Tumor Catalytic-Photothermal Therapy with Yolk-Shell Gold@Carbon

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Tumor Catalytic-Photothermal Therapy with Yolk-Shell Gold@Carbon Nanozymes Lei Fan, Xiangdong Xu, Chunhua Zhu, Jie Han, Lizeng Gao, Juqun Xi, and Rong Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17916 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 17, 2018

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ACS Applied Materials & Interfaces

Tumor Catalytic-Photothermal Therapy with Yolk-Shell Gold@Carbon Nanozymes Lei Fan1, Xiangdong Xu,1 Chunhua Zhu,2 Jie Han,1 Lizeng Gao,*2 Juqun Xi,*2,3 Rong Guo1

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School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, Jiangsu, China.

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Institute of Translational Medicine, Department of Pharmacology, School of Medicine, Yangzhou University,

Yangzhou, 225001 Jiangsu, China. 3

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,

Yangzhou, 225009, Jiangsu, China.

KEYWORDS: nanozymes, yolk-shell structure, carbon/Au composite, ROS, thermal therapy

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ABSTRACT：：Nanozymes, as a new generation of artificial enzymes, offer great opportunities in biomedical engineering and disease treatment. Synergizing the multiple intrinsic functions of nanozymes can improve their performance in biological systems. Here, we report a novel nanozyme with yolk-shell structure fabricated by combining a single gold nanoparticle core with a porous hollow carbon shell nanospheres (Au@HCNs). Au@HCNs exhibited enzyme-like activities similar to horseradish peroxidase and oxidase under an acidic environment, showing the ability of ROS generation. More importantly, the ROS production of Au@HCNs was significantly improved upon 808-nm light irradiation by the photothermal effect, which is often used for tumor therapy. Cellular and animal studies further demonstrated that the efficient tumor destruction was achieved through the combination of light-enhanced ROS and photothermal therapy. These results implied that the intrinsic enzyme-like activity and photothermal conversion of nanozymes can be synergized for efficient tumor treatment, providing a proof-of-concept of tumor catalytic-photothermal therapy based on nanozymes.

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INTRODUCTION In the past few decades, the design and construction of multifunctional artificial enzymes, as promising alternatives to natural enzymes, has received much attention.1,2 Nanozymes are nanomaterials possessing intrinsic enzyme-like activity, such as metalorganic frameworks,3 metallic materials4 and carbon materials,5,6 representing a new generation of artificial enzymes and showing promising potential in bionics, biomedical applications and environmental science. In particular, nanozymes with peroxidase-mimic activity were reported to catalyze the hydrogen peroxide (H2O2) decomposition and produce reactive oxygen species (ROS) to thwart harmful organisms.7,8 It is well known that ROS, such as superoxide radicals and hydroxyl radicals, if present at abnormal levels, will induce mitochondrial dysfunction and cause cells to undergo apoptosis or necrosis.9,10 This principle has been used for tumor treatment with chemotherapy, radiotherapy and photodynamic therapy, which can elevate ROS levels in situ.11 However, the major challenge for this concept is how to produce sufficient ROS to induce cancer cell death, while minimizing the damage to normal cells.12,13 Nanozymes that have intrinsic enzyme-like activity and other nanoscale effects may provide a feasible strategy for tumor therapy by synergistically combining their diverse catalytic activities. Au nanoparticles are one of the most widely studied nanozymes that perform ROS regulation by their intrinsic enzyme-like activities.14-16 For example, Au nanoparticles capped by cysteamine show peroxidase-like activity,17 and Au nanoparticles capped by citrate exhibit glucose oxidase-like activity.18 However, the unsupported Au nanoparticles always show low stability, which will cause a decreased catalytic activity. The ideal strategy is to use solid supports to confine Au nanoparticles and improve their activity with synergistic effects.19-21 Core@carbon yolk-shell spheres have attracted lots of attention among scientists, because the small molecules can pass through the permeable carbon shells to reach the active center and make the catalytic reaction happen, and the carbon shells also prevent aggregation of the active catalytic cores.22-25 In addition, carbon spheres have high stability even in harsh environments and the possibility of long-term storage. More importantly, carbon spheres can also carry out intrinsic enzyme-like activities similar as those for Au nanoparticles.26,27 Therefore, we propose to use a carbon shell as the support to confine a gold core in a yolk-shell structure, which can synergistically increase the catalytic activity for ROS generation. Herein, we prepared a typical yolk-shell structure in which an Au nanoparticle core was encapsulated within a hollow carbon nanospheres with porous shell (Au@HCNs). The Au@HCNs performed high peroxidase-like and oxidase-like activity enzyme functions due to the cooperative effect of Au nanoparticles and the carbon shell in the 3



yolk-shell structure. Meanwhile, carbon-based materials28-30 and Au nanoparticles31-32 are also excellent near-infrared light (NIR) absorbing agents which can transform light into heat for tumor photothermal therapy (PTT). We found that the photothermal effect significantly enhanced the enzyme-mimicking activities, including those of peroxidase and oxidase, resulting in high levels of ROS to kill cancer cells. In this work, a new strategy was provided for the first time to regulate nanozyme activity with light radiation, and a proof-of-concept tumor catalytic-photothermal therapy based on yolk-shell Au@HCNs was proposed. RESULTS AND DISCUSSION Preparation and characterization of Au@HCNs. An illustration of the synthesis of yolk-shell Au@HCNs is shown in Figure 1A. Firstly, a wine-red solution of Au nanoparticles was prepared according to the previous literature,33 which were capped with a SiO2 shell by tetraethyl orthosilicate (TEOS) hydrolysis through the Stöber process, yielding Au@SiO2 nanospheres. Next, the Au@SiO2 nanospheres were impregnated with dopamine in Tris-HCl buffer (pH=8.5). After that, the Au@SiO2@polydopamine (PDA) nanocomposites were obtained by polymerization of dopamine molecules on the surface of Au@SiO2 slowly. Subsequently, the obtained Au@SiO2@PDA nanoparticles were carbonized in N2 at 800 °C for 2 hours. Finally, the silica core was etched with NaOH, giving rise to a yolk-shell structure of Au particles encapsulated by hollow carbon nanospheres, abbreviated as Au@HCNs. It is worth emphasizing that the dopamine used in our experiments provides a nitrogen source, and the nitrogen atoms incorporated in carbon shell are considered to be an effective method to regulate the properties of carbon, which expands the potential applications of the carbon materials.22, 24 For purposes of comparison, we also synthesized N-doped hollow carbon nanospheres (HCNs) as the control group (Figure S1).

Figure 1. (A) Schematic diagram of Au@HCNs synthsis. (B) TEM images of Au nanoparticles (a), Au@SiO2 nanospheres (b), Au@SiO2@PDA nanospheres (c) and Au@HCNs (d). 4


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Figure 1B and Figure S1 show the morphology evolution from Au nanoparticles to Au@HCNs. As shown in these Figures, Au@HCNs were uniformly distributed spheres with an average diameter about 180 nm, where almost every hollow carbon nanosphere contained an Au nanoparticle embedded near the inner carbon shell. The high-resolution transmission electron microscopy (HRTEM) image (Figure S2) demonstrates that the diameter of Au nanoparticle was about ~ 10 nm and the carbon shell thickness was 6 ~ 7 nm. The lattice spacings of Au nanoparticles were 0.235 nm and 0.204 nm, which were consistent with Au (111) and Au (200) planes, respectively.34 The energy-dispersive spectroscopy (EDS) line scan of Au@HCNs, shown in Figure S2, suggested that the shell mainly contained C and N, and the dark nanoparticle inside the shell was Au. Additionally, we used the dynamic light scattering (DLS) analysis to determine the particle size distributions. The DLS results revealed that the hydrated particle sizes of AuNPs, HCNs and Au@HCNs were 23.24 ± 0.116 nm, 273 ± 0.316 nm and 275 ± 0.355 nm, respectively. The successful encapsulation of Au in the hollow carbon spheres was further confirmed by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) measurements. Figure S3 shows the detailed analysis of XPS spectra. The divided four peaks at 284.8, 286.1, 286.7, and 288.7 eV showed in the C 1s spectrum could be assigned to C=C, C–O, C=O and C–N, respectively.35 The high-resolution N 1s spectrum could be divided into four peaks (398.3, 400.1, 401.2 and 404.0 eV), which corresponded to pyridinic-N (N-6), pyrrolic-N (N-5), quaternary N (N-Q) and oxidized N (N-X), respectively.36 The two peaks in Au 4f spectrum were located at 84.4 and 88.1 eV with a splitting of 3.7 eV, indicating the Au was in a metal state. The XRD pattern shown in Figure S4 (a) reveals the face-centered cubic crystal structure of Au, which is in agreement with the PDF card No. 04-0784. It was noteworthy that no characteristic diffraction peaks for the carbon shell were observed, possibly due to the amorphous structure and strong diffraction from crystalline Au. The typical D and G bands in the Raman spectrum appearing at 1360 and 1580 cm-1 confirmed the existence of carbon in the products (Figure S4 (b)). The intensity ratio of ID/IG for Au@carbon nanospheres was 0.92, suggesting the successful transformation of polydopamine to carbon through pyrolysis. The porous nature of the yolk-shell Au@carbon nanospheres was also characterized by measuring the nitrogen adsorption-desorption isotherm. The results shown in Figure S4 (c) exhibit a type IV curve, where the surface area was about 178.8 m2/g and the pore size was about 3.7 nm (Table S1), confirming that the carbon shell was permeable. Thermogravimetric analysis (TGA) was used to measure the content of Au in the Au@HCNs (Figure S4 (d)), and the result indicates that after calcination in O2, the Au content in the yolk-shell structure was about 4.46%. Importantly, Au@HCNs dispersed well in various physiological buffers and maintained 5



for a long time without any obvious aggregation (Figure S5). Taken together, the characterization results demonstrate that yolk-shell Au@HCNs were synthesized through our synthetic route. This structure of the Au/carbon composites may provide a possibility to obtain a synergetic catalytic effect with high activity and stability, and thereby act as an efficient artificial enzyme. Mimicking enzymatic activities. The peroxidase-like activity of Au@HCNs was first measured by catalyzing the oxidation of the peroxidase substrate tetramethylbenzidine (TMB).37 HCNs were chosen as the control group. As shown in Figure 2a, a deep blue color with maximum absorption peak located at 652 nm demonstrated that both HCNs and Au@HCNs were able to catalyze the oxidation of TMB in the presence of H2O2. But H2O2 alone could not cause detectable color change. By comparison, the yolk-shell Au@HCNs produced more significant color change than that induced by the HCNs. As shown in Figure 2b, the time-dependent absorbance changes for different nanozymes proved that Au@HCNs had better peroxidase-like activity than HCNs. The results of comparative studies showed that Au@HCNs possessed an intrinsic peroxidase mimetic activity similar to that of horseradish peroxidase (HRP), and the improved peroxidase-like activity was from the intrinsic catalytic properties of AuNPs encapsulated in HCNs. Similar to the other nanomaterial-based peroxidase mimics, the catalytic activity of Au@HCNs was also affected by temperature (Figure 2c), pH (Figure 2d) and concentration of substrates (Figure S6). The optimal temperature was about 40 °C and the optimal pH was about 4.5, similar to those of HRP.38 The steady-state kinetic behavior was investigated at 25 °C and the resulting curves followed the Michaelis-Menten equation (Figure S6 (c) and (d)). On the basis of the calculated kinetic parameters (Table S2), the Km values for Au@HCNs (0.0323 mM for TMB and 210 mM for H2O2) were lower than those for HCNs (0.0386 mM for TMB and 289 mM for H2O2). Taking the above observations into account, we concluded that the HCNs had peroxidase-like enzyme activity and that this activity could be enhanced by introduction of Au nanoparticles.

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Figure 2. (a) Absorbance spectra and visual color changes of TMB in different reaction systems: (1) TMB + H2O2, (2) TMB + H2O2 + HCNs and (3) TMB + H2O2 + Au@HCNs in pH 4.5 NaAc buffer (0.1 M) at 25°C after 3 min incubation. (b) The time-dependent absorbance changes at 652 nm with of HCNs and Au@HCNs. (c, d) The peroxidase mimetic activity of HCNs and Au@HCNs was dependent on temperature (c) and pH (d). The concentrations of TMB, H2O2, HCNs and Au@HCNs were 0.416 mM, 0.2646 M, 50 µg/mL and 50 µg/mL, respectively.

Figure 3. (a) Absorbance spectra and visual color changes of TMB in different reaction systems: (1) TMB, (2) TMB + HCNs, and (3) TMB + Au@HCNs in pH 4.5 NaAc buffer (0.1 M) at 25°C after 30 min incubation. (b) The time-dependent absorbance changes at 652 nm with HCNs and Au@HCNs. (c, d) The oxidase mimetic activity of HCNs and Au@HCNs was dependent on temperature (c) and pH (d). The concentrations of TMB, HCNs and Au@HCNs were 0.416 mM, 50 µg/mL and 50 µg/mL, respectively. 7



Apart from the peroxidase mimetic activity, another feature of Au@HCNs nanozyme was that it also acted as an effective oxidase mimic. As shown in Figure 3a, the oxidation of TMB by HCNs and Au@HCNs without H2O2 also produced a blue color with maximum absorption wavelength at 652 nm, indicating that both HCNs and Au@HCNs had oxidase-like catalytic activity. It is also important to highlight the fact that HCNs incorporating Au nanoparticles could increase the TMB oxidation rate, and consequently much more intense colorimetric responses were observed in contrast to the system without Au nanoparticles. After that, the catalytic activity of Au@HCNs was investigated as a function of reaction time at different reaction time (Figure 3b), temperature (Figure 3c), pH (Figure 3d) and concentration of nanozymes (Figure S7). The results showed that the catalytic activity reached a plateau at ~30 min at 25 °C, and the optimal temperature and pH were approximately 40 °C and 4.5. Similar to the peroxidase-like activity, Au@HCNs exhibited better oxidase-like catalytic efficiency than HCNs regarding to the kinetic parameters of Km (0.170 mM and 0.201 mM for Au@HCNs and HCNs respectively) and Vmax (4.92×10-8 M/s and 3.78×10-8 M/s for Au@HCNs and HCNs, respectively) (Figure S7 and Table S3). These results demonstrated that Au nanoparticles could enhance the oxidase-like activity to improve the catalytic efficiency of carbon materials. In order to clarify the importance of the yolk-shell structure, the enzyme-like catalytic activities of Au@HCNs and free AuNPs were compared. AuNPs with diameter around 10 nm (Figure 1B (a)) were prepared. As shown in Figure S8, compared with Au@HCNs, AuNPs exhibited much lower peroxidase-like and no oxidase-like activity for the same content of Au. It is easy to understand that free Au nanoparticles will lose catalytic activity due to the aggregation.39 However, for yolk-shell Au@HCNs, the catalytic ability remained at a high level. There are three main reasons for this result. To begin with, the sp2, sp3 carbon atoms and N atoms in the shells usually exhibit good catalytic activity to catalyze the conversion of small molecules. In addition, the permeable carbon shells of Au@HCNs allow H2O2 and TMB to access Au nanoparticles for effective catalytic reaction. Last, these carbon shells can prevent coagulation of Au nanoparticles, preventing deactivation of the Au nanoparticles. Therefore, the unique structure of Au@HCNs resulted in excellent enzyme-like activities. Photothermal conversion of Au@HCNs. To verify the potential use of HCNs and Au@HCNs in photothermal therapy, HCNs and Au@HCNs solutions were exposed to an 808-nm NIR laser, where the water was used as a control. As seen from Figure 4a, the temperature trends of different materials obtained in our experiments indicated that a photothermal heating effect could be obtained. It was a remarkable fact that the temperature increased 8


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quickly for the suspension containing Au@HCNs, suggesting that the photothermal effect of HCNs could be improved by introducing Au nanoparticles. Further detailed research on the photothermal conversion of Au@HCNs was carried out. As illuminated in Figure 4b, the temperature rise of the Au@HCNs at different concentrations was recorded under continuous laser irradiation (2.0 W/cm2, 808-nm). With Au@HCNs concentrations increasing from 50 µg/mL to 150 µg/mL, the solution temperature was rapidly increased by 9.5 to 17.2 °C after 10 min laser irradiation. More importantly, the Au@HCNs remained stable even after irradiation. After three cycles of on-off irradiation, Au@HCNs still maintained high photothermal conversion efficiency (Figure 4c). IR photographs of the suspensions visualized the photothermal effect, verifying the same conclusion (Figure 4d). On the basis of the quantification method reported by Roper,40 the photothermal conversion efficiency value (η) of Au@HCNs (26.8%) was higher than most of the Au photothermal agents, such as Au nanoshells (13%),41 Au vesicles (18%)42 and Au nanorods (22%).43 Thus, the above results demonstrate that Au@HCNs had excellent photothermal conversion efficiency, and that they would be a promising candidate for PTT.

Figure 4．．Photothermal property and stability of Au@HCNs. (a) Temperature changes of water, SiO2@PDA, Au@SiO2@PDA, HCNs and Au@HCNs at the same concentration (100 µg/mL) under 808-nm laser irradiation. (b) The temperature of Au@HCNs suspension at various concentrations under 808-nm laser irradiation. (c) Photothermal conversion of Au@HCNs suspension (100 µg/mL) over three on/off cycles under 808-nm laser irradiation. (d) IR images recorded with an IR camera after laser irradiation at different time versus the Au@HCNs suspension (100 µg/mL). The power of NIR was 2.0 W/cm2.

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Effects of NIR on enzyme-mimicking activities. The above results demonstrated that our yolk-shell Au@HCNs showed excellent enzyme-mimicking activity and photothermal conversion efficiency. We wondered if the photothermal conversion could affect the enzyme-like activity of Au@HCNs. Figure 5a and 5b demonstrated that the absorbance at 652 nm of TMB with and without H2O2 was increased when the nanozyme system was irradiated by an 808-nm laser, and the absorbance at 652 nm also enhanced with NIR power intensity (Figure 5c and 5d). These results indicated that the NIR irradiation was beneficial for the oxidation of TMB. At first, we considered that this effect might come from the rise in temperature due to NIR irradiation. However, the peroxidase-like and oxidase-like activities of Au@HCNs shown in Figure 2c and Figure 3c maintained almost the same levels in the range 25 °C to 45 °C. With further increase of temperature, the enzyme-like activities were decreased. Therefore, we concluded that the rise in temperature caused by NIR irradiation was not the major factor enhancing the reaction rate of TMB oxidation.

Figure 5. The effect of NIR on enzyme-like activity of HCNs and Au@HCNs (HCNs: 50 µg/mL, Au@HCNs: 50 µg/mL). (a, c) Peroxidase-like activity (reaction time: 3 min, laser irradiation time: 1 min); (b, d) Oxidase-like activity (reaction time: 30 min, laser irradiation time: 5 min). The power of NIR was 2.0 W/cm2. In order to determine out the mechanism by which NIR irradiation was able to enhance the reaction rate of TMB oxidation, we hypothesized that the benefits were obtained through light-enhanced reactive oxygen species (ROS) generation. As reported,44-47 carbon-based nanostructures, metals, metal oxides and metal sulfides, recognized as enzyme mimetics, have been reported to possess intrinsic peroxidase and oxidase mimetic activities, 10


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and the production of ROS in enzyme catalytic reactions has been studied by indirect spectroscopy method. Here, we used electron spin resonance (ESR) technology to directly confirm the production of superoxide and hydroxyl radicals induced by HCNs and Au@HCNs. In this experiment, BMPO was used as a spin trap.48 Figure 6a shows the ESR spectra for the solutions with and without of nanozyme at pH 4.5. In presence of H2O2 and BMPO, the typical ESR spectra of spin adduct due to hydroxyl radical were weak. On the contrary, strong hydroxyl radical spin adduct ESR signals appeared when the HCNs and Au@HCNs were added. As shown in Figure 6b, in the absence of H2O2, the HCNs and Au@HCNs could also interact with O2 to produce superoxide radicals, whose signals were also captured by BMPO.49 Interestingly, when we used an 808-nm laser to irradiate the Au@HCNs system with and without H2O2, the ESR signals of hydroxyl radicals and superoxide radicals were both enhanced. This means that a higher level of ROS was produced. In order to further test our hypothesis, we investigated the impact of a ROS inhibitor (hypotaurine) on the enzyme catalysis.50 As shown in Figure 6c, after treatment with hypotaurine, the absorbance values at 652 nm of TMB in HCNs/TMB/H2O2 and Au@HCNs/TMB/H2O2 systems with and without laser irradiation were all decreased. Similar effects were also obtained in HCNs/TMB and Au@HCNs/TMB systems (Figure 6d). We also considered the influence of the thickness of carbon shell on the stability, heat and ROS conversion efficiencies. The results indicated that the slighting increasing of the carbon shell thickness did not lead to an obvious change in the performance of the products. Thus, all above results confirmed that the enzyme-like activity of Au@HCNs could generate hydroxyl radicals and superoxide radicals and that NIR irradiation could enhance the production of ROS to accelerate the enzyme reaction. Our findings lead us to conclude that the oxidase-like and peroxidase-like activities of Au@HCNs are dependent on ROS produced by the nanozyme system; the NIR irradiation of the nanozyme system is beneficial to the production of ROS to accelerate the catalytic reaction. We believe that two factors account for the light-enhanced ROS generation. First, exposure of Au@HCNs to a laser irradiation not only produces shock photoacoustic waves, but also activates the surface of the carbon shells to influence electron transport and increases their capacity to accept or donate electrons, which inhibits the quick recombination of electron-hole pairs and thus enhances the production of ROS.51 Second, Au nanoparticles are typical noble metal, they can improve the photoenergy conversion efficiency of carbon materials by extending the spectral absorption and promoting the generation of electron-hole pairs.52 These properties make Au@HCNs more easily catalyze the conversion of small molecules, showing highly enzyme-mimicking activities and triggering ROS generation.

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Figure 6. (a) ESR signals of hydroxyl radicals generated by HCNs and Au@HCNs in the presence of H2O2 (0.5 mM) at pH 4.5. (b) ESR signals of superoxide radicals generated by HCNs and Au@HCNs in CH3OH/buffer at pH 4.5. The sample suspensions contained 25 mM BMPO and 0.1 mg/mL nanomaterials. Spectra were recorded at 3 min after sample mixing. For Au@HCNs + Laser sample, laser irradiation time was 1 min. (c) The hypotaurine (4%) inhibited peroxidase-like activity of HCNs (50 µg/mL) and Au@HCNs (50 µg/mL) with and without laser irradiation (reaction time: 3 min, irradiation time: 1 min). (d) The hypotaurine (4%) inhibited oxidase-like activity of HCNs (50 µg/mL) and Au@HCNs (50 µg/mL) with and without laser irradiation (reaction time: 30 min, irradiation time: 5 min). The power of NIR was 2.0 W/cm2. In vitro cytotoxicity. Given the promising photothermal transition and light-enhanced ROS generation under NIR irradiation, we wondered whether our yolk-shell gold@carbon nanozymes could be used in tumor catalytic-photothermal therapy. To verify our hypothesis, we first investigated the photothermal conversion and ROS generation of Au@HCNs in mouse colon cancer cells (CT26). HCNs were also chosen as the control. Before the cytotoxicity experiments, we tested if the Au@HCNs could bound to cancer cells. As shown in Figure S9, when the CT26 were incubated with Au@HCNs at 37 °C for 12 h, most of the cells nonspecifically bonded to the carbon spheres. It was also found that the Au@HCNs were not only located at extracellular sites, but also migrated into CT26 cells. If the Au@HCNs penetrated into cells, it was not easy for them to escape from the cells.53 This was beneficial for long-term photothermal therapy and ROS production in tumor cells. 12


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We used an MTT assay to test the viability of CT26 cells treated with HCNs and Au@HCNs. Figure 7a and 7b show that the cell viability at 200 µg/mL HCNs was 66.1%, while the value was 58.9% at 200 µg/mL Au@HCNs. This phenomenon illustrated that HCNs or Au@HCNs with enzyme-like activity represented a novel system with apparent anticancer activity using for tumor catalytic therapy. The reason for this result was that HCNs or Au@HCNs with dual enzyme-like activities could exert ROS mobilization, transferring oxygen to free radicals accompanied with O2 consumption and catalyzing H2O2 (over-expressed in tumor cells) into free radicals, which thus synergistically induced cell death. Importantly, the cell cytotoxicity of the nanozyme was related to its enzyme-like activity. The higher enzyme-like activity the artificial enzyme had, the stronger tumor cell growth inhibition would be. Under the same condition of laser irradiation (10 min, 2.0 W/cm2), HCNs or Au@HCNs could also show PTT efficiency. When the concentration of HCNs or Au@HCNs was higher than 50 µg/mL, the growth of the cancer cells was significantly inhibited. Therefore, cancer cell death due to Au@HCNs under NIR irradiation was not only due to the photothermal effect of the nanoparticles, but also a result of toxic ROS burst by nanozymes.

Figure 7. Relative viabilities of CT26 cells incubated with HCNs (a) and Au@HCNs (b) with and without 808-nm laser irradiation for 10 min. (c) Fluorescence intensity of DCFH determining the concentration of ROS (808-nm, 3 min). (d) Fluorescence images of cells treated with Au@HCNs and control. (e) Absorbance of WST-1 showing the presence of superoxides in cells (808-nm, 3 min). The concentrations of HCNs and Au@HCNs were both 50

µg/mL. The power of NIR was 2.0 W/cm2.

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To further explore this, we used DCFH-DA, a fluorescent probe that specifically identifies ROS,54 to verify our conclusion. As shown in Figure 7c, the rapidly increased fluorescence intensity of DCFH-DA over 3 h demonstrated the generation of ROS. Additionally, Au@HCNs generated higher intracellular ROS level than that of HCNs, indicating that Au@HCNs could produce higher levels of ROS. To confirm this effect, confocal microscopy was used to observe the change in fluorescence intensity directly. Cells treated with Au@HCNs exhibited strong fluorescence (Figure 7d), which confirmed the great ability of Au@HCNs to induce ROS production. In contrast, little fluorescence could be observed in the control cells. Furthermore, a WST-1 probe was used to specifically detect superoxides (Figure 7e).55 The cells treated with HCNs or Au@HCNs exhibited high absorbance values in the assays for superoxides compared with the cells in control groups, indicating the production of superoxides induced by HCNs or Au@HCNs. Notably, when the cells incubated with HCNs or Au@HCNs were irradiated for 1 min using 808-nm laser, the intensities of the signals (DCFH-DA and WST-1) were all increased, in agreement with the ESR experiments and the results of enzyme activities in vitro. Thus, the Au@HCNs nanozyme provided a catalytic-photothermal anti-tumor platform, combining light-enhanced ROS toxicity and photothermal therapy. These features endowed the Au@HCNs nanozyme with good potential for application in cancer treatment. Anticancer activity of Au@HCNs in vivo. Encouraged by the promising results of Au@HCNs in vitro, the experiments in vivo were carried out to evaluate the tumor growth inhibition. Firstly, we investigated the biodistribution of Au@HCNs following intravenous injection into tumor-bearing mice; the amounts of Au in major organs (spleen, liver, kidney, lung, heart and brain) and the tumor tissues were measured by ICP-AES. As shown in Figure S10, the content of Au in the tumor was over 20.6 % of the injected dose, showing a relatively high targeting efficiency for the Au@HCNs, and thus the enhanced performance of catalytic-photothermal therapy against tumors could be achieved. After that, CT26 tumor-bearing mice were divided into five groups randomly: (1) PBS (intravenous injection), (2) HCNs (intravenous injection), (3) Au@HCNs (intravenous injection), (4) HCNs (intravenous injection) + laser and (5) Au@HCNs (intravenous injection) + laser. The tumor sites were irradiated by the laser (808-nm, 2.0 W/cm2, 10 min) at the 12 h post i. v. injection (dose = 12.5 mg/kg) time point. The temperature increase of the tumor site was measured and recorded by an IR thermal camera every two minutes (Figure 8a). As shown in this Figure, the tumor temperature of group (5) rapidly raised from 33 to 52.9 °C in 10 min under NIR irradiation. In comparison, the tumor temperature of group (1) only increased slightly from 33 to 42 °C under the same conditions. The tumor volumes of different groups were recorded every other day during the subsequent 21 days (Figure 8b). It was found that group (4) and (5) showed efficient tumor restraint and gradual 14


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disappearance, leaving a benign scar at the primary tumor position after 21 days. In contrast, the tumor in mice treated with PBS only grew up progressively. Although the treatment with HCNs or Au@HCNs without NIR irradiation could also inhibit the tumor growth, the therapeutic effect was not as good as that of group (4) and group (5). The body weights, tumor weights, tumor photographs, and CT26 tumor-bearing mouse photographs in each group on 21 days are shown in Figure 8c-f. The tumor volume and weight in group (5) were the lowest values among all the groups, revealing that the tumors were completely eradicated without recurrence during the treatment. Compared with the previously reported materials for tumor photothermal therapy, our yolk-shell structure Au@HCNs possesses two prominent features. One is excellent photothermal conversion property. Second, the Au@HCNs can generate ROS, which could be enhanced under 808-nm laser irradiation. So the synergistic catalytic-photothermal therapy using nanozymes will accelerate the destruction of tumor cell.

Figure 8. In vivo catalytic-photothermal therapy of CT26 tumor bearing mice. (a) IR thermal images of CT26 tumor-bear mice with the NIR laser irradiation (808 nm, 2.0 W/cm2, 10 min) after intravenous injection with PBS, HCNs and Au@HCNs. (b) Tumor growth curves of different groups after treatment. (c) The body weight after various treatments during 21 days. (d) Photos of tumors from (1) control, (2) HCNs, (3) Au@HCNs, (4) HCNs + Laser, (5) Au@HCNs + Laser. (e) The tumor weight after various treatments indicated over 21 days. (f) Representative photos of tumors on mice after various treatments indicated on 21day. 15



It is noteworthy that the enzyme-mimicking activities of Au@HCNs will be lost under neutral conditions (Figure 2d and Figure 3d), which means that the Au@HCNs will not produce ROS in the blood circulation system (pH=7.0). Once the Au@HCNs enter into the acidic tumor microenvironment, ROS will be generated due to the recovery of enzyme-mimicking activities. This acid-responsive feature ensures the biological safety of Au@HCNs. To verify this, mice treated with different doses of Au@HCNs were sacrificed after thirty days of supervision. The major organs and tissues were analyzed by H&E. The histology analysis in Figure S11 revealed that the tissue structures had no obvious organ damage or inflammatory lesions. Moreover, we demonstrated that the Au@HCNs were degradable in physiological conditions (Figure S12). The carbon shell was broken and the spheres had gradually collapsed, leading to the shrinkage of the nanospheres. The degradation became more obvious on 12 day. These preliminary studies confirmed the biosafety of the Au@HCNs. Taken together, the results described above indicate that Au@HCNs exhibited high photothermal effects and light-enhanced ROS damage in vitro and in vivo, which makes this nanozyme an ideal candidate for highly efficient catalytic-photothermal tumor therapy. CONCLUSION In summary, we developed a rational approach to construction of yolk-shell gold@carbon nanospheres, which possess excellent enzyme-mimicking activities and photothermal conversion properties. The peroxidase-like and oxidase-like activities originating from Au@HCNs enable the generation of ROS during the catalytic process. Importantly, the enzyme-mimicking activities, including the ROS generation, could be enhanced by an 808-nm laser irradiation. Finally, the obtained Au@HCNs are capable of inducing cancer cell death through light-enhanced ROS generation and photothermal conversion both in vitro and in vivo, which substantially inhibited CT26 tumor growth by intravenous administration. Thus, it could be concluded that the combined catalytic-photothermal tumor therapy using nanozymes shows good potential for biological applications. EXPERIMENTAL METHODS Materials. HAuCl4·3H2O, tetraethylorthosilicate (TEOS), hypotaurine, 3, 3’, 5, 5’-tetramethylbenzidine (TMB) and dopamine hydrochloride (DA) were purchased from Sigma-Aldrich (USA). Ethanol, isopropyl alcohol and ammonia solution were purchased from Sinohparm Chemical reagent (Shanghai, China). Trisodium citrate dehydrate (99%), polyvinypyrrolidone (PVP) and H2O2 were purchased from Aladdin (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay were purchased from Thermo Fisher Scientific (USA). Dichlorofluorescein diacetate (DCFH-DA) 16


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and water-soluble tetrazolium-1 (WST-1) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) was obtained from Radical Vision (Marseille, France). All chemicals were of analytical grade. The distilled water was from a Millipore water purification system. Peroxidase-like activity of Au@HCNs and kinetic studies. The major absorbance peaks of blue color produced by the oxidation of TMB by Au@HCNs/H2O2 in 0.1 M NaAc buffer (pH 4.5) located at 370 and 652 nm. In a typical experiment, reagents were added into 1.0 mL buffer solution in the order of Au@HCNs (in varying amounts), 10 µL TMB (final concentration 0. 416 mM), and 30 µL H2O2 (final concentration 0.2646 M). Kinetic measurements were carried out by monitoring the absorbance change at 652 nm on a UV-vis 2550 spectrophotometer (Shimadzu, Japan). The Lineweaver-Burk plot was used to calculate the Michaelis-Menten constant. An experiment using HCNs/H2O2/TBM was also carried out as a control. Oxidase-like activity of Au@HCNs and kinetic studies. Similar to the studies of peroxidase-like activity, chemicals were added into 1.0 mL buffer solution in the order Au@HCNs (in varying amounts) and 10 µL TMB (final concentration 0.416 mM). Kinetic measurements of the oxidase reactions of Au@HCNs were performed using a UV-vis 2550 spectrophotometer (Shimadzu, Japan) at 652 nm. The Michaelis-Menten constant was calculated using the Lineweaver-Burk plot. An experiment with HCNs/TMB was also carried out as a control. Photothermal therapy in vitro. HCNs or Au@HCNs were diluted in DMEM medium before testing. Their cytotoxicity to CT26 cells was tested by seeding them in 96-well plates at a density of 1×104/cells per well. The cells were kept in culture for 12 h before replacing the medium with media containing the nanozymes at different concentrations. The following MTT assay and PTT evaluation were similar to our previous work,56 except that the NIR irradiation powder density was 2.0 W/cm2. Mouse tumor therapy. The tumor-bearing mice were randomly divided into the following five groups (n = 3 per group) for various treatments: (1) PBS control, (2) HCNs, (3) Au@HCNs, (4) HCNs + NIR irradiation and (5) Au@HCNs + NIR irradiation. HCNs or Au@HCNs were intravenously injected into mice bearing CT26 tumors at concentrations of 15 mg/kg (100 µL). On day 1, 3, 6, 9, 12, 15, 18 and 21, each group was intravenously injected with different nanozyme solutions. After intravenous injection for 12 h, laser irradiation was introduced in group (4) and group (5), and the laser power was constant at 2.0 W/cm2 with an irradiation time of 10 min. Body weights and tumor volumes were measured every other day, and the volume (V) of each was calculated as V = (tumor length) × (tumor width)2/2. The relative tumor volumes were calculated as V/V0, where V0 was the initial volume of the 17



tumor). The tumor volumes were recorded as ‘zero’ when the tumors disappeared. ASSOCIATED CONTENT Supporting Information Additional figures about material. This information is available free of charge via the Internet at http://pubs.acs.org/. Synthesis method and Characterizations, photothermal evaluation, electron spin resonance measurement, hypotaurine inhibition on enzyme-like activity, cell culture, observation of the Au@HCNs binding to cancer cells, intracellular ROS assay, mouse tumor model. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Lizeng Gao) and [email protected] (Juqun Xi)

ACKNOWLEDGMENTS The authors acknowledge the final support from the National Natural Science Foundation of China (No. 21673202, 81671810 and 21703198), the University Natural Science Foundation of Jiangsu Province (16KJD150004), the Social Development Project of Yangzhou City (YZ2016074), the Innovation Foundation of Yangzhou University (No. 2015CXJ069) and the Interdisciplinary Subject Construction Foundation of Yangzhou University (No. jcxk 2015-19). The work was also supported by the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions and Priority Academic Program Development of Jiangsu Higher Education Institutions. We would also like to thank the Testing Centre of Yangzhou University for their technical support. REFERENCES (1) Duan, D.; Fan, K.; Zhang, D.; Tan, S.; Liang, M.; Liu, Y.; Zhang, J.; Zhang, P.; Liu, W.; Qiu, X. Nanozyme-Strip for Rapid Local Diagnosis of Ebola. Biosens. Bioelectron. 2015, 74, 134-141. (2) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S. Intrinsic Peroxidase-Like Activity of Ferromagnetic Nanoparticles. Nat. Nanotechnol. 2007, 2, 577-583. (3) Zhao, M.; Deng, K.; He, L.; Liu, Y.; Li, G.; Zhao, H.; Tang, Z. Core-Shell Palladium Nanoparticle@Metal-organic Frameworks as Multifunctional Catalysts for Cascade Reactions. J. Am. Chem. Soc. 2014, 136, 1738-1741. 18


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Yolk-shell gold@carbon nanozymes show the intrinsic enzyme-like activity and photothermal conversion, which can be synergized for efficient tumor treatment, providing a proof-of-concept of tumor catalytic-photothermal therapy based on nanozymes.

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Tumor Catalytic-Photothermal Therapy with Yolk-Shell Gold@Carbon

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