Hollow Cu2Se Nanozymes for Tumor Photothermal-Catalytic Therapy

Subscriber access provided by BUFFALO STATE

Article

Hollow Cu2Se Nanozymes for Tumor Photothermal-Catalytic Therapy Xianwen Wang, Xiaoyan Zhong, Huali Lei, Yuehao Geng, Qi Zhao, Fei Gong, Zhijuan Yang, Ziliang Dong, Zhuang Liu, and Liang Cheng Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.9b01958 • Publication Date (Web): 29 Jul 2019 Downloaded from pubs.acs.org on July 29, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials

Hollow Cu2Se Nanozymes for Tumor Photothermal-Catalytic Therapy Xianwen Wang1, Xiaoyan Zhong2, Huali Lei1, Yuehao Geng1, Qi Zhao1, 3*, Fei Gong1, Zhijuan Yang1, Ziliang Dong1, Zhuang Liu1, and Liang Cheng1* 1Institute

of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for

Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, China 2

National Engineering Research Centre for Nanomedicine, College of Life Science and Technology,

Huazhong University of Science and Technology, Wuhan 430074, China. 3

Je & Na Biotechnology Co., Ltd., 1198 Fenhu Dadao, Wujiang, Suzhou, Jiangsu 215215, China.

Corresponding Author: *E-mail: [email protected] (Q. Zhao), Phone: +86-512-65884616 *E-mail: [email protected] (L. Cheng), Phone: +86-512-65882097

ABSTRACT: Tumor microenvironment (TME)-mediated cancer therapy, such as chemodynamic therapy (CDT) based on Fenton reaction, has attracted extensive attention in recent years. However, efficient Fenton reactions usually require stringent reaction conditions (low pH value and sufficient H2O2). Therefore, there is an urgent need to improve the efficiency of Fenton reaction within the tumor for enhanced CDT in cancer treatment. Herein, Cu2Se hollow nanocubes (HNCs) are successfully prepared via an anion exchange method using Cu2O nanocubes (NCs) as the template. This method is also successfully used to synthesize Cu2S or CuSSe HNCs with the similar structure. By tuning the reaction time in the process of transforming Cu2O NCs into Cu2Se HNCs, Cu2Se HNCs with optimized performances in high NIR II photothermal conversion efficiency (50.89 %) and good Fenton-like properties are obtained. After surface coating, PEGylated Cu2Se HNCs show good water-dispersibility and biocompatibility. Importantly, in vitro and in vivo experiments demonstrate the significant synergistic effect of combining photothermal therapy (PTT) and CDT based on PEG-Cu2Se HNCs, achieving greatly enhanced efficacy than that obtained by PTT or CDT alone. Moreover, such PEG-Cu2Se HNCs appear to be rather safe for treated animals without noticeable long-term toxicity.

1 ACS Paragon Plus Environment

Chemistry of Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

INTRODUCTION The specific production of highly toxic reactive oxygen species (ROS) with the assistance of nanomaterials in the tumor to destroy the cellular constituents has been recognized as an effective strategy for cancer treatment and attracted wide attention,1-5 especially the recently developed chemodynamic therapy (CDT), which employs the Fenton agents (generally iron-based materials) through Fenton reaction to produce lots of toxic •OH in the presence of H2O2.4, 6-10 Owing to the existence of excessive endogenous H2O2 in the tumor microenvironment (TME) instead of normal tissues, these nanomaterials could respond to the characteristics of TME and produce •OH only at the tumor site, thus having low invasiveness and high therapeutic specificity.11 Recently, iron-based materials (e.g., FeS2 nanocubes,3 Fe2P nanorods,12 Fe5C2@MnO2 nanocrystals,13 and Fe nanoparticles11) have been reported to kill cancer as CDT agents. However, the Fenton reaction based on ferrous materials is only effective in relatively strong acidic condition (pH 3.0-5.0), while inefficient Fenton reaction occurs in neutral physiological conditions and weakly acidic TME. Moreover, even at suitable pH, the Fenton reaction efficiency of iron-based materials is relatively lower (～63 M-1 s-1), resulting in the slow formation of •OH and limited efficacy in CDT.2 Therefore, how to enhance the efficiency of Fenton reaction at the tumor site would be critical in the further development of CDT. One of the most common strategies for improving the efficiency of the Fenton reaction in CDT is to increase the local temperature at the tumor site, which has been widely employed due to its simplicity and practicability.3,

5-6, 8, 14

Photothermal therapy (PTT) uses photothermal agents to

convert near-infrared (NIR) light energy into hyperthermia to destroy cancer cells.15-19 Despite the rapid development of photothermal nanomaterials in the field of nanomedicine in recent years, 20-21

16,

however, most of the current research on PTT focused on the first near-infrared window (NIR I,

750 - 980 nm). Compared with NIR I window, the second near infrared (NIR II, 1000-1350 nm) window can provide stronger penetration depth owing to lower tissue background and reduced photon scattering, and would be more attractive for practical PTT applications.22 In addition, NIR II windows could allow higher maximum allowable exposure (MPE) lasers. The skin exposure for MPE was 0.33 W/cm2 at 808 nm, while the value as high as 1.0 W/cm2 at NIR II window.22-23 Therefore, it is meaningful to explore a Fenton reagent with excellent NIR-II optical absorbance, 2 ACS Paragon Plus Environment

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


which has been relatively less explored to our best knowledge. Another strategy to improve the efficiency of Fenton reaction in CDT is to develop new Fenton reagents with better catalytic efficiency under neutral physiological conditions and weak acidic conditions.2,

14, 24

As typical catalysts used to degrade pollutants and organic dyes, copper-based

nanomaterials may present tremendous potential as a Fenton-like reagent for CDT.25-26 Comparatively, the Cu+-catalyzed Fenton-like reaction is superior to Fe2+/Fe3+ in both kinetics and energy. It has been reported that Cu+-catalyzed Fenton-like has high efficiency in neutral and weakly acidic conditions, and the reaction rate can reach～160 fold higher than that of Fe2+/Fe3+.2 However, copper-based nanomaterials used for CDT of cancer had been seldom reported except the recent self-assembled copper-amino acid nanoparticles.2,

27

In addition, copper-based nanomaterials have

become one of the research hot-spots because of their strong absorption in the NIR region, good biocompatibility, and excellent photothermal conversion efficiency.25 Compared with other copper-based materials, Cu2Se nanomaterials are particularly interesting not only because they have similar optical absorption in NIR window and great photothermal performance, but also owing to the fact that Cu and Se are important trace elements in the human body.28-29 Herein, Cu2Se hollow nanocubes (Cu2Se HNCs) with strong NIR II absorbance were explored for PTT-CDT synergistic therapy of cancer. The Cu2Se HNCs were successfully synthesized via the anion exchange method by using Cu2O nanocubes (Cu2O NCs) as the template (Scheme 1a). In the process of transforming Cu2O NCs into Cu2Se HNCs, the optical absorption of the samples in the NIR II window gradually increased, while the Fenton-like properties of the samples gradually decreased. The optimum reaction time at 1.5 h was selected to form Cu2Se HNCs with excellent photothermal conversion efficiency (50.89 %) in the NIR II window and good Fenton-like properties. After being modified by thiolated

polyethylene

glycol

(SH-PEG),

the

final

PEG-Cu2Se

HNCs

showed

good

water-dispersibility and stability. As shown by in vitro and in vivo experiments, PEG-Cu2Se HNCs could produce high levels of •OH through Fenton-like reaction in the presence of H2O2 to cause effective apoptosis of cancer cells. Under the laser irradiation, cancer cells were completely eradicated owing to the fact that the mild photothermal effect generated under 1064-nm NIR-II laser irradiation would further accelerate the Fenton-like reaction, so as to achieve a synergistic effect in combined CDT-PTT (Scheme 1b). 3 ACS Paragon Plus Environment


Page 4 of 29

MATERIALS AND METHODS Chemicals. All chemicals and reagents were analytical grade and used without any further purification. Copper chloride dihydrate (CuCl2∙2H2O), polyvinylpyrrolidone (PVP K30, MW=40000 Da), sodium hydroxide (NaOH), ascorbic acid (AA), sodium sulfide nonahydrate (Na2S∙9H2O), sodium borohydride (NaBH4, 99%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Selenium powder (Se, ∼100 mesh, ≥ 99.5%) was obtained from Aladdin Chemistry Co., Ltd. Mercapto polyethylene glycol (SH-PEG, MW=5000) was achieved from Shanghai Advanced Vehicle Technology Co., Ltd. Deionized water (DI water, 18.2 MΩ·cm) obtained from a Milli-Q water system was used for all the experiments. Preparation of Cu2O NCs. Cu2O NCs were prepared by adding AA into Cu2+ and NaOH aqueous using PVP as capping agents. In a typical process, 0.6 g PVP and 0.5 mmol CuCl2∙2H2O were dissolved in 20 mL DI water under magnetic stirring, followed by addition of 8.0 mmol NaOH aqueous solution. After stirring for 10 min, 1.0 mmol AA was added to the above mixture dropwise and stirred for another 10 min. Upon the addition of AA, orange Cu2O NCs were formed immediately. The obtained Cu2O NCs were collected by centrifugation and washed thoroughly with DI water and ethanol for several times. All experiments were carried out at room temperature. Preparation of Cu2X (S, Se) HNCs. In a typical synthesis, selenium source (NaHSe solution) was first prepared by mixing 0.125 mmol Se power and 0.375 mmol NaBH4 with 5.0 mL of DI water under N2 atmosphere at room temperature. For the formation of Cu2Se HNCs, the as-prepared selenium source was added into the above Cu2O NCs suspension and stirred for 1.5 h at room temperature to get phase-pure Cu2Se HNCs. In a similar way, when only the selenium source was replaced by equivalent Na2S∙9H2O or mixed solution (NaHSe: Na2S∙9H2O = 1: 1), the Cu2S HNCs or Cu2SSe HNCs were obtained. The prepared samples were collected by centrifugation and washed repeatedly with DI water and ethanol, and then stored at 4 oC for further use. Surface modification of Cu2Se HNCs. For PEG modification, 10 mg Cu2Se HNCs and 50 mg SH-PEG were dispersed in 10 mL DI water under vigorous stirring for 12 h at room temperature. Then, the prepared Cu2Se-PEG HNCs were purified by repeatedly centrifugation (12000 rpm, 8 min) and washed with DI water, and finally stored 4 oC for further use. Characterization. The crystallographic structure and morphology of products was examined by 4 ACS Paragon Plus Environment

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


power X-ray diffraction (XRD, PANalytical) and field-emission scanning electron microscopy (FESEM, Gemini 500, Germany). Transmission electron microscopy (TEM) and elemental mapping were recorded using a HRTEM (JEM-2100F, Tokyo, Japan, 200 kV accelerating voltage). The electron binding energy and surface chemical composition of samples were performed with X-ray photoelectron spectrometer (XPS, ESCALab 250Xi, Thermo Fisher Scientific, USA). The UV-vis-NIR spectra of samples was detected using a spectrophotometer (PerkinElmer Lambda 750, USA). Surface area of samples was determined by Brunauer-Emmet-Teller (BET, ASAP 2020, Quantachrome, USA) method. Photothermal performance of PEG-Cu2Se HNCs. To investigate the photothermal performance of PEG-Cu2Se HNCs, DI water and PEG-Cu2Se HNCs aqueous dispersions of different concentrations (10, 20, 40, 60, and 120 μg/mL) were illuminated for 5 min by a continuous 1064 nm laser (0.75 W cm-2). Moreover, five Laser On/Off cycles were used to evaluate the photothermal stability of Cu2Se HNCs. The temperature of the solutions was monitored by using an IR thermal camera (Fortric 225) and recorded every 10 s. The photothermal conversion efficiency (η) of Cu2Se HNCs was calculated according to the previous reported papers. 30-31 Fenton-like properties of Cu2Se HNCs. The Fenton-like properties of Cu2Se HNCs were firstly evaluated by the catalytic oxidation of o-phenylenediamine (OPDA) in the presence of H2O2. Typically, reagents were added into 1.0 mL PBS (pH 7.0) in the order of Cu2Se HNCs (final concentration 40 μg/mL), OPDA, H2O2. Unless otherwise stated, the concentrations of OPDA and H2O2 were 1.0 and 1.0 mM respectively for the oxidation of OPDA at room temperature (25 oC). In addition, to study the effect of temperature on Fenton-like efficiency, the reaction was placed at different temperature in dark environment for 0.5 h. Analysis of the amount of •OH generation was conducted, using the classical colorimetric method which was based on the methylene blue (MB) degradation after selective •OH capture. Briefly, the absorbance at λ= 664 nm of MB solution (10 μg/mL) at PBS (pH= 7.0) with or without 1.0 mM H2O2 were examined before and after the addition of Cu2Se HNCs. Besides, enhancing the temperature to 45 oC to simulate heat during PTT, to evaluate the effect of temperature on the production of •OH. The potential influence from Cu2Se HNCs on the absorbance was avoided by centrifugation before measurement.8 5 ACS Paragon Plus Environment


Page 6 of 29

In vitro hemolysis assay. Hemolysis test was carried out using mice blood to evaluate the hematotoxicity of the prepared PEG-Cu2Se HNCs in vitro. Typically, 0.5 mL mice blood cell solution was obtained after anticoagulation and washed with PBS solution for three times, and then diluted by 5.0 mL PBS. After that, 0.2 mL diluted red blood cells (RBCs) were with 0.8 mL PEG-Cu2Se HNCs in PBS (25, 50, 75, and 100 μg/mL). DI water was used as a positive control group and PBS was used as a negative control. Then the samples were shaken and kept stable in 37 oC for 4 h, the mixtures were centrifugated at 10, 000 rpm for 10 mins. The absorbance of the supernatant was measured at 546 nm. The hemolyis ratio was calculated using the follow equation: Hemolysis percent (%) = [(Asample-RBCs+-Asample RBCs-) - Anegative] / (Apositive - Anegative) × 100 %, where Asample, Anegative, and Apositive are the absorbance of the samples, the negative control and the positive control, respectively.32 Cell experiments. For cytotoxicity evaluation, Human umbilical vein endothelial cells (HUVECs) and 4T1 cells were seeded into the 96-well culture plate (1 × 104 cells per well, 96-well plate) for 24 h and then cultured with PEG-Cu2Se HNCs at varying concentrations (0, 5, 10, 15, 20, 30, 50, 75, and 100 μg/mL) and incubated for 24 h. After that, the solutions were exchanged with fresh RMPI 1640

medium

and

the

cell

viabilities

was

measured

by

using

the

standard

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay according to the manufacturer’s protocol.30 To study the chemodynamic therapy (CDT) effect of PEG-Cu2Se HNCs, 4T1 cells (1 × 104 cells per well, 96-well plate) were incubated with PEG-Cu2Se HNCs (0 or 100 μg/mL) and H2O2 at gradient concentrations (0, 10, 25, 50, 75, and 100 μM) for 24 h, and then washed repeatedly with PBS to remove the excess medium. Afterwards, the cell viability was measured by using the standard MTT assay. For the in vitro photothermal-CDT, 4T1 cells (1 × 104 cells per well, 96-well plate) were incubated with H2O2 (0 or 50 μM) and PEG-Cu2Se HNCs at various concentrations (0, 5, 10, 20, 30, and 50 μg/mL). After 24 h incubation, the cells were treated with 1064 nm laser (0.75 W/cm2) for different times (3 or 5 min). Finally, the cell viabilities were measured by standard MTT assay. To evaluate the efficient generation of intracellular •OH, 4T1 cells were incubated with PEG-Cu2Se HNCs (100 μg/mL), H2O2 (50 μM or 100 μM) or Cu2Se HNCs + H2O2 for 4 h. The 4T1 6

ACS Paragon Plus Environment

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cells were then washed several times with PBS and further incubated with fresh RMPI 1640 medium containing DCFH-DA (20 μM) for 0.5 h. The intracellular ROS was then examined by recording the fluorescence of DCF (λex = 488 nm, λem =525 nm) by a confocal microscope (Zeiss LSM800), and the fluorescence intensity of each photo was measured by ImageJ software. Localized photothermal ablation and chemodynamic effect of PEG-Cu2Se HNCs was evaluated using 4T1 cells. Typically, 4T1 cells were respectively were seeded on 12-well plates at a density of 1×105 cells per well cultured in 37 oC for 24 h. After that, the 4T1 cells were washed several times with PBS and incubated with PEG-Cu2Se HNCs (100 μg/mL), H2O2 (50 μM) or Cu2Se HNCs + H2O2 for 2 h. Then, the cells were irradiated with NIR laser (1064 nm, 0.75 W/cm2) for 0 or 5 min. After the treatment, the cells were rinsed again with PBS and stained by using calcein acetoxymethyl ester (calcein AM) and propidium iodide (PI) for separate visualization of live cells and dead cells to verify the photothermal/chemodynamic ablation on the cells. The confocal microscope (Zeiss LSM800) was applied to observe the green fluorescence caused by calcein AM and red fluorescence caused by PI indicating live and dead cells, respectively. Tumor model. The female Balb/c mice were obtained from Suzhou Belda Biopharmaceutical Co. Ltd and performed following the protocols approved by the Soochow University Laboratory Animal Center. The 4T1 tumors were generated by subcutaneous injection of 4T1 cells (2 ×106) suspended in 50 μL PBS on the back of each Balb/c mice. When the tumor volume reached～100 mm3, the mice can be used for the following in vivo experiments. In vitro and in vivo PA imaging. For PA imaging, the PEG-Cu2Se HNCs dispersions at various concentrations (0, 0.25, 0.5, 1.0, 2.0, and 4.0 mg/mL) were recorded by a PA imaging instrument (Visual Sonics, Vevo LAZR). For in vivo PA imaging, the PEG-Cu2Se HNCs dispersions (2.0 mg/mL, 50 μL) was intratumorally injected into 4T1 tumors of mice for PA imaging scans under laser irradiation with different wavelength (700, 808 and 900 nm). PA imaging of the mice before PEG-Cu2Se HNCs injection were also taken as the control. Photothermal-enhanced CDT in vivo. 4T1 tumor-bearing Balb/c mice were randomly divided into the following five groups (n= 5 per group): (1) PBS; (2) PBS + laser; (3) PEG-Cu2Se HNCs; (4) PEG-Cu2Se HNCs + laser; (5) PEG-Cu2Se HNCs + laser. For groups 3, 4, and 5, PEG-Cu2Se HNCs was intratumorally injected into the mice at a dose of 5.0 mg/kg. After that, the tumors of mice in 7 ACS Paragon Plus Environment


Page 8 of 29

groups 2 and 5 were exposed to 1064 nm NIR laser (1.0 W/cm2) for 10 min, while in group 4 was exposed to 1064 nm NIR laser (0.75 W/cm2) for 10 min. During the irradiation, the IR thermal camera (Fortric 225) was used to monitor the tumor temperature changes at different time points. After different treatment, the tumor length and width of the mice were recorded every 2 days using a digital caliper and the tumor volumes were calculated according the following equation: tumor volume (V) = (tumor length) × (tumor width)2/2. After 1 day, one mouse from each group was sacrificed and related tumor tissues were collected for hematoxylin and eosin (H&E) staining and ROS staining to further evaluate the therapeutic effect and ROS generation of each group. To check the in vivo biocompatibility of the Cu2Se HNCs, after 16 days, mice of each group were randomly chosen and euthanized to retrieve organs (including the heart, liver, spleen, lung, and kidney). The obtained organs were collected for H&E staining histology analysis.

RESULTS AND DISCUSSION Cu2Se hollow nanocubes (HNCs) were synthesized using Cu2O nanocubes (NCs) as the template by a sacrificial template method based on anion exchange. Firstly, uniform Cu2O NCs were synthesized by adding ascorbic acid (AA) into a solution of Cu2+ and NaOH using PVP (polyvinyl pyrrolidone, MW= 40000 Da) as the capping agent. The chemical composition and phase structure of as-prepared Cu2O NCs were confirmed by powder X-ray diffraction (XRD) analysis. All of the reflection peaks were consistent with cubic Cu2O (JCPDS No. 74-1230) (Figure 1a). The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the as-prepared Cu2O NCs clearly showed a regular cubic morphology and good mono-dispersity with an average diameter size of 86.89 ± 19.93 nm (Figure 1b-c and Figure S1a, Supporting Information). The lattice spacing was measured to be ~0.245 nm, which was in good agreement with the (222) interplanar spacing of the cubic Cu2O crystal (inset of Figure 1c). It is worth mentioning that the effect of PVP had an important influence on the formation of Cu2O NCs. Without PVP, Cu2O NCs would grow into heterogeneous morphology with aggregation (Figure S2, Supporting Information). In addition, energy dispersive spectroscopy (EDS) (Figure 1d), EDS-mapping (Figure 1g), and X-ray photoelectron spectroscopy (XPS) (Figure 1j-l) of Cu2O NCs further demonstrated that the synthesized Cu2O NCs were pure without any impurities. Owing to the solubility product constant (Ksp) of Cu2Se is much lower than that of Cu2O, thus 8


Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cu2Se HNCs could be formed when selenium source was added to the solution containing Cu2O NCs via an anion exchange process.33 The phase structure and chemical composition of the as-prepared Cu2Se NCs were characterized by XRD analysis, showing that all diffraction peaks containing the (111), (220), (311), and (222) faces could clearly correspond to the cubic Cu2Se phase (JCPDS no. 88-2043) (Figure 1a). No obvious impurities were found on XRD pattern, indicating the high purity of the as-prepared Cu2Se HNCs. From SEM and TEM images, it could be observed that the synthesized Cu2Se nanomaterials showed a hollow structure with an average size of 102.95 ± 30.39 nm and the shell thickness at about 11.61 ± 2.67 nm (Figure 1e-f and Figure S1b, S3, Supporting Information). The average diameter of Cu2Se HNCs was slightly increased compared to Cu2O NCs, which should be due to the Cu2Se nanoparticles formed on the surface during the selenization process.33 In the process of transforming Cu2O NCs into Cu2Se HNCs, the shell thickness of Cu2Se HNCs became thicker (0 to 14.28 ± 1.81 nm), and the corresponding Cu2Se HNCs had a better photothermal effect, while their Fenton reaction properties gradually decreased. The lattice spacing was measured to be ~0.389 nm, which was ascribed from the (111) interplanar spacing of the cubic Cu2Se crystal (inset of Figure 1f). Due to the hollow structure, the Brunauer-Emmett-Teller (BET) surface area of Cu2Se HNCs was measured to be 117.2 m2/g (Figure 1h), much higher than that of the previously reported hollow structure, potentially useful as drug delivery systems. In addition to the signals of Ni, O, and C elements which were mainly from the substrate, only Cu and Se elements could be detected, further verified by the results of elemental mapping. The molar ratio was measured to be 2: 1 (Figure 1d, i). The XPS survey spectra (Figure 1l) showed that and Cu2Se HNCs were mainly composed of Cu and Se. Figure 1j showed the core level spectra of the Cu 2p region, the value of binding energies at 932.6 eV and 952.6 eV correspond to Cu 2p3/2 and Cu 2p1/2, respectively.34-35 Moreover, no satellite peak was observed in the Cu 2p region, thus indicating that the valence state of Cu in Cu2Se HNCs was +1. Figure 1k showed the core level spectra of the Se 3d region, the binding energies at 54.5 and 53.9 eV correspond to Se 3d5/2 and Se 3d3/2, respectively, which showed that the valence state of Se in Cu2Se HNCs was -2.34-35 Based on the above characterization, Cu2Se HNCs were successfully prepared by an anion exchange method using Cu2O NCs as the template. In order to investigate the formation mechanism, the process of the Cu2Se HNCs was investigated by observing the products obtained at different time points (Figure 2a and Figure S4, Supporting 9 ACS Paragon Plus Environment


Page 10 of 29

Information). After Cu2O NCs were reacted with selenium source (NaHSe) for about 0.25 h, the sample changed from the cubic morphology to the distinct core-shell structure. With the reaction proceeding to 1 h, the core of the sample became smaller, and the shell became larger. After 4 h, the core completely disappeared, and the hollow structure of Cu2Se was obtained. In addition, the process of converting Cu2O NCs into Cu2Se HNCs was also monitored by XRD (Figure 2d). As the reaction proceeded, the diffraction peaks of Cu2O NCs gradually decreased, and the characteristic diffraction peak of Cu2Se NCs increased, indicating that the Cu2O NCs were gradually transformed into Cu2Se HNCs. Very interestingly, by this similar anion exchange method, the Cu2S HNCs (Figure 2b, e and Figure S5, Supporting Information) or Cu2SSe HNCs (Figure 2c, f and Figure S6, Supporting Information) could also be also successfully synthesized when the selenium source was replaced by equivalent sulfur source (Na2S∙9H2O) or mixed solution (NaHSe: Na2S∙9H2O = 1: 1). According to a serious of materials characterization (SEM, TEM, and XRD), Cu2S HNCs and Cu2SSe HNCs also were successfully prepared (Figure S7-12, Supporting Information). Based on the above experimental results, the following relevant chemical equation may be involved: Cu2+ + 2OH- = Cu(OH)2↓ (1) Cu(OH)2 + C6H8O6 (AA) = Cu2O + 2H2O +C6H6O6

(2)

Cu2O + X2- + H2O = 2OH- + Cu2X (3) The formation mechanism of Cu2X (X= Se, S) was presented and illustrated in Figure 2g, which could be explained by the Kirkendall effect.33-34 According to this mechanism, vacancies would be formed owing to different diffusion rates between the two components in a diffusion couple. When Cu2O NCs are dispersed into X2- solution, X2- ions will react with Cu2O NCs, and then a thin shell layer of Cu2X would be formed on the surface of Cu2O NCs. The thin layer is made up of many Cu2X nanocrystals with a large number of grain boundaries, which allow possible the diffusion of materials through the Cu2X layer and further reaction between X2- and Cu2O. According to the Kirkendall effect, the different diffusion rates of the components in a diffusion couple may lead to the formation of Kirkendall voids near the interface. Therefore, when Cu2O NCs is reacting with X2solution, the Cu2X shell will be gradually increased and the Cu2O core will gradually disappear, attributing to the fact that the outward transport rate of the Cu2+ ions is faster than the inward transport rate of X2- ions through the formed Cu2X. Finally, the Cu2X HNCs are successfully prepared through a sacrificial template method based on anion exchange strategy. Compared with 10 ACS Paragon Plus Environment

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


other methods for the synthesis of hollow copper chalcogenides, the sacrificial template method based on anion exchange strategy not only is simple and convenient, but also could avoid successfully the removal of templates as the templates are consumed during the preparation process. In addition, it should be noted that the research of copper-based nanomaterials is mainly focused on copper sulfide nanoparticles, while only a few studies have been reported in the literature about copper selenides, which is mainly due to the difficulty in preparation of water-soluble, biocompatible copper selenides.35 Based on this, Cu2Se HNCs was selected to explore its related properties and tumor inhibition experiments in vitro and in vivo. During the conversion of Cu2O NCs into Cu2Se HNCs, the optical absorption of the samples in the NIR I and NIR II windows gradually increased with the prolongation of the reaction time. It is noteworthy that the sample had strong optical absorption in the NIR II window at 1.5 h, and remained constant after 3 h (Figure 3a). In addition, copper-based materials are also good catalysts and have a wide range of applications including nanoenzyme.30 We wondered whether our synthesized Cu2Se HNCs would have the catalytic performance for application in nanomedicine. The Fenton-like activity of the samples was examined by the catalytic oxidation of o-phenylenediamine (OPDA), a widely used •OH production indicator that gradually changed from colorless to yellow by time-dependent oxidization. OPDA will display a characteristic absorption band at ～415 nm owing to the formation 2,3-diaminophenazine (OPD), indirectly indicating the generation of •OH.36 During the anion exchange process, the absorption peak at 415 nm was gradually weakened as the reaction time prolonged, which indicated that the Fenton-like properties of the sample were gradually decreased (Figure 3b). In order to balance the absorbance and catalytic properties, the optimal anion exchange reaction time was at 1.5 for the formation of Cu2Se HNCs with good optical absorption and excellent Fenton-like effect (Figure 3c). Therefore, we selected Cu2Se HNCs at this reaction time point for the following study. In order to further improve the stability and biocompatibility of them, PEG-Cu2Se HNCs with good dispersibility in the different media were prepared by modification of Cu2Se HNCs with SH-PEG (Figure S13). Owing to the strong optical absorbance of as-prepared Cu2Se HNCs in the NIR-II window, the photothermal conversion performance of Cu2Se HNCs was investigated. The temperature of Cu2Se HNCs (120 μg/mL) increased from 30.2 to 58.4 oC, while water only increased 11 ACS Paragon Plus Environment


Page 12 of 29

by 5.0 oC, demonstrating the excellent photothermal effect of Cu2Se HNCs (Figure 3d, e, and Figure S14, Supporting Information). Similar to other photothermal agents, the enhanced temperature not only depended on the concentration of the Cu2Se HNCs (Figure S16a, Supporting Information), but also on the power densities of the laser (Figure S15, 16b, Supporting Information). The photothermal conversion efficiency (η) of the Cu2Se HNCs was calculated to be 50.89 % (Figure 3g, h), much higher than the previous reported other photothermal agents, such as Cu3BiS3 nanorods (40.7 %),37 V2C quantum dots (45.05 %),38 Au-Cu9S5 nanoparticles (37 %),39 MoOx nanoparticles (37.4 %),40 black TiO2-x nanoparticles (39.8 %)41 and WO3−x@γ-PGA nanoparticles (25.8 %) (Table 1).14 Moreover, by comparing temperature variation of PEG-Cu2Se HNCs irradiated by 808 nm and 1064 nm, it can be observed that PEG-Cu2Se HNCs have better photothermal performance under 1064 nm laser irradiation than that under 808 nm laser irradiation, likely attributing to the higher optical absorbance of PEG-Cu2Se HCN in the NIR II window (Figure 3i). Moreover, the prepared Cu2Se HNCs showed excellent photothermal stability after multiple cycles of laser irradiation (Figure 3f), indicating the great potential of the Cu2Se HNCs as a durable and reusable photothermal agent. Next, OPDA was also used to detect the generation of •OH of the PEG-Cu2Se HNCs in the presence of H2O2. With the increasing of H2O2 and OPDA, the characteristic peak at 415 nm became higher, indicating that more •OH was produced (Figure 3j and Figure S17, Supporting Information). In addition, the catalytic activity of PEG-Cu2Se HNCs was not significantly changed compared with Cu2Se HNCs without thiol surface coating (Figure S18, Supporting Information). Based on the previous study, mild photothermal performance can affect the catalytic rate of Fenton reaction.3, 5-6, 8, 14 With the increase of temperature, it was observed that Cu2Se HNCs had a higher catalytic rate of Fenton-like reaction (Figure 3k). Thus, Cu2Se HNCs could produce lots of •OH through an effective Fenton-like reaction in the presence of H2O2, and the temperature could obviously improve the efficiency of Fenton-like reaction to produce •OH. In order to further study the Fenton-like properties of Cu2Se HNCs, methylene blue (MB), another probe of the •OH detection was used to evaluate the generation of •OH under different conditions.24 The distinct characteristic peak of MB at 664 nm was gradually decreased with the generation of •OH. By comparing the degradation rates of MB in different groups (Figure 3l), it was further demonstrated that Cu2Se NCs could produce •OH in the presence of H2O2, and catalytic rate of 12


Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fenton-like reaction would be further increased with the increase of temperature. The experimental results showed that PEG-Cu2Se HNCs had a strong catalytic rate at neutral pH of 7.0 as a good catalyst for CDT of cancer, different from iron-based nanomaterials depended on strong acidity (pH 3.0 - 5.0),37 making those our PEG-Cu2Se HNCs promising in TME-responsive CDT for effective cancer treatment. Utilizing its high photothermal conversion efficiency and considerable •OH generation ability, in vitro anticancer effect of PEG-Cu2Se HNCs was evaluated. No observable hemolysis effect was found even at the high concentration at 100 μg/mL, indicating their good biosafety when circulating in the blood pool (Figure S19, Supporting Information). We then assessed the cytotoxicity of PEG-Cu2Se HNCs by using both human umbilical vein endothelial cells (HUVECs) and 4T1 breast cancer cells. No obvious cytotoxicity of PEG-Cu2Se HNCs towards non-cancerous HUEVC cells was found, indicating that PEG-Cu2Se HNCs have good biocompatibility (Figure 4a and Figure S20, Supporting Information). However, the viabilities of 4T1 cancer cells showed a gradual decreased with the increase of Cu2Se HNCs concentrations under the same condition. Such an interesting difference may be ascribed to the •OH generation through Fenton-like reaction in cancer cells with a relatively high level of H2O2 (Figure 4a).12 Therefore, CDT based on Fenton like reaction can kill cancer cells to some extent. To further study in vitro CDT with PEG-Cu2Se HNCs, the effect of H2O2 on cell viabilities under different conditions was explored. The cell viabilities of 4T1 cells in the group of H2O2 + PEG-Cu2Se HNCs were found to be significantly lower than that in the groups of H2O2 alone and PEG-Cu2Se HNCs alone. As expected, and the viabilities of cells treated with H2O2 + PEG-Cu2Se HNCs gradually decreased with the increasing H2O2 concentrations, due to the fact that more •OH would be produced by PEG-Cu2Se HNCs through the Fenton-like reaction at high concentrations of H2O2 (Figure 4b). In addition of the function of CDT, PEG-Cu2Se HNCs could also be employed as a PTT agent owing to their strong NIR II absorbance. The photothermally enhanced CDT cell-killing ability of PEG-Cu2Se HNCs was shown in a dose-dependent manner (Figure 4c). The viabilities of 4T1 cells were not obviously decreased with or without 1064 nm laser irradiation in the presence of H2O2 alone. Moreover, in the presence of PEG-Cu2Se HNCs containing H2O2, the cell survival rate irradiated with laser was significantly lower than that of cells without laser irradiation (Figure 4c). These results demonstrated that photothermally enhanced CDT based on PEG-Cu2Se HNCs could 13



Page 14 of 29

offer a good cancer therapeutic effect in the presence of H2O2. In order to further verify the Fenton-like reaction of PEG-Cu2Se HNCs in cancer cells, a common fluorescent probe 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect •OH generation. No obvious green fluorescence was observed in the groups of control, H2O2, and PEG-Cu2Se HNCs. In contrast, obvious green fluorescence was found in H2O2 + PEG-Cu2Se HNCs group. More importantly, the green fluorescence was further enhanced with the increase of the concentration of H2O2 (Figure 4d-e), indicating the •OH generation through the Fenton-like reaction of PEG-Cu2Se HNCs in cancer cells. Subsequently, calcein acetoxymethyl ester (Calcein-AM) and propidine iodide (PI) co-staining fluorescence imaging were performed to further verify that the in vitro synergistic treatment of PEG-Cu2Se HNCs on ablating cancer cells. 4T1 cells were treated with PEG-Cu2Se HNCs and incubation for 3 h and then stained to verify the living (green) and dead cells (red) (Figure 4f). The strong green fluorescence signal was distinctly observed in the groups of control, NIR, H2O2, H2O2 + laser and PEG-Cu2Se HNCs, indicating that the laser illumination, H2O2, or PEG-Cu2Se HNCs alone would not kill those cells. However, bright red fluorescence was evidently appeared in the group of PEG-Cu2Se HNCs + H2O2 + laser, indicating that most of the 4T1 cells were completely died after such treatment. Notably, both red and green fluorescence was observed in the groups of PEG-Cu2Se HNCs + laser and PEG-Cu2Se HNCs + H2O2, and the red fluorescence in the former group was much lower than that in the later one, suggesting that the photothermal performance and Fenton-like properties of PEG-Cu2Se HNCs had significant inhibitory effects on cancer cells, although could not completely kill those cells under this condition. The excellent NIR absorbance of PEG-Cu2Se HNCs endowed them great potential for photoacoustic (PA) imaging, which is a non-invasive biomedical imaging modality with reasonable penetration depth and good spatial resolution/sensitivity.30 Obvious PA signals were detected for PEG-Cu2Se HNCs samples and the enhancement of PA signals monotonously increased depending on the nanoparticle concentration (Figure S21a, Supporting Information). Then we explored in vivo PA imaging of PEG-Cu2Se HNCs using 4T1 tumor-bearing mice. While only a weak PA signal could be observed at the tumor site before injection, strong PA signals at the tumor site showed up after injection and the PA signals were ～ 3.13, ～ 3.43, and ～ 4.05 fold higher than that of the 14 ACS Paragon Plus Environment

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


control group when exposed to 700, 808, and 900 nm laser excitation, respectively (Figure S21b, c, Supporting Information). The results showed the great potential of the PEG-Cu2Se NCs as an outstanding agent for PA imaging. Encouraged by the satisfactory photothermal-enhanced CDT effect in vitro, we further evaluated the synergistic therapeutic effects of PEG-Cu2Se HNCs in vivo by utilizing the high concentration of H2O2 within solid tumors (50～100 μM).37, 42-43 The 4T1 tumor-bearing mice were randomly divided into the following five groups: (1) PBS, (2) PBS + laser (1.0 W/cm2), (3) PEG-Cu2Se HNCs, (4) PEG-Cu2Se HNCs + laser (1, 0.75 W/cm2), and (5) PEG-Cu2Se HNCs + laser (2, 1.0 W/cm2). The intratumoral injection dose of nanoparticles was 5.0 mg/kg in the treatment groups (4, 5, and 6). The tumor temperatures were monitored by an IR thermal camera. The results showed that the temperature in the tumor significantly increased under 1064-nm NIR II laser irradiation (0.75 W/cm2 or 1.0 W/cm2), while there was no significant temperature change in the tumors of mice after PBS injection (Figure 5a, b). The tumor volumes of different groups were recorded every 2 days. It was found that the tumors of mice in groups 4 and 5 showed efficient tumor restraint and gradual disappearance (Figure 5c). In contrast, the tumors of mice in the PBS and PBS + laser groups grew quickly. Although the treatment with PEG-Cu2Se HNCs (CDT) could inhibit the tumor growth, the therapeutic effect was not as good as that achieved in group 4 (CDT +PTT, 0.75 W/cm2) and group 5 (CDT +PTT, 1.0 W/cm2) (Figure 5d). Moreover, the mice in the treated groups (4, 5) exhibited obviously 100% survival within 16 days, which was much different from the other three groups (Figure S22, Supporting Information). After the various treatments, tumor sections stained with hematoxylin and eosin (H&E) were further conducted to validate the therapeutic effect. As expected, cancer cells showed severe damage and necrosis appeared in the synergistic treatment group of CDT and PTT (groups 4 and 5), while no or little damage of cancer cells was noted in other groups (Figure 5g). The results showed that CDT alone had limited therapeutic effect and could not completely eradicate tumors. However, after being combined with the mild photothermal effect, CDT could completely eradicate tumors, indicating that photothermal-enhanced CDT could significantly improve the therapeutic efficiency in tumor treatment. In order to further verify that PEG-Cu2Se HNCs can produce •OH in tumors, DCFH-DA was also 15 ACS Paragon Plus Environment


Page 16 of 29

used to detect the generation of •OH in vivo. No obvious green fluorescence was observed in the groups of PBS and PBS + NIR, while obvious green fluorescence was detected in PEG-Cu2Se HNCs group, and the fluorescence intensity became stronger with the increase of temperature after PTT (Figure 5f, h). Finally, it was observed that the •OH level was significantly increased in PEG-Cu2Se HCN under laser irradiation. This phenomenon can be attributed to the fact that the mild photothermal effect generated by the PEG-Cu2Se HCN under 1064 nm laser irradiation could accelerate the Fenton-like reaction, resulting in more •OH production in the tumor site. Good biocompatibility and low toxicity of nanomaterials are prerequisites for their application in the field of nanomedicine,25, 44 we also carefully investigated the long-term toxicity of the mice after injection of PEG-Cu2Se HNCs. Within 16 days of the treatment, the average body weights of mice in all groups showed no obvious change (Figure 5e), indicating no acute in vivo toxicity of PEG-Cu2Se HNCs. The corresponding histological changes of major organs such as heart, liver, spleen, lung, and kidney were analyzed using H&E staining (Figure 5i and Figure S23, Supporting Information). No observable organ damage or inflammation was observed after various treatments, further indicating no obvious side effects or toxicity of PEG-Cu2Se HNCs. Next, we further studied the bioelimination behaviors of PEG-Cu2Se HNCs in vivo. Firstly, PEG-Cu2Se HNCs (10 mg/kg) were i.v. injected into healthy mice, then they were sacrificed on the 1st, 7th, and15th days, with their main organs (heart, liver, spleen, lung, and kidney) harvested for biodistribution analysis. There was no significant difference in the Cu level in all tested organs after 15 days injection, which indicated that PEG-Cu2Se HNCs could not be effectively removed from mice (Figure S24, Supporting Information). In order to further study the biosafety of PEG-Cu2Se HNCs, blood parameters including complete blood panel assays and blood biochemical parameters exhibited that PEG-Cu2Se HNCs had no meaningful changes compared with the control group within the injection dose range (Figure S25&S26, Supporting Information). Furthermore, from the histological examination by H&E staining in mice at different days (0, 1, 7, and 15 days), no obvious signs of inflammation or tissue damage were found in the main organs (heart, liver, spleen, lung, and kidney) (Figure S27, Supporting Information). All the above results indicated that the biosafety use of PEG-Cu2Se HNCs in cancer therapy.

CONCLUSIONS 16 ACS Paragon Plus Environment

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In summary, Cu2Se HNCs using Cu2O NCs as the template was successfully synthesized, and the anion exchange growth mechanism of Cu2Se HNCs was proposed. During the anion exchange process, the optical absorption of the Cu2Se HNCs in NIR II window gradually increased while the Fenton-like properties of the samples gradually decreased. At the optimal reaction condition, the synthesized Cu2Se HNCs have excellent optical absorption in the NIR II window and strong Fenton-like catalytic properties. This anion exchange method could be used to synthesize Cu2S or CuSSe HNCs with similar structures. After modification by SH-PEG, the final PEG-Cu2Se HNCs showed good water-dispersibility. As evidenced by in vitro and in vivo experiments, PEG-Cu2Se HNCs could be employed for combined PTT-CDT treatment of cancer, which presented superior antitumor efficacy compared to PTT or CDT alone. This work not only provides a paradigm for the design of Fenton-like copper-based CDT agents, but also demonstrates the great potential of multifunctional PEG-Cu2Se HNCs with photothermal-enhanced CDT efficiency for theranostic applications.

ASSOCIATED CONTENT Supporting Information. Supplementary Figure S1~S27. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Q. Zhao), Phone: +86-512-65884616 *E-mail: [email protected] (L. Cheng), Phone: +86-512-65882097 The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT



This

article

was

partially

supported

by

the

National

Page 18 of 29

Research

Programs

of

China

(2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041, 51572180), Collaborative Innovation Center of Suzhou Nano Science and Technology, a Jiangsu Natural Science Fund for Distinguished Young Scholars (BK20130005, BK20170063), and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and a Project Funded for Postgraduate Research & Practice Innovation Program of Jiangsu Province (2019 SJKY19_2282). This work was also supported by the State Key Laboratory of Radiation Medicine and Protection (GZK1201810). L. Cheng was supported by the Tang Scholar of Soochow University.

REFERENCES 1.

Fan, K.; Xi, J.; Fan, L.; Wang, P.; Zhu, C.; Tang, Y.; Xu, X.; Liang, M.; Jiang, B.; Yan, X.; Gao,

L., In Vivo Guiding Nitrogen-Doped Carbon Nanozyme for Tumor Catalytic Therapy. Nat. Commun. 2018, 9, 1400. 2.

Ma, B.; Wang, S.; Liu, F.; Zhang, S.; Duan, J.; Li, Z.; Kong, Y.; Sang, Y.; Liu, H.; Bu, W.; Li,

L., Self-Assembled Copper Amino Acid Nanoparticles for in Situ Glutathione "AND" H2O2 Sequentially Triggered Chemodynamic Therapy. J. Am. Chem. Soc. 2019, 141, 849-857. 3.

Tang, Z.; Zhang, H.; Liu, Y.; Ni, D.; Zhang, H.; Zhang, J.; Yao, Z.; He, M.; Shi, J.; Bu, W.,

Antiferromagnetic

Pyrite

as

the

Tumor

Microenvironment-Mediated

Nanoplatform

for

Self-Enhanced Tumor Imaging and Therapy. Adv. Mater. 2017, 29, 1701683. 4.

Zhao, P.; Tang, Z.; Chen, X.; He, Z.; He, X.; Zhang, M.; Liu, Y.; Ren, D.; Zhao, K.; Bu, W.,

Ferrous-Cysteine-Phosphotungstate Nanoagent with Neutral pH Fenton Reaction Activity for Enhanced Cancer Chemodynamic Therapy. Mater. Horiz. 2019, 6, 369-374. 5.

Chen, H.; Gu, Z.; An, H.; Chen, C.; Chen, J.; Cui, R.; Chen, S.; Chen, W.; Chen, X.; Chen, X.;

Chen, Z.; Ding, B.; Dong, Q.; Fan, Q.; Fu, T.; Hou, D.; Jiang, Q.; Ke, H.; Jiang, X.; Liu, G.; Li, S.; Li, T.; Liu, Z.; Nie, G.; Ovais, M.; Pang, D.; Qiu, N.; Shen, Y.; Tian, H.; Wang, C.; Wang, H.; Wang, Z.; Xu, H.; Xu, J.-F.; Yang, X.; Zhu, S.; Zheng, X.; Zhang, X.; Zhao, Y.; Tan, W.; Zhang, X.; Zhao, Y., Precise Nanomedicine for Intelligent Therapy of Cancer. Sci. China: Chem. 2018, 61, 1503-1552. 6.

Guan, G.; Wang, X.; Li, B.; Zhang, W.; Cui, Z.; Lu, X.; Zou, R.; Hu, J., "Transformed" Fe3S4 18


Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Tetragonal Nanosheets: a High-Efficiency and Body-Clearable Agent for Magnetic Resonance Imaging Guided Photothermal and Chemodynamic Synergistic Therapy. Nanoscale 2018, 10, 17902-17911. 7.

Lin, H.; Chen, Y.; Shi, J., Nanoparticle-Triggered in Situ Catalytic Chemical Reactions for

Tumour-Specific Therapy. Chem. Soc. Rev. 2018, 47, 1938-1958. 8.

Feng, W.; Han, X.; Wang, R.; Gao, X.; Hu, P.; Yue, W.; Chen, Y.; Shi, J.,

Nanocatalysts-Augmented and Photothermal-Enhanced Tumor-Specific Sequential Nanocatalytic Therapy in Both NIR-I and NIR-II Biowindows. Adv. Mater. 2019, 31, 1805919. 9.

Liu, C.; Wang, D.; Zhang, S.; Cheng, Y.; Yang, F.; Xing, Y.; Xu, T.; Dong, H.; Zhang, X.,

Biodegradable

Biomimic

Copper/Manganese

Silicate

Nanospheres

for

Chemodynamic/Photodynamic Synergistic Therapy with Simultaneous Glutathione Depletion and Hypoxia Relief. ACS Nano 2019, 13, 4267-4277. 10. Gao, S.; Lin, H.; Zhang, H.; Yao, H.; Chen, Y.; Shi, J., Nanocatalytic Tumor Therapy by Biomimetic Dual Inorganic Nanozyme-Catalyzed Cascade Reaction. Adv. Sci. 2019, 6, 1801733. 11. Zhang, C.; Bu, W.; Ni, D.; Zhang, S.; Li, Q.; Yao, Z.; Zhang, J.; Yao, H.; Wang, Z.; Shi, J., Synthesis of Iron Nanometallic Glasses and Their Application in Cancer Therapy by a Localized Fenton Reaction. Angew. Chem. Int. Ed. 2016, 55, 2101-2106. 12. Liu, Y.; Zhen, W.; Wang, Y.; Liu, J.; Jin, L.; Zhang, T.; Zhang, S.; Zhao, Y.; Song, S.; Li, C.; Zhu, J.; Yang, Y.; Zhang, H., One-Dimensional Fe2P Acts as a Fenton Agent in Response to NIR II Light and Ultrasound for Deep Tumor Synergetic Theranostics. Angew. Chem. Int. Ed. 2019, 58, 2407-2412. 13. Feng, L.; Xie, R.; Wang, C.; Gai, S.; He, F.; Yang, D.; Yang, P.; Lin, J., Magnetic Targeting, Tumor Microenvironment-Responsive Intelligent Nanocatalysts for Enhanced Tumor Ablation. ACS Nano 2018, 12, 11000-11012. 14. Liu,

P.;

Wang,

Y.;

An,

L.;

Tian,

Q.;

Lin,

J.;

Yang,

S.,

Ultrasmall

WO3-x@gamma-poly-L-glutamic Acid Nanoparticles as a Photoacoustic Imaging and Effective Photothermal-Enhanced Chemodynamic Therapy Agent for Cancer. ACS Appl. Mater. Interfaces 2018, 10, 38833-38844. 15. Cheng, L.; Wang, C.; Feng, L.; Yang, K.; Liu, Z., Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869-10939. 19 ACS Paragon Plus Environment


Page 20 of 29

16. Gu, Z.; Zhu, S.; Yan, L.; Zhao, F.; Zhao, Y., Graphene-Based Smart Platforms for Combined Cancer Therapy. Adv. Mater. 2019, 31, 1800662. 17. Liu, Y.; Bhattarai, P.; Dai, Z.; Chen, X., Photothermal Therapy and Photoacoustic Imaging via Nanotheranostics in Fighting Cancer. Chem. Soc. Rev. 2018, 48, 2053-2108. 18. Tang, Q.; Xiao, W.; Huang, C.; Si, W.; Shao, J.; Huang, W.; Chen, P.; Zhang, Q.; Dong, X., pH-Triggered and Enhanced Simultaneous Photodynamic and Photothermal Therapy Guided by Photoacoustic and Photothermal Imaging. Chem. Mater. 2017, 29, 5216-5224. 19. Wang, X.; Wang, C.; Wang, X.; Wang, Y.; Zhang, Q.; Cheng, Y., A Polydopamine Nanoparticle-Knotted Poly(ethylene glycol) Hydrogel for On-Demand Drug Delivery and Chemo-photothermal Therapy. Chem. Mater. 2017, 29, 1370-1376. 20. Liang, C.; Diao, S.; Wang, C.; Gong, H.; Liu, T.; Hong, G.; Shi, X.; Dai, H.; Liu, Z., Tumor Metastasis Inhibition by Imaging-Guided Photothermal Therapy with Single-Walled Carbon Nanotubes. Adv. Mater. 2014, 26, 5646-5652. 21. Reina, G.; Miguel Gonzalez-Dominguez, J.; Criado, A.; Vazquez, E.; Bianco, A.; Prato, M., Promises, Facts and Challenges for Graphene in Biomedical Applications. Chem. Soc. Rev. 2017, 46, 4400-4416. 22. Wang, X.; Ma, Y.; Sheng, X.; Wang, Y.; Xu, H., Ultrathin Polypyrrole Nanosheets via Space-Confined Synthesis for Efficient Photothermal Therapy in the Second Near-Infrared Window. Nano Lett. 2018, 18, 2217-2225. 23. Zhu, P.; Gao, S.; Lin, H.; Lu, X.; Yang, B.; Zhang, L.; Chen, Y.; Shi, J., Inorganic Nanoshell-Stabilized Liquid Metal for Targeted Photonanomedicine in NIR-II Biowindow. Nano Lett. 2019, 19, 2128-2137. 24. Lin, L.-S.; Song, J.; Song, L.; Ke, K.; Liu, Y.; Zhou, Z.; Shen, Z.; Li, J.; Yang, Z.; Tang, W.; Niu, G.; Yang, H.-H.; Chen, X., Simultaneous Fenton-like Ion Delivery and Glutathione Depletion by MnO2-Based Nanoagent to Enhance Chemodynamic Therapy. Angew. Chem. Int. Ed. 2018, 57, 4902-4906. 25. Wang, X.; Miao, Z.; Ma, Y.; Chen, H.; Qian, H.; Zha, Z., One-Pot Solution Synthesis of Shape-Controlled Copper Selenide Nanostructures and Their Potential Applications in Photocatalysis and Photothermal Therapy. Nanoscale 2017, 9, 14512-14519. 26. Garcia-Bosch, I.; Siegler, M. A., Copper-Catalyzed Oxidation of Alkanes with H2O2 under a 20


Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fenton-like Regime. Angew. Chem. Int. Ed. 2016, 55, 12873-12876. 27. Fu, J.-j.; Chen, M.-y.; Li, J.-x.; Zhou, J.-h.; Xie, S.-n.; Yuan, P.; Tang, B.; Liu, C.-c., Injectable Hydrogel Encapsulating Cu2MnS2 Nanoplates for Photothermal Therapy Against Breast Cancer. J. Nanobiotechnol. 2018, 16, 83. 28. Rayman, M. P., Selenium in Cancer Prevention: A Review of The Evidence and Mechanism of Action. Proc. Nutr. Soc. 2005, 64, 527-542. 29. Uriu-Adams, J. Y.; Keen, C. L., Copper, Oxidative Stress, and Human Health. Mol. Asp. Med. 2005, 26, 268-298. 30. Wang, X.; Li, F.; Yan, X.; Ma, Y.; Miao, Z.-H.; Doug, L.; Chen, H.; Lu, Y.; Zha, Z., Ambient Aqueous Synthesis of Ultrasmall Ni0.85Se Nanoparticles for Noninvasive Photoacoustic Imaging and Combined Photothermal-Chemotherapy of Cancer. ACS Appl. Mater. Interfaces 2017, 9, 41782-41793. 31. Roper, D. K.; Ahn, W.; Hoepfner, M., Microscale Heat Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles. J. Phys. Chem. C 2007, 111, 3636-3641. 32. He, G.; Ma, Y.; Zhou, H.; Sun, S.; Wang, X.; Qian, H.; Xu, Y.; Miao, Z.; Zha, Z., Mesoporous NiS2 Nanospheres as a Hydrophobic Anticancer Drug Delivery Vehicle for Synergistic Photothermal–Chemotherapy. J. Mater. Chem. B 2019, 7, 143-149. 33. Cao, H.; Qian, X.; Zai, J.; Yin, J.; Zhu, Z., Conversion of Cu2O Nanocrystals into Hollow Cu2-xSe Nanocages with the Preservation of Morphologies. Chem. Commun. 2006, 4548-4550. 34. Wang, Z.; Peng, F.; Wu, Y.; Yang, L.; Zhang, F.; Huang, J., Template Synthesis of Cu2-xSe Nanoboxes and Their Gas Sensing Properties. CrystEngComm 2012, 14, 3528-3533. 35. Zhang, S.; Sun, C.; Zeng, J.; Sun, Q.; Wang, G.; Wang, Y.; Wu, Y.; Dou, S.; Gao, M.; Li, Z., Ambient Aqueous Synthesis of Ultrasmall PEGylated Cu2-xSe Nanoparticles as a Multifunctional Theranostic Agent for Multimodal Imaging Guided Photothermal Therapy of Cancer. Adv. Mater. 2016, 28, 8927-8936. 36. Yin, W.; Yu, J.; Lv, F.; Yan, L.; Zheng, L. R.; Gu, Z.; Zhao, Y., Functionalized Nano-MoS2 with Peroxidase Catalytic and Near-Infrared Photothermal Activities for Safe and Synergetic Wound Antibacterial Applications. ACS Nano 2016, 10, 11000-11011. 37. Li, A.; Li, X.; Yu, X.; Li, W.; Zhao, R.; An, X.; Cui, D.; Chen, X.; Li, W., Synergistic Thermoradiotherapy Based on PEGylated Cu3BiS3 Ternary Semiconductor Nanorods with Strong 21



Page 22 of 29

Absorption in the Second Near-Infrared Window. Biomaterials 2017, 112, 164-175. 38. Cao, Y.; Wu, T.; Zhang, K.; Meng, X.; Dai, W.; Wang, D.; Dong, H.; Zhang, X., Engineered Exosome-Mediated Near-Infrared-II Region V2C Quantum Dot Delivery for Nucleus-Target Low-Temperature Photothermal Therapy. ACS Nano 2019, 13, 1499-1510. 39. Ding, X.; Liow, C. H.; Zhang, M.; Huang, R.; Li, C.; Shen, H.; Liu, M.; Zou, Y.; Gao, N.; Zhang, Z.; Li, Y.; Wang, Q.; Li, S.; Jiang, J., Surface Plasmon Resonance Enhanced Light Absorption and Photothermal Therapy in the Second Near-Infrared Window. J. Am. Chem. Soc. 2014, 136, 15684-15693. 40. Yin, W.; Bao, T.; Zhang, X.; Gao, Q.; Yu, J.; Dong, X.; Yan, L.; Gu, Z.; Zhao, Y., Biodegradable MoOx Nanoparticles with Efficient Near-Infrared Photothermal and Photodynamic Synergetic Cancer therapy at the Second Biological Window. Nanoscale 2018, 10, 1517-1531. 41. Han, X.; Huang, J.; Jing, X.; Yang, D.; Lin, H.; Wang, Z.; Li, P.; Chen, Y., Oxygen-Deficient Black Titania for Synergistic/Enhanced Sonodynamic and Photoinduced Cancer Therapy at Near Infrared-II Biowindow. ACS Nano 2018, 12, 4545-4555. 42. Van de Bittner, G. C.; Dubikovskaya, E. A.; Bertozzi, C. R.; Chang, C. J., In Vivo Imaging of Hydrogen Peroxide Production in a murine Tumor Model with a Chemoselective Bioluminescent Reporter. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 21316-21321. 43. Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.; Petros, J.; Taylor, W. R.; Murthy, N., In Vivo Imaging of Hydrogen Peroxide with Chemiluminescent Nanoparticles. Nat. Mater. 2007, 6, 765-769. 44. Jasim, D. A.; Menard-Moyon, C.; Begin, D.; Bianco, A.; Kostarelos, K., Tissue Distribution and Urinary Excretion of Intravenously Administered Chemically Functionalized Graphene Oxide Sheets. Chem. Sci. 2015, 6, 3952-3964.


Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Schematic illustration of (a) the preparation process of the PEG-Cu2Se HNCs and (b) the proposed synergistic antitumor mechanism of PEG-Cu2Se HNCs for photothermal-enhanced CDT in the NIR II window.



Page 24 of 29

Figure 1. Characterization of the Cu2O NCs and Cu2Se HNCs. (a) XRD pattern and (d) EDS of Cu2O NCs and Cu2Se HNCs. SEM images of (b) Cu2O NCs and (e) Cu2Se HNCs. TEM images of (c) Cu2O NCs and (f) Cu2Se HNCs, the insets were the corresponding HRTEM images. EDS elemental mapping of (g) Cu2O NCs and (i) Cu2Se HNCs. (h) Nitrogen adsorption-desorption isotherm of Cu2Se HNCs. XPS spectra of the (j) Cu 2p region, (k) Se 3d (O 1s) and (l) survey spectra of the Cu2O NCs and Cu2Se HNCs.


Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. The formation mechanism of Cu2X HNCs. TEM images and XRD pattern of the as-prepared (a, d) Cu2Se HNCs, (b, e) Cu2Se HNCs and (c, f) Cu2SSe HNCs obtained at different reaction times. (g) Schematic illustration of growth mechanism of Cu2X HNCs.



Page 26 of 29

Figure 3. Characterization of photothermal and Fenton-like properties of the as-prepared Cu2Se HNCs. (a) UV-vis-NIR spectra of Cu2Se HNCs obtained at different reaction times points (Cu: 40 μg/mL). (b) The Fenton-like properties by OPDA probe in the presence of Cu2Se HNCs at different reaction time points (Cu: 20 μg/mL, OPDA: 1.0 mM, H2O2: 1.0 mM). (c) Absorption at 1064 nm and Fenton-like properties of Cu2Se HNCs at different reaction time points. (d) Photothermal heating curves and (e) the corresponding thermal images of PEG-Cu2Se HNCs under 1064 nm laser irradiation (0.75 W/cm2, 5 min). (f) Photothermal conversion cycling test of PEG-Cu2Se HNCs (40 μg/mL, 0.75 W/cm2, 5 min). (g) the photothermal profile of PEG-Cu2Se HNCs (40 μg/mL) irradiated by NIR laser for 10 min, followed by natural cooling to room temperature; (h) Linear time data versus −ln(θ) acquired from the cooling period of (g). (i) Temperature variation of PEG-Cu2Se HNCs with different concentration irradiated by 808 and 1064 nm laser at power density of 0.75 W/cm2 for 5 minutes. (j) The effects of H2O2 concentration on the Fenton-like activity of PEG-Cu2Se HNCs (PEG-Cu2Se HNCs: 20 μg/mL, H2O2: 1.0 mM). (k) The effects of different temperature on the catalytic activity of PEG-Cu2Se HNCs. (l) MB degradation under different experimental conditions (PEG-Cu2Se HNCs: 40 μg/mL, MB: 10 μg/mL, H2O2: 1.0 mM). 26 ACS Paragon Plus Environment

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. In vitro photothermal-enhanced CDT therapy of PEG-Cu2Se HNCs. (a) Relative viability of HUVECs and 4T1 cells with different concentrations of Cu2Se HNCs. (b) Viability of 4T1 cells incubated with H2O2 at different concentrations and treated with PEG-Cu2Se HNCs (50 μg/mL). (c) The viability of 4T1 cells incubated with different concentrations of PEG-Cu2Se HNCs and treated with NIR, H2O2 (50 μM) + NIR. (d) Confocal images of 4T1 cells stained by DCFH-DA treated for different conditions. (e) Quantitative analysis of the fluorescence intensity of intracellular •OH. (f) Confocal images of calcein AM/PI co-stained 4T1 cells after incubation with different formulations.



Page 28 of 29

Figure 5. In vivo photothermal-enhanced CDT therapy of PEG-Cu2Se HNCs. (a) IR thermal images and (b) the corresponding temperature changes at the tumor sites of the 4T1 tumor-bearing mice in different groups at different time intervals. (c) Relative tumor growth curves, (d) relative tumor weight at the end of the experiment and (e) body weight of the 4T1 tumor bearing mice in different groups. (f) •OH and (g) H&E staining in tumor tissues from different groups. (h) Quantitative analysis of the fluorescence intensity of the 4T1 tumor bearing mice in different groups. (i) H&E staining of the main organs (heart, liver, spleen, lung, kidney) of in the groups of PBS and PEG-Cu2Se HNCs + laser (2).


Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC

Multifunctional Cu2Se hollow nanocubes with excellent optical absorption in the NIR II window and strong Fenton-like catalytic activity are prepared via anion exchange method and applied for photothermal-enhanced chemodynamic cancer therapy.


Hollow Cu2Se Nanozymes for Tumor Photothermal-Catalytic Therapy

Recommend Documents