Gold Nanoparticle-Decorated g-C3N4 Nanosheets for Controlled

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Biological and Medical Applications of Materials and Interfaces

Gold Nanoparticles Decorated g-C3N4 Nanosheets for Controlled Generation of Reactive Oxygen Species upon 670 nm Illumination Jiayong Dai, Jibin Song, Yuan Qiu, Jingjing Wei, Zhongzhu Hong, Lei Li, and Huanghao Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01307 • Publication Date (Web): 28 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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ACS Applied Materials & Interfaces

Gold Nanoparticles Decorated g-C3N4 Nanosheets for Controlled Generation of Reactive Oxygen Species upon 670 nm Illumination Jiayong Dai,

†,‡

Jibin Song,‡ Yuan Qiu,‡ Jingjing Wei,‡ Zhongzhu Hong,‡ Lei Li*,† and

Huanghao Yang*,‡

†

Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing

211166, P. R. China. E-mail: [email protected]

‡

MOE Key Laboratory for Analytical Science of Food Safety and Biology, State Key

Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China. E-mail: [email protected]

ACS Paragon Plus Environment

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KEYWORDS: g-C3N4, gold nanoparticles, photocatalysis, water splitting, reactive oxygen species, photodynamic therapy

ABSTRACT: Conventional photosensitizers based photodynamic therapy (PDT) is triggered by UV-light irradiation and depends on oxygen. However, it is hard to be applied for the deep and hypoxia tumor. To address this issue, we reported a new kind of g-C3N4 nanosheets decorated with gold nanoparticles (AuNPs), which could generate high amount of reactive oxygen species (ROS) under 670 nm laser irradiation in an oxygenfree environment. This synthesized semiconductor-metal heterojunction was served as a superior photodynamic agent, showing prominent cancer cell killing and tumor growth suppressing effects in the presence of 670 nm light and the g-C3N4-AuNPs composites,


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and its excellent ROS generation property was also validated by further bactericidal experiment.

1. Introduction

Photodynamic therapy (PDT), which was proposed several decades ago, represents a non-invasive and oxygen-dependent-featured approach for cancer treatment by taking


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advantages of the non-toxic photosensitizers and therapeutic properties of light.1-3 Cytotoxic reactive oxygen species (ROS) generated during the light illumination can lead to cell apoptosis and/or necrosis, tumor microvasculature damage, stimulation of nonspecific immune response, and finally tumor ablation.4 However, there is convincing evidence that the highly localized mode with low systemic toxicity and fewer side effects often results in unsatisfactory outcomes due to the abnormal hypoxia and acidulated tumor microenvironment (TME), which promotes tumor metastasis, aggressiveness, and resistance.5 Most current PDT agents mainly operate through an oxygen-dependent type II mechanism, which leads to severe hypoxia in the tumor microenvironment while limiting the therapeutic benefits of PDT.6 Hence, the exploration of the oxygen-independent PDT is among the current challenges in cancer therapy. Due to their particular physicalchemical properties, the nanomaterials know a broad spectrum of applications in biomedicine.7 Meanwhile, a growing number of published works showed that materials with (photo-) catalytic property, such as manganese dioxide (MnO2) and titanium dioxide (TiO2),8-9 can be creatively used as oxygen-independent PDT agents.


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Graphitic carbon nitride (g-C3N4), a layered metal-free semiconductor nanomaterial with excellent photocatalytic activity, has already attracted considerable attention for its varied applications including energy storage,10 photocatalysis,11 sensing,12 bioimaging13, therapy,14 electrochemistry15 as well as adsorbtion.16 This widely available material displays high chemical and thermal stability and a moderate bandgap (2.7 eV) corresponding to an optical wavelength of 459 nm. Good stability along with the low biotoxicity makes g-C3N4 a promising nanomaterial for biomedical and clinical applications, as well. However, the enhancement of the performance while extending the range of the light wavelengths still requires further exploration.

The effective generation of ROS is a crucial property of an ideal PDT agent. Usually, this property is improved by introduction of heteroatoms into the carbon framework,17 dye sensitization,18 and morphology control.19 And it has been shown that the formation of heterostructure can greatly enhance the separation efficiency of photogenerated electron-hole pairs and improve the light harvesting, which implies the enhanced generation of ROS.20 In this respect, quantum dots (QDs) of narrow-bandgap


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semiconductors, noble metal nanoparticles, and transition metal oxides had been coupled with g-C3N4 to achieve this primary goal.21 Light source also plays an equally key role in improving the therapeutic indices. Considering that long-term UV light irradiation of living cells and tissues may cause DNA damage and cell death while near-infrared (NIR) light can minimize photic injury and achieve deeper penetration effect with less attenuation, the use of NIR light has significant advantages for phototherapy.22-27 Unsatisfactorily, gC3N4 can hardly take use of light > 459 nm even if it exhibits extraordinary photocatalytic quality under visible light. Inversely, numerous researches had indicated that nanoscale gold materials are NIR light-sensitive with excellent catalytic character meantime,28 so it is easy to anticipated that AuNPs could widen the response range of g-C3N4 towards light.

Herein, we synthesized g-C3N4 nanosheets decorated with 5 - 10 nm gold nanoparticles (g-C3N4-AuNPs), which could generate high amount of ROS under 670 nm laser irradiation in an oxygen-free environment. To demonstrate the feasibility of g-C3N4AuNPs for PDT application, three cancer cell lines (A549, MCF-7, and HeLa) and bacteria (i.e., E. coli) were co-cultured with g-C3N4-AuNPs followed by light treatment. The results


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showed the synthesized g-C3N4-AuNPs can kill cancer cells and bacteria by producing ROS under 670 nm light treatment, which can stimulate a new thinking in the exploitation of PDT agents. Tumor bearing animal model experiments proved that in the present of gC3N4-AuNPs and light treatment, the tumor growth can be effectively suppressed. (Figure 1).

Figure 1. Schematic illustration of the hybrid g-C3N4-AuNPs based photodynamic cancer therapy by producing ROS under 670 nm light treatment.


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2. Experimental Section

2.1. Reagents and materials. Melamine, methanol, nitric acid (HNO3, 65-68%) and glutaraldehyde were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Chloroauric acid hydrate (HAuCl4·3H2O, ≥99.9%), 1-pyrenebutanoic acid (1-Py, ≥97%), sodium hydroxide (NaOH, ≥98%), 2, 7-dichloroﬂuorescein diacetate (DCFH-DA, ≥97%), Calcein-AM, propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-FITC Apoptosis Detection Kit was from Beyotime Biotechnology Co., Ltd. (Shanghai, China). LIVE/DEADTM BacLightTM Bacterial Viability Kit for fluorescence microscope was from Thermo Fisher Scientific Inc. (Shanghai, China). All solutions were prepared using ultrapure water (18.2 MΩ resistivity) produced by the MilliQ system.

2.2. Apparatus. The HT7700 (100 kV) and Tecnai G2 F20 S-TWIN (200 kV) transmission electron microscope, Nova NanoSEM 230 scanning electron microscope (FEI Company, USA), ESCALAB 250Xi (Thermo Scientific Inc., USA) x-ray photoelectron


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spectroscopy (XPS, monochromatic Al Kα radiation), X’pert3 and Empyrean x-ray powder diffraction spectroscopy (XRD, PANalytical B.V., Netherlands), Mulitmode 8 microscope atomic force microscope (Bruker, USA), UH4150 UV-Vis spectrophotometer (Hitachi, Japan) and F4600 fluorescence spectrophotometer (Hitachi, Japan), the Corona SH1000Lab absorbance microplate reader (Japan), Nicolet iS50 FT-IR spectrometer (Thermo Scientific), Zetasizer Nano particle analyser series (Malvern Instruments Ltd., England), super-resolution confocal laser scanning microscope (CLSM, Nikon N-SIM, Japan) were employed for characterization and measurement.

2.3. Synthesis of g-C3N4 and g-C3N4-AuNPs. Graphitic carbon nitride was synthesized using melamine as precursor in a muffle furnace (600 °C, 2 h, heating speed: 3 °C/min,). After being naturally cooled to room temperature, the light yellow colored bulk products were ground into powders further use. Then we dispersed about 1 g of g-C3N4 powders in 100 mL 5 M HNO3, and the mixture was immersed in a 130 °C oil bath for 24 h. The acidized products were centrifuged and washed to neutral with water. Followed by ultrasonication for ~16 h, highly water-dispersible g-C3N4 nanosheets can be obtained by


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two step centrifugation (3000 rpm for 30 min to remove the large unexfoliated nanoparticles, then 8000 rpm for 15 min to get the sediment). Finally, the g-C3N4 nanosheets solution of good dispersion was obtained after redispersing the sediment and stored at room temperature. The gold nanoparticles were successfully loaded onto gC3N4 nanosheets at the content of 0.5, 1.0, 2.0 wt% through a photodeposition process and corresponding hybrid samples are labeled as g-C3N4-0.5% Au, g-C3N4-1.0% Au, gC3N4-2.0% Au, respectively. Briefly, 3 mL methanol was added into 12 mL H2O containing 10 mg g-C3N4 nanosheets. Bubbled with N2 gas for 30 min, different volume of HAuCl4 (10 mM) solution was added and stirred for 30 min. The suspension was photoirradiated by a 300 W Xe lamp (constant-current mode, 15.0 A) for 30 min and repeatedly rinsed with water and then characterized.

2.4. 1-Py modification. Added 1.0 mL 1-Py (0.1 M in 0.5 M NaOH) into 10 mg g-C3N41.0% Au in 19.0 mL water, the aqueous solution was kept stirring for 12 h. Repeatedly rinsed with water, the final material was dissolved in water for the follow-up experiments.

2.5. In vitro Cytotoxicity Assay. For cytotoxicity assay, L02, A549, MCF-7, and HeLa


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cell lines were separately grown in 100 μL medium (DMEM or RPMI-1640 containing 10% fetal bovine serum, 37 °C, 5% CO2) for more than 12 h. And then incubated with 100 µL of varying concentrations of g-C3N4-1.0% Au (0 - 200 µg mL-1) for another 24 h. The cytotoxicity study was performed with CCK-8 in accordance with the manufacturer’s protocol.

2.6. In vitro PDT Effects. Respectively, A549, MCF-7, and HeLa cells were seeded in 96-well plates and incubated for more than 12 h. The cells were then subjected to g-C3N41.0% Au (200 µg mL-1) for 6 h in hypoxic atmosphere or normoxia condition. By irradiation at 0.15 W cm-2 using 670 nm light source for different times (0 - 20 min) in a hypoxic chamber, CCK-8 assays were performed after another 24 h incubation.

2.7. In vivo PDT Effects. Animal experiments were executed according to the protocol approved by the Institutional Animal Care and Use Committee of Fuzhou University, and all procedures were consistent with the guide for the care and use of laboratory animals (Ministry of Science and Technology of China, 2006). MCF-7 tumor-bearing BALB/c nude mice models (weight ≈ 20 g) were prepared by subcutaneously injecting of 5 × 106 MCF-


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7 cells (100 μL) into the back of the hind leg and randomly divided into six groups named Control, PBS + Light, g-C3N4, g-C3N4 + Light, g-C3N4-1.0% Au, g-C3N4-1.0% Au + Light, respectively. Therapy study was performed when the tumor volume reached ~30 mm3. Administrated groups were tail intravenous injected with corresponding material solution at the dose of 10 mg kg-1 every other day. Light treatment (670 nm, 0.15 W cm-2, 20 min) was carried out after 24 hours of each single injection. After the corresponding treatments, the body weight and tumor size of each animal were measured every two days till the 14th day to sacrifice the mice. Tumor size was calculated using the formula that tumor volume = 1/2 (length × width × width). Major organs and the tumors from each animal were sectioned into slices then H&E stained for histological analysis. TUNEL staining was also performed after the light treatment.

2.8. In vitro Antibacterial Effects. The antibacterial activity of g-C3N4-1.0% Au was evaluated by using E. coli bacteria as the model according to a literature procedure.29 In the presence of g-C3N4-1.0% Au (200 µg mL-1), E. coli (1×105 CFU mL-1) was treated under 670 nm light (0.15 W cm-2) for different times. The bacterial suspensions were


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cultured for 12 h at 37 °C to determine the optical density at 600 nm (OD600) by taking the culture medium without bacteria as background. Live and dead staining of bacteria was further studied using LIVE/DEADTM BacLightTM Bacterial Viability Kit on CLSM. Plate counting was also used to check the antibacterial ability of g-C3N4-1.0% Au under 670 nm light irradiation. The number of the bacterial colonies can be easily determined by spreading the bacterial suspensions on the solid medium and cultured for 24 h at 37 °C. To investigate the morphological changes of bacteria, SEM characterization were performed using glutaraldehyde for bacterial immobilization.

3. Results and Discussion

3.1. Morphology and Characterization. Pyrolysis method was used to fabricate g-C3N4 while the liquid phase exfoliation method was used to produce nanosheets with single or few layers. In the TEM images (Figure 2a, Figure S1a-b), a layered structure of g-C3N4 nanosheets with irregular morphology can be clearly seen. As-synthesized g-C3N4 nanosheets were about 200 nm, which was in agreement with the hydrodynamic size


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measurement results (about 204 nm, Figure S5). This results was confirmed by the atomic force microscopy (AFM) (Figure 2d), by which representative g-C3N4 nanoflakes of 170 -180 nm in width and about 4 nm in height were noticed.

Methanol, as sacrificial reagent, was used to capture holes and inject electrons for AuNPs’ deposition. By light irradiation of the g-C3N4 suspensions containing Au3+ salt, spherical gold nanoparticles (AuNPs) with size of 5 - 10 nm were uniformly generated onto the surface of the g-C3N4. Changing the amount of gold precursor from 0 to 2.0%, g-C3N4-AuNPs nanostructures with varying content of AuNPs were successfully formed, and the incremental amount of the AuNPs generated on g-C3N4 nanosheets was obvious (Figure S1b-e). HRTEM image of Au nanoparticle was also taken, and the interplanar distances of 0.24 and 0.20 nm were in line with the lattice spacing of the (111) and (200) planes of gold (Figure 2b). The HRTEM image (Figure S2), high angle annular dark fieldSTEM (HAADF-STEM) images (Figure 2c, Figure S3b-c), element mapping images (Figure S3d-h), AFM image (Figure S6), and SEM image (Figure S7) of g-C3N4-1.0% Au, and the XRD (Figure S8), XPS (Figure S9, S10) and thermal stability (Figure S12)


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characterization results also confirmed that the successful photodeposition of AuNPs. The residual Au content in the supernatant of each reaction solution was measured by ICP-MS after the Xe lamp irradiation (Figure 2h). The Au amounts deposited onto g-C3N4 were calculated to be 47.443, 94.911, 193.748 μg for 0.5%, 1.0%, and 2.0% doping, respectively.


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Figure 2. Characterizations of g-C3N4 and g-C3N4-AuNPs. a) TEM image of g-C3N4-1.0% Au, scale bar = 200 nm. b) HRTEM image of the AuNPs on g-C3N4-1.0% Au, scale bar = 5 nm. c) HAADF-STEM image of g-C3N4-1.0% Au, scale bar = 200 nm. d) AFM pattern of g-C3N4 nanosheets. e) The UV-vis absorption spectra of g-C3N4-AuNPs (100 μg mL-1). f) The electron-hole energy transfer schematic diagram of g-C3N4-AuNPs. g) FL spectra of DCFH after oxidized by the generated ROS (670 nm, 0.15 W cm-2, 30 min). h) Residual Au in the supernatant of different reaction systems.

The optical absorption of pure g-C3N4 and g-C3N4-AuNPs was characterized (Figure 2e). The introduction of AuNPs into the system can increase the optical absorption in the UV-vis range of g-C3N4, which is essential for improving performance of g-C3N4. The surface charge is a crucial property of the nanomaterials that ensures a stable dispersion in different media with low ionic strength (Figure S11). Generally, a value of zeta-potential higher than 30 mV is required to provide enough electrostatic repulsive force to counterbalance the van der Waals attraction which leads to particle aggregation.30 Zetapotential of the g-C3N4 nanosheets used in this work is +39.4 mV while the gold decoration


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enhanced this value even more (about +46.0 mV). Hence, the nanomaterials investigated in this study manifested good dispersibility in water. On the other hand, the positively charged nanomaterials can be endocytosed by cancer cells with higher efficiency after the adsorption-mediated cell binding and internalization.31

3.2. ROS Generation. 2, 7-dichloroﬂuorescein diacetate (DCFH-DA) is frequently used for ROS detection. Under 670 nm irradiation, the generated ROS could quickly oxidize DCFH to DCF, which has a green emission under light excitation at 488 nm (Figure 2g). Pure DCFH was also selected for this study based on its intrinsic photo instability. Bare g-C3N4 nanosheets exhibited a weak response to 670 nm light. However, the ability to generate ROS was apparently enhanced by the increased amount of Au loaded on the support, reinforcing the idea of the facilitated photo-induced charge separation. Au content of 1.0% manifested the best ability to generate ROS in comparison to 2.0%, which may due to the formation larger aggregates and the increased concentration of recombination centers. The enhanced ability of the resulting Au/C3N4 heterostructure to produce ROS should be attributed to the plasmon-enhanced photocatalytic water splitting


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(Figure 2f). AuNPs absorbed the 670 nm light energy, and the excited electron was injected into g-C3N4 nanosheets, prolonged the photoinduced charge separation process and delayed the recombination of electron-hole pairs, hence generating reactive oxygen based radicals. It was also proposed that H2O molecule can bridge the triangular pore between three tri-s-triazine units by forming hydrogen bonds with the N atoms of g-C3N4 while the adsorption of H2O on the (0001) surface of g-C3N4 was not significantly affected by the layer stacking, fact demonstrated by the combination of first-principles and semiempirical studies.32 To determine which kind of ROS was generated, electron spin resonance (ESR) measurement was used, and we found that hydroxyl radical (•OH) and superoxide anion (O2-) were massively generated under illumination in the hypoxia environment (Figure S14). Furthermore, the efficiency of ROS generation by g-C3N4-1.0% Au was tested for different periods (Figure S13). It was noticed that for longer periods, the fluorescent intensity at 522 nm was increased gradually, indicating the continuous production of ROS. Then g-C3N4-1.0% Au was chemically modified with 1-pyrenebutanoic acid (1-Py) for biological applications (Figure S15, Figure S16). On account of that the Py-COOH (sodium salt) could absorb onto the surface of g-C3N4 through π-π interaction


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for stabilization and pyrenebutyrate’s lack of toxicity was confirmed by the results of previous reports as well.33-35

3.3. In vitro Cytotoxicity Assay and Hemolysis Assay. Biocompatibility is an essential concern when develop nanomaterials for biomedical applications. Cytotoxicity induced by g-C3N4-1.0% Au was determined by the CCK-8 assay (Figure S21). Normal human embryo hepatocellular line L02, lung adeno-carcinoma A549 cell line, breast cancer cell line MCF-7, as well as HeLa cervical carcinoma cells were selected as cell line models. At 0 - 200 µg mL-1 of g-C3N4-1.0% Au, the cell viability of the aforementioned four cell lines was superior to 80%, indicating a good biocompatibility. This should be attributed to the chemical compositions, where g-C3N4 was of low biotoxicity and AuNPs had been already proved to be ideal computed tomography (CT) contrast agents and photoacoustic (PA) imaging probes in theragnostic applications.36 Regarding the influence of g-C3N41.0% Au on the hemolytic behavior of red blood cells (RBCs), deionized water and PBS were used as positive and negative controls, respectively. Negligible hemolysis of RBCs was observed in the presence of g-C3N4-1.0% Au, which indicates a great blood


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compatibility of the tested nanomaterial (Figure S17). Therefore, it can be stated that the prepared nano-construct based on gold decorated carbon nitride is a safe and promising nanomaterial for biomedical applications.

Figure 3. In vitro PDT effects of 1-Py modified g-C3N4-1.0% Au in a) normoxia condition and b) hypoxic atmosphere. c) Live/dead cell staining results of MCF-7 in hypoxia atmosphere. A. MCF-7. B. MCF-7 + g-C3N4-1.0% Au. C. MCF-7 + g-C3N4-1.0% Au + 10


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min light irradiation. D. MCF-7 + g-C3N4-1.0% Au + 20 min light irradiation. Scale bar = 100 µm. d) Flow cytometry images of MCF-7 after different treatment in hypoxia atmosphere. ① MCF-7; ② MCF-7 + Light; ③ MCF-7 + g-C3N4-1.0% Au; ④ MCF-7 + g-C3N41.0% Au + 20 min light irradiation. All cell experiments were carried out at the concentration of 200 μg mL-1.

3.4. Intracellular ROS Generation and PDT Effects. Intracellular ROS production with 670 nm laser irradiation was further tested (Figure S27). Cells treated either with g-C3N4, AuNPs with 670 nm light irradiation showed negligible fluorescence signal. However, after treatment with g-C3N4-1.0% Au and light irradiation, bright green fluorescence emissions can be observed on confocal laser scanning microscope (CLSM) for all three tumor cell lines, illustrating the ROS generation through the photocatalytic splitting water, which can cause an oxidative stress to cells and thus, induce the cell death. To validate the feasibility of PDT with g-C3N4-1.0% Au, in vitro PDT assays in normoxia condition and hypoxic atmosphere were performed (Figure 3a, b). For hypoxic atmosphere, the MCF-7 cells, both live and dead, were stained with Calcein-AM and propidium iodide (PI) (Figure 3c).


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It can be observed that the cells co-cultured with g-C3N4-1.0% Au remained alive, which is in well agreement with the aforementioned good biocompatibility. Triggering the intracellular ROS production process by 670 nm light, the cell apoptosis could be distinguished after a period of incubation at 37 ºC. The flow cytometry assay also confirmed the cell apoptosis inducing effect of our heterostructure (Figure 3d). Additionally, the photothermal effect of 1-Py modified g-C3N4-1.0% Au was also evaluated (Figure S23). After irradiation for 20 min with 670 nm light (0.15 W cm-2), the temperature increment of the sample (200 μg mL-1) was as low as ~2.4 ℃. So the photothermal effect of 200 μg mL-1 1-Py modified g-C3N4-1.0% Au was neglectable and the anticancer effect was truly induced by efficient ROS generation.

3.5. In vivo Anticancer Efficacy. To demonstrate the tumor therapy in vivo, the mice were randomly divided in six groups, including 1) Control, 2) PBS + Light, 3) g-C3N4, 4) g-C3N4 + Light, 5) g-C3N4-1.0% Au, 6) g-C3N4-1.0% Au + Light, respectively. When the tumor volume reach 30 mm3, the samples were intravenously injected into the MCF-7 tumor-bearing mice through tail vein. At 24 h post-injection, light was applied to irradiate


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to the whole tumor region. The length and width of the tumor were recorded to calculate the tumor size at each other day. As shown in Figure 4b, time-related tumor volume increase can be observed in each group. The PBS + light group showed significant increases of the tumor volumes by ~20-fold, suggesting that only 670 nm light irradiation had no influence on tumor growth. Free g-C3N4 with or without light irradiation exhibited a similar tumor growth with that of the PBS group, showing that g-C3N4 nearly has no tumor therapy efficiency. In the g-C3N4-1.0% Au group, the tumor also has a fast growth speed, indicating that no anticancer effect without laser irradiation. Distinctly, 14 days treatment remarkably suppress the tumor growth of the g-C3N4-1.0% Au + Light group in comparison with the others groups, due to high efficient of ROS was produced in the tumor region, which is consistent with the in vitro therapy effect. Moreover, no apparent loss of body weight of all the groups was observed during the therapy process, confirming the biocompatibility of the nanoplatform (Figure 4a). Representative tumor picture of different groups was captured after the tumor-bearing BALB/c nude mice were sacrificed (Figure S28). And the body distribution results revealed that the hybrid nanosheets mainly


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accumulated in liver and spleen, but there are still part of the materials can reach tumor area to function as the PDT agent (Figure 4c).

For investigating the tumor suppression mechanism, fluorescent TUNEL assay was performed to examine apoptosis level in the tumors from the mice receiving different treatments (Figure 4d). The cancer cell nuclei were blue stained by 4’, 6-diamidino-2phenylindole (DAPI), and only the cell in the g-C3N4-1.0% Au + Light treated group exhibited green stained signal, which illustrated that DNA fragmentation appeared after light treatment. This phenomenon agree well with the cell experiment result that g-C3N41.0% Au can generation ROS under 670 nm light illumination to induce cell death of MCF7. H&E stained histological images of tumor sections and major organs (heart, liver, spleen, lung, and kidney) told that no apparent ablation of tumor tissue and no other form of organ toxicity was found (Figure 4e, Figure S29).


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Figure 4. Results of in vivo PDT Effects. a) Body weight curves of the MCF-7 tumorbearing mice after different treatments. b) Tumor growth curves of the MCF-7 tumorbearing mice of different groups (*p < 0.05). c) Body distribution of g-C3N4-1.0% Au after tail intravenous injection (24 h). d) TUNEL assay. Typical fluorescence microscope scanning images of tumor sections, respectively. Scare bar = 100 μm. e) Representative H&E stained histological images of tumor sections after different treatments. Scale bar = 100 μm. (Pictures were of Control, PBS + Light, g-C3N4, g-C3N4 + Light, g-C3N4-1.0% Au, and g-C3N4-1.0% Au + Light for d and e).


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3.6. In vitro Antibacterial Effects. Several predominant mechanisms such as nanoknives,37 oxidative stress,38 photothermal effect,29 and wrapping or trapping39 have been proposed to explain the antimicrobial activities of two-dimensional nanomaterials. In this study, we rationalized the results of the antimicrobial activity based on the ROS generated over the noble metal/semiconductor hybrid nanomaterials, which were able to induce the oxidative stress to kill cells. To highlight this property, mixtures of E. coli and g-C3N4-1.0% Au were treated with 670 nm light for different periods. By comparing the OD values, it was clear that the viability of the bacteria continuously decreased with prolonging the irradiation time. Nevertheless, bacteria did not show apparent response to 670 nm light treatment in the absence of g-C3N4-1.0% Au (Figure S30). Secondly, LIVE/DEADTM BacLightTM Bacterial Viability Kit, a commonly dye to stain live and dead bacteria, was used to stain the bacteria in dark (Figure 5). CLSM images showed that gC3N4-1.0% Au killed E. coli bacteria when exposed to 670 nm light irradiation. On the contrary, neither light nor g-C3N4-1.0% Au alone can affect the bacteria viability. Thirdly, using the dilution-plate method, the light treated mixtures were spread onto a solid medium, and allowed for 12 h incubation at 37 ºC. Dramatically, the bacterial colony


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decreased to an easily countable level in accordance with the OD values and CLSM images. Finally, SEM images (Figure S31) of the treated E. coli demonstrated that the generated ROS can destroy their morphological structure. Inside cell membrane was damaged even if the outside cytoderm remained nearly intact due to the high mechanical strength of the peptidoglycan.

Figure 5. Live/dead staining and plates smearing results of E. coli bacteria. a) E. coli bacteria. b) E. coli bacteria + g-C3N4-1.0% Au. c) E. coli bacteria + g-C3N4-1.0% Au + 10 min light irradiation. d) E. coli bacteria + g-C3N4-1.0% Au + 20 min light irradiation. Scale


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bar = 25 µm. Bactericidal experiments were carried out at the concentration of 200 μg mL1.

4. Conclusions

In summary, we have successfully prepared a hybrid nanomaterial of g-C3N4 coated with AuNPs through liquid phase exfoliation of g-C3N4 combined with photodeposition of AuNPs. The incorporation of AuNPs into the nansystem significantly enhanced the electron/hole separation due to the improved efficiency of 670 nm light absorption, which caused effectively ROS generation via photocatalytic water-splitting reaction. Particularly, the g-C3N4-AuNPs also had preferable 670 nm light triggered ROS generation property in biomedical applications. Consequently, the generated ROS were utilized for both cancer treatment and bacteria killing with excellent performance. The outcomes of this works open up new avenues for the development of other interesting heterostructures with high potential in biomedical applications.


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Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website. Supplementary experimental section, material characterization such as HRTEM, HAADFSTEM, element mapping images, AFM, SEM, XRD, XPS, DLS, zeta potential, thermal stability, ESR and FT-IR, cell experiment results such as cytotoxicity assay and intracellular ROS detection, H&E stained histological images of major organs and some antibacterial effect results were provided for better understanding.

AUTHOR INFORMATION

Corresponding Author

*[email protected], ORCID ID: https://orcid.org/0000-0002-0014-7892.

*[email protected], ORCID ID: https://orcid.org/0000-0001-5894-0909.

Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENT

We acknowledge financial support from the National Natural Science Foundation of China (81673228, 81473020 and 21874024), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2014) and Center for Global Health of Nanjing Medical University.

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Gold Nanoparticle-Decorated g-C3N4 Nanosheets for Controlled

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