BSA-CuFeS2 NPs only) (Figure 3e), confirming the efficient generation of much •OH. These results indicate that BSA-CuFeS2 NPs can be used in a synergetic PTT/CDT cancer therapy. Cytotoxicity and In Vitro PTT/CDT inhibitory of Cancer Cells. Encouraged by excellent photothermal property and catalytic performance of BSA-CuFeS2 NPs, the potential toxicity and the anticancer effect in vitro were further surveyed by a Cell Counting Kit-8 (CCK-8) assay. Various concentrations (100, 50, 25, 12.5, 0 ppm) of BSA -CuFeS2 NPs were cultured with 4T1 cells. The BSA-CuFeS2 NPs show the negligible adverse effects on the viability of 4T1 cells, demonstrating their good cytocompatibility (Figure 4a). It is found that the 4T1 cell viability declines with elevated concentration (0, 12.5, 25, 50, 100 ppm) of BSA-CuFeS2 NPs irradiated by the laser. Additionally, either the group of BSA-CuFeS2 NPs in the presence of H2O2 or upon


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laser, the obviously decreased cell viability could also be observed (Figure 4b). Especially, much more significant declining of cell viability could be found upon two treatments of combining PTT with CDT, indicating the synergistically strengthened anticancer effect. To further confirm and visually observe the cell apoptosis induced by photothermal and ROS ablation, the living and dying cells with respectively green fluorescence and red fluorescence were observed by a confocal laser scanning microscope (CLSM) (Figure 4c). No obvious changes could be found in the 4T1 cell of control groups (e.g., 4T1 cell treated with PBS, only BSA-CuFeS2 NPs, H2O2 and NIR laser irradiation) by the CLSM images. However, a portion of the 4T1 cell was damaged after treated with BSA-CuFeS2 NPs under an H2O2 condition or upon NIR laser irradiation. Comparatively, when simultaneously under H2O2 and NIR laser irradiation, the majority of 4T1 cells treated with BSA-CuFeS2 was damaged, which could be confirmed by nuclei shrinkage and cell-membrane damage, etc. These results, in accord with the results of the CCK8 assay, further prove the significant photothermal and ROS effect of BSACuFeS2 NPs in the synergistic therapy tumor. MR Imaging Performance. Magnetic resonance (MR) imaging, providing excellent 3D detail and soft-tissue tomographic information, is a powerful technique for the guidance of tumor therapy in vivo. The relaxation property of the BSA-CuFeS2 NPs was examined to investigate whether BSA-CuFeS2 NPs could act as MR contrast agents. It is observed that BSA-CuFeS2 NPs possess negative contrast effects (T2-weighted MRI) (Figure S6). The transverse relaxivities r2 and the longitudinal relaxivity r1 are calculated to be 5.06 mM-1 s-1 , and 0.05 mM-1 s-1), respectively (Figure S6), resulting in an extraordinary high r2/r1 ratio (r2/r1 = 101.2), which potentially enable the BSA-CuFeS2 NPs serving as a efficient T2 contrast agent (Figure S6)63-65. To further confirm the MR imaging in vivo, tumor-bearing mice were intravenous injection with


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BSA-CuFeS2 NPs (15 mg kg-1) and imaged by a 3T clinical MR system. The dramatical dark effect for the whole tumor area (Figure S7), and the tumor MR signal remarkably and quickly decreases to 43.2% (Figure S8), suggesting BSA-CuFeS2 can be used for effective T2-weighted MRI to guide tumor ablation in vivo. In Vivo Toxicity, Pharmacokinetics and Biodistribution Studies. Investigating systemic toxicity is critical for nanomaterials to clinical research.66-68 Mice treated with BSA-CuFeS2 NPs do not have a significant symptom of adverse reaction, such as neurological status, activity, eating, drinking. The weight of all the mice slightly increased (Figure S9). To further explore long-term biosafety, the normal hematology parameters and standard blood biochemical indexes were measured in BSA-CuFeS2 NPs treated mice and the healthy mice (the control group) to evaluate their toxicity in vivo. Hematological parameters and blood biochemical indexes (Figure 5), such as mean red blood cell hemoglobin (MCH), mean corpuscular volume (MCV), red blood cells (RBC), lymphocyte (LYM), intermediate cells (MID), mean platelet volume (MPV), RDESD, white blood cell count (WBC), and, mean corpuscular hemoglobin concentration (MCHC), etc., are all in the normal range. Moreover, Liver function indicators, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), are no obviously abnormal, suggesting no hepatic dysfunctions caused by BSA-CuFeS2 NPs (Figure 5). Creatinine (CREA) and blood urea nitrogen (BUN) as renal function indexes show no abnormity (Figure 6a). In addition, H&E examination shows no obvious pathological toxicity and an inflammatory lesion in the major organs (Figure 6b), indicating high biocompatibility. Besides, identifying the long-term biodistribution and clearance of nanomaterials are significantly important to in vivo translation potential. The Cu concentrations in solubilized main organs were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES)


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(Figure 7a). It is clear that at 1h post-injection, liver, spleen, and kidney have a significant Cu uptake, i.e., with Cu uptake of 27.6 ± 4.8% in the liver, 16.1 ± 6.6% ID/g in the spleen, and 6.2 ± 0.4%ID/g in the kidney. After 5 d, the Cu concentration significantly decreased to 15 ± 4.7%ID/g and 3.3 % ID/g in the liver and spleen, suggesting that BSA-CuFeS2 NPs can be quickly clearable from the body, as confirmed by the relative short circulation time (Figure 7b). Notably, the Cu concentration in the kidney quickly decreased and closed to the level of that of the control group after 5 days, indicating the ultrasmall size of BSA-CuFeS2 NPs could be lead to renal clearance. Excitingly, the Cu level of main organs shows no obvious differences with that of the control group after 1 month, indicating almost utter clearance and high biosafety. The fluorescence imaging shows that the BSA-CuFeS2 NPs mainly accumulated in the liver, spleen, and kidney in accordance with the results of ICP-AES (Figure 7c and 7d). The fluorescence signal growth rate (FSGR) of tumor at 30 min post-injection increased to 8.8 times compared with before injection, indicating high passive accumulation efficiency of BSA-CuFeS2 NPs through permeation retention effect (Figure S10). Therapy of Tumor by BSA-CuFeS2 NPs. Inspired by the high photothermal-conversion efficiency, excellent catalytic performance, efficient T2-weighted MRI and long-term biosafety, in vivo CDT/PTT experiments were further implemented. Mice were assigned to four groups: (1) saline solution (the control); (2) BSA-CuFeS2 NPs only; (3) 808 nm NIR laser only; (4) BSACuFeS2 NPs plus 808 nm laser. The tumor-site temperatures in the group of CuFeS2 NPs rapidly increased from 36.7 to ∼51.8 °C in 5 min of 808 nm NIR laser irradiation, which is enough to ablate the tumor (Figure 8a and Figure S11). Compared with the 808 nm NIR laser only group, no obvious temperature increase. To evaluate therapeutic effects and possible damage from CDT/PTT treatment, the tumor volume (Figure 8b) were measured and analyzed. The weight of


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all the mice slightly increased, confirming low systemic toxicity of these BSA-CuFeS2 NPs. Remarkably, Compared with the tumor volumes of the control group, reaching to ~600 mm3, the tumor growth was apparently suppressed in the single CDT group. Specifically, the tumor treated by CDT/PTT was almost thoroughly ablated after the treatment (Figure 8c and Figure S12). H&E, TUNEL, and Ki-67 antibody staining results indicate the obvious necrosis and strongly inhibitive proliferation of tumor cells in the CuFeS2 NPs with 808 nm NIR laser group (Figure 8d). While the control group shows no obviously inhibitive the viability of tumor cells. CONCLUSIONS In summary, a synergistic nanotheranostic agent for efficient MRI-guided enhanced PTT/CDT based on a novel kind of ultrasmall BSA-CuFeS2 NPs through an eco-friendly aqueous biomineralization strategy. This approach utilizes pH-independent Fenton-like properties to efficiently boost the production of •OH for CDT, thus overcoming traditional CDT based Fenton reaction which is only energetic at a narrow pH range (pH = 3−4). Moreover, extraordinarily high photothermal conversion efficiency of BSA-CuFeS2 NPs not only shows a dramatical PTT effect but also considerably enhances the CDT efficiency, thus presenting distinctively synergistic enhanced of PTT/CDT efficiency for suppressing and ablation of tumor in vivo. More importantly, the ultrasmall size (~4.9 nm) and biocompatible surface chemistry enable BSACuFeS2 NPs to be readily excreted from the body through the kidneys and liver, thus effectively avoiding long-term toxicity and systemic toxicity. It is extremely expected that such pHindependent and photothermal-enhanced CDT strategy based on Fenton-like reaction will open a new door to design efficient CDT nanotheranostic agent and enhance the clinically translatable potential of CDT/PTT. EXPERIMENTAL METHODS


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Chemicals and reagents. Bovine serum albumin (BSA), copper(II) chloride dihydrate (CuCl2·2H2O), sodium hydroxide (NaOH), hydrogen peroxide solution (H2O2), ferrous (II) sulfate heptahydrate (FeSO4·7H2O), 5, 5-Dimethyl-1-pyrroline N-oxide (DMPO), 2’,7’dichlorofluorescein diacetate, and sodium sulfide nonahydrate (Na2S·9H2O) were bought from Sigma-Aldrich Inc. Synthesis of BSA-CuFeS2 NPs. BSA-CuFeS2 NPs were synthesized via an eco-friendly aqueous biomineralization strategy at physiological temperature (37 °C). Firstly, 50 mL of water was heated to 90 °C at ambient conditions remove dissolved oxygen. Then, a NaOH solution (2M, 1.2 mL) was added into 50 mL aqueous solution containing 250 mg BSA, 17.00 mg CuCl2·2H2O, and 55.60 mg FeSO4·7H2O to adjust the pH to ∼12 at ambient conditions. Afterward, 97 mg Na2S·9H2O was rapidly injected to the above system immediately to generate a black solution. Finally, the resulting BSA-CuFeS2 NPs was purified by ultrafiltration through a membrane filter (MWCO, 10 kDa), washed with water three times. CuFeS2 without BSA modification was synthesized via the similar synthesis procedures of BSA-CuFeS2 NPs, with the difference that BSA was not added. Material Characterization. UV-Vis spectra was measured by the UV-3600 Shimadzu spectrometer. TEM images were obtained with a JEM-2100F transmission electron microscope (TEM). Element concentration was analyzed by ICP-AES. Malvern Instrument (Nano ZS90, Ltd.) was used to measure Dynamic light scattering (DLS). The temperature and the thermal image were respectively recorded by the thermo-camera (FLIR A325SC camera) and 808 nm NIR laser.


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Study of catalytic performance. The conventional colorimetric method based on oxidation of TMB was used to quantitative analysis of •OH generation. Briefly, the absorbance at λ = 652 nm of TMB solution (816 μM，3 mL in the cuvette ) with or without 400 μM of H2O2, 5 ppm BSACuFeS2 NPs in varying pH conditions (e.g., 7.4, 6.5, 5.4, 4 and 3) was measured. Besides, to evaluate the enhancement of •OH generation, enhancing the temperature (e.g., 25, 37, 45, 55 ℃) simulate heat during PTT. The absorbance was standardized to the control. ESR was then applied to further prove the ability of pH-independent production •OH with DMPO trapping •OH. 0.1 mL DMPO solution (0.1 M), including 40 μL H2O2 (2 mM) + 1 μg BSA-CuFeS2 NPs in varying pH conditions (e.g., 7.4, 6.5, 5.4) was instantly added to a capillary tube. Then ESR spectrum was acquired by Bruker Elexsys580 spectrometer with the settings of the previous report.9 In Vitro Cytotoxicity Assay. Experiments in cell culture accord with the previous report.8, 69 A CCK-8 assay was used to evaluate the cytotoxicity of BSA-CuFeS2 NPs. The cells in DMEM medium were added to culture plates until adherent. Then the cells were incubated with DMEM medium contained BSA-CuFeS2 NPs various concentration (12.5, 25, 50,100 ppm, ). After 24 h, the CCK-8 assay was used for analyzing the cytotoxicity. PTT/CDT inhibitory In Vitro. DMEM medium contained BSA-CuFeS2 NPs at various concentration (12.5, 25, 50,100 ppm, ) with 100 μM H2O2 or without H2O2 was cultured with the 4T1 cells and then irradiated for 5 min by the laser (1 W/cm2). Finally, the cytotoxicity was analyzed by the CCK-8 assay.


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Confocal laser scanning microscope. DMEM medium contained 4T1 cell was seeded in the CLSM-specific media

culture

contained

disk

until

adherent.

DCFH-DA

(0.01

After

cultured

mM),

with then

DMEM the

culture culture

media was substituted by DMEM medium containing the following samples: 100 μM H2O2, 100 ppm BSA-CuFeS2 and a mixture of 100 μM H2O2 and 100 ppm BSA-CuFeS2. After incubated 1 h, the ROS was observed by the CLSM. The 4T1 cells in DMEM medium were added to the CLSM-specific culture disk. After 12 h, the cells were cultured with DMEM medium contained BSA-CuFeS2 NPs at 100 ppm with 100 μM H2O2 or without H2O2. Then the cell was beamed with the laser (5 min, 1 W/cm2). After staining with calcein-AM and PI, the cells were observed by the CLSM. MR Imaging. The in vitro MR imaging and the relaxation rate r2 were obtained by a 3.0 T clinical MRI instrument (GE Signa 3.0 T). Solutions of BSA-CuFeS2 NPs containing varying Fe concentrations were dispersed in centrifuge tubes for MR scanning. The T2-weighted FR-FSE sequence and relaxivities rate (r2) was in accord with the previous method.69, 70 The mice bearing 4T1 tumor xenograft were used for T2-MRI study in vivo. BSA-CuFeS2 NPs saline solution (15 mg kg-1) were intravenously injected. At given time intervals, the T2-MRI was acquired by the T2-weighted FR-FSE sequence. In Vivo Therapy of Tumor.Tumor-bearing mice were assigned to four groups (n = 5, per group): (1) saline solution (200 μL); (2) BSA-CuFeS2 NPs only (15 mg kg-1); (3) 808 nm NIR laser only; (4) BSA-CuFeS2 NPs (15 mg kg-1) plus 808 nm laser (1.5 W/cm2, 5 min). All mice were intravenously injected with BSA-CuFeS2 NPs or PBS solution before NIR laser ， the thermal images and temperature of tumor sites were monitored with an infrared thermal imaging


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camera. The normal equation: tumor volume = (tumor width)2×(tumor length)/2 was used to calculate the tumor volume. Thereafter, the histopathological analysis was evaluated by TUNEL, H&E, and Ki-67. In Vivo Toxicity. The protocols of the animal experiment were approved by Fudan University Laboratory Animal Center. The female Kunming mice of each group were intravenous injection different doses of BSA-CuFeS2 NPs (0, 5, 10, and 15 mg/kg). The blood biochemical indexes, histological, and hematological were collected at 30 days after intravenous administration. Pharmacokinetics, Biodistribution Studies. After BSA-CuFeS2 NPs (15 mg/kg) was intravenously injected, a 20 μL blood was collected in the given time points (2, 5, 10, 15 and min, 0.5, 0.75, 1, 2, 4, 8, and 24 h). Then Cu concentrations were determined by ICP-AES. The onecomponent pharmacokinetic model was used to calculate the blood circulation lifetime of BSACuFeS2 NPs. The biodistribution of BSA-CuFeS2 NPs in vivo was analyzed by female mice. The main organs were extracted, weighed and digested by aqua regia solution, after intravenous injection of BSA-CuFeS2 NPs (15 mg kg-1) at predesignated time intervals (0 h, 1 h, 1 d, 5 d, and 30 d). Cu uptake in different tissues was quantified by ICP-AES. The biodistribution of BSA-CuFeS2 NPs in the main organs was also investigated by the fluorescence imaging. A near-infrared fluorescent DiR-labeled BSA-CuFeS2 NPs was intravenously injected. Then the mice were imaged using the Caliper IVIS Lumina II spectrum imaging system (PerkinElmer, USA). After fluorescence imaging, their major organs were collected for ex vivo imaging.


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Statistical Analysis. One-way ANOVA statistical analysis was used to analyze differences between datum. 0.05 was thought significant, and the data were defined with (*) for p < 0.05, (**) for p < 0.01, and (***) for p < 0.001, respectively. SCHEMES AND FIGURES

Scheme 1. Schematic illustration of the synthesis of BSA-CuFeS2 NPs and BSA-CuFeS2 NPsmediated synergetic pH-independent CDT/PTT.


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Figure 1. (a, b) TEM image, (c) high-resolution TEM image, (d) size distribution histogram, (e) XRD patterns, (f) the hydrodynamic size distribution, and (g, h, i) the XPS spectra of the BSACuFeS2 NPs.


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Figure 2. (a) UV-Vis spectra of the BSA-CuFeS2 aqueous suspensions at varying Cu concentrations (2, 4, 6, 12, 25 and 50 ppm). (b) Mass extinction coefficient. (c) Photothermal curves of BSA-CuFeS2 at different Cu concentrations. (d) Photothermal effect of the BSACuFeS2 aqueous suspensions under irradiation, and then the laser was shut off. (e) Heating stability curves of the BSA-CuFeS2 solution for five on/off cycles. (f) The calculation time constant (τs), and calculation the photothermal-conversion efficiency.


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Figure 3. (a) UV-Vis spectra and (b) colorimetric analysis of the TMB aqueous with or without H2O2 or BSA-CuFeS2 at varying pH values. Inset: Corresponding different color reactions of


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samples. (c) ESR spectra. (d) The •OH concentration of different reaction systems calculated by ESR. (e) Confocal images of 4T1 cells after various treatments with H2O2 only, BSA-CuFeS2 only, H2O2 and BSA-CuFeS2 stained with DCFH-DA. The scale bar is the same (50 μm).


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Figure 4. (a) Assay of the viability of 4T1 cell cultured with varying BSA-CuFeS2 concentrations. (b) 4T1 cells viability after various treatments with H2O2 only, 808 nm laser


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only, and both, with different BSA-CuFeS2 concentrations. (c) Confocal imaging of 4T1 cells after various treatments (control, BSA-CuFeS2 only, 808 nm laser only, BSA-CuFeS2 + H2O2, BSA-CuFeS2 + 808 nm laser, and BSA-CuFeS2 + H2O2 + 808 nm laser group). The scale bar is the same (50 μm).


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Figure 5. Hematological index and biochemical blood analysis of mice after intravenous injection with varying BSA-CuFeS2 doses.


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Figure 6. (a) Renal function indexes of mice treated by varying BSA-CuFeS2 doses. (b) Pathological H&E stained images of the major organs sections of mice treated with varying BSA-CuFeS2 doses. The scale bar is the same (100 μm).


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Figure 7. (a) Biodistribution of Cu in the major organs at a varying time. (b) Blood circulation lifetime of BSA-CuFeS2. (c) In vivo fluorescence images of tumor-bearing mice injected intravenously of BSA-CuFeS2 at the varying time. (d) Ex vivo fluorescence images the major tissue at a varying time.


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Figure 8. (a) Representative IR thermal images of mice bearing 4T1 tumor treated by BSACuFeS2 or with saline during laser irradiation at a varying time. (b) Tumor growth volume curves after different treatments. (c) Photographs of nude mice bearing transplanted 4T1 tumors and tumor region on the 16th day. (d) H&E, TUNEL, and Antigen Ki-67 immunofluorescence staining of 4T1 tumor regions after different treatments in 12 h. The scale bar is the same (100 μm).


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ASSOCIATED CONTENT Supporting Information Available: Supplementary figures from Figure S1 to Figure S12 as supporting information are included, and this material is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Hangrong Chen: 0000-0003-0827-1270 Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (Grant No. 2017YFB0702602), the National Natural Science Foundation of China (Grant No. 51772316, 51602334), the Key Projects of International Cooperation and Exchanges of NSFC (No.81720108023), the Natural Science Foundation of Shanghai (Grant No. 18ZR1444800), Shanghai Rising-Star Program (No. 19QA1410300). REFERENCES 1.

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