Exploration of Graphitic-C3N4 Quantum Dots for Microwave-Induced

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Exploration of Graphitic‑C3N4 Quantum Dots for Microwave-Induced Photodynamic Therapy Xiao Chu,# Kang Li,# Hongyu Guo,‡ Huibin Zheng,‡ Suzanne Shuda,|| Xiaolan Wang,# Junying Zhang,*,‡ Wei Chen,*,|| and Yu Zhang*,# #

Guangdong Key Lab of Orthopaedic Technology and Implant Materials, Key Laboratory of Trauma & Tissue Repair of Tropical Area of PLA, General Hospital of Guangzhou Military Command of PLA, Guangzhou 510010, China ‡ Key Laboratory of Micro-nano Measurement, Manipulation and Physics (Ministry of Education), Department of Physics, Beihang University, Beijing 100191, China || Department of Physics, The University of Texas at Arlington, Arlington, Texas 76019-0059, United States S Supporting Information *

ABSTRACT: Here, we report our new observations on graphitic-phase carbon nitride (g-C3N4) quantum dots (QDs) as agents for microwave induced photodynamic therapy (MIPDT). For the first time, we observed that singlet oxygen is produced in g-C3N4 QDs by microwave irradiation, which can be used for tumor destruction. The results of live/dead staining and flow cytometry show that g-C3N4 QDs based MIPDT can effectively kill cancer cells and promote tumor cell death. In addition, the cell viability and hemolysis tests in vitro indicate that g-C3N4 QDs have very low cell toxicity and possess excellent biocompatibility in the physiological environments. All these indicate that g-C3N4 QDs are promising for MIPDT, a new potential modality for cancer treatment. KEYWORDS: microwave, microwave induced photodynamic therapy (MIPDT), graphitic-C3N4 quantum dots, tumor, singlet oxygen a number of ways.17,18 Microwaves can propagate through all types of tissues and nonmetallic materials, enabling MIPDT a new therapy for deep cancer treatment.12,18,19 Here, for the first time, we report graphitic carbon nitride (g-C3N4), a well-known photocatalyst for microwave induced PDT. g-C3N4 has been investigated as a new photosensitizer for PDT20−22 and as a luminescence agent for cell imaging.23 However, g-C3N4 has never been studied as an agent for MIPDT. g-C3N4 with twodimensional frameworks of the tris- triazine connected via tertiary amines has a high thermal and chemical stability. gC3N4 has been investigated extensively in the past few years as an efficient metal-free photocatalyst, which can effectively absorb visible light to produce reactive oxygen species (ROS).24,25 g-C3N4 exhibits a high photocatalytic activity for water splitting,24,26,27 organic decomposition,28 CO2 reduction,29 and bacterial inactivation.30 More interestingly, g-C3N4 has been extensively investigated for biological applications because of its biocompatibility and no or low toxicity. Waterdispersible g-C3N4 ultrathin nanosheets have a strong blueemission which can be used for cell imaging.23 g-C3N4 nanosheets show a potential for Aβ-targeted Alzheimer

1. INTRODUCTION Photodynamic therapy (PDT) has been widely used for skin cancer treatment,1,2 but rarely for deep-seated cancers or solid tumor because of the difficulty of light delivery for activation. Possible solutions to the light delivery problem have been focused on exploiting long-wavelength irradiation. Great progress has been achieved on developing photosensitizers activated by near-infrared (NIR) light due to its deeper penetration into tissues than ultraviolet (UV) or visible light.3,4 Likewise, up-conversion nanoparticles have been used for PDT, because they can be excited at NIR and emit light in the UV− visible range.5,6 Scintillation or afterglow nanoparticles have also been utilized for PDT treatment because of the ability of treating deep-seated tissues.7−9 Cerenkov light was also reported for PDT activation.10 All the above solutions have some kinds of limitations. NIR light cannot penetrate deeper than 10 mm in tissue while retaining enough energy to activate photosensitizers (PSs).11 Xray has a deep penetration but it can easily damage the healthy tissues. Cerenkov light is not very effective and it is not convenient to produce and control. Most recently, we proposed and reported microwave induced photodynamic therapy (MIPDT)12 for cancer treatment, which is based on copper cysteamine (Cu-Cy), a new photosensitizer.13,14 Microwave energy is distinct from other energies for thermal therapy15,16 in © 2017 American Chemical Society

Received: February 14, 2017 Accepted: June 1, 2017 Published: June 1, 2017 1836

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering

buffered saline (PBS) three times to remove particles that were not internalized in cells, fixed again with 1% osmium tetroxide for 2 h. The cells were treated with a grade series of ethanol (30, 50, 70, 80, 90, and 100%) at each concentration for 10 min, then embedded with Epon812 and cut with an ultramicrotome (Leica Ultracut, Germany). The ultrathin sections were then stained with uranylacetate and lead citrate. The nanoparticles entered into the cells were observed using the transmission electron microscope (TEM, H-7650, Hitachii, Japan). Cellular Internalization Assay of g-C3N4 QDs. UMR-106 cells were seeded in 24-well plate at a density of 2 × 104 cells per mL, and 0.5 mL each well for 24 h to allow attachment. Then different concentrations of g-C3N4 QDs (0, 0.1, and 0.4 mg/mL) were added into a 24-well plate after discarding the medium and continued to incubate for 24 h. Finally, these samples were observed under UV light by fluorescence microscope (DMI4000, Leica, Japan), and the excitation wavelength is 340−380 nm. In Vitro MW Heating Experiment. To evaluate the hyperthermia efficiency of g-C3N4 QDs under microwave irradiation in vitro, we added 0.5 mL as-prepared samples into a 48-well plate. The microwave probe was inserted below the liquid level, and then exposed to MW (5 W, 2450 MHz, Baoxing medical equipment, Co.LTD) irradiation. The temperature was detected using fiber thermometers (Xi′an Heqiguang technology, ThermAgile-RD, HQ-FTS-D1D00−001). The heating curves with time were plotted to measure the temperature change of the g-C3N4 QDs solution under microwave irradiation. Singlet Oxygen Test in Vitro under MW Irradiation. To measure the production of singlet oxygen when g-C3N4 QDs solution was under MW irradiation, as the literature35 reported, the method of RNO-ID (p-nitrosodimethylaniline (RNO)-imidazole (ID)) was used to test the release of singlet oxygen in solution. The RNO-ID solution was prepared as follows: 0.225 mg RNO (Sigma, USA) and 16.34 mg ID (Sigma, USA) were added to 30 mL of double distilled water, and stirred until completely dissolved. 100 μL of g-C3N4 QDs solution was added into 200 μL of as-prepared RNO-ID solution, then the mixture solution was irradiated under different powers including 0, 5, 10, 15, 20, 30, 35, and 40 W for 5 min, using the RNO-ID solution as control. Then we detected the singlet oxygen production in g-C3N4 QDs solution under 5 and 10 W microwave powers with the increase of time. The optical density (OD) was measured using a microplate reader (Thermo, Multiskn Go) at 440 nm. Every group was repeated triplicate and data are presented as the mean ± standard deviation. ROS Detection in UMR-106 Cells. The 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA, Sigma, USA) was used to measure the intracellular ROS.38,39 DCFH-DA passively enters the cell and was then hydrolyzed into nonfluorescence DCFH, which can react with singlet oxygen to form the highly fluorescent compound dichlorofluorescein (DCF).38,39 A 20 μM working solution was diluted with 0.1 M of DCFH-DA stock solution (in methanol) in DMEM without serum or other additive; 400 μL of UMR-106 cell suspension (2 × 104 cells/mL) was seeded into two 24-well cell culture plates (A and B) in a humidified 5% CO2 atmosphere at 37 °C for 24 h; 400 μL of different concentrations (0, 0.1, or 0.4 mg/mL) of g-C3N4 QDs solution were added to the plates. The cells were cultured for 24 h and then washed twice with sterile PBS, added to 400 μL of DCFH-DA working solution at 37 °C for 30 min, and washed gently three times with PBS. Then, 400 μL of DMEM was added into the plates. The cells in culture plate A were washed twice with the PBS before applying MW at a dose of 5 W for 5 min. Plate B was as control without any treatment. The fluorescence microscope (Olympus BX51, Japan) was used to observe the fluorescence intensity of the cells in order to qualitatively measure the singlet oxygen produced within the cells. Also the value of fluorescence was calculated quantitatively with the fluorescence microplate (VarioskanFlash 4.00.53) at 488 nm/525 nm. Cell viability Test in Vitro by MTT Assays. The potential cytotoxicity of g-C3N4 QDs to UMR-106 cells was measured by using 3-(4, 5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) assay after the cells were cocultured with g-C3N4 QDs for 1, 3, and 5 days. Cells were seeded into the 96-well plate at a density of 2 × 104 cells per mL and 100 μL every well for 24 h to allow attachment. Then

treatments because they can generate ROS under visible-light illumination to oxidize Aβ peptides for further fibrillation.31 Further reducing the nanosheet into quantum dots (QDs) could achieve an economic probe for two-photon fluorescence imaging of cellular nucleus.32 The pH-sensitive emission of gC3N4 QDs causes high photocytotoxicity under acidic conditions mimicking those of tumor tissue, and thus has a potential to be utilized as a cancer-selective photodynamic therapy (PDT) agent.20 In this study, for the first time, we demonstrate that g-C3N4 QDs can release singlet oxygen under low-power microwave irradiation. A series of cell experiments in vitro showed that g-C3N4 QDs can enter into osteosarcoma UMR-106 cells and produce singlet oxygen under microwave irradiation, which enhanced the killing effect of microwave on tumor cells. Under microwave irradiation g-C3N4 QDs can produce the singlet oxygen for cancer destruction. This study is concentrated on osteosarcoma, which is the most common type of bone cancer and one of the cancers that are most difficult to cure. The preliminary results show that g-C3N4 QDs are excellent agents for MIPDT.

2. MATERIALS AND METHODS All the chemical reagents used in the experiments were obtained from commercial sources as guaranteed-grade reagents and used without further purification. Preparation and Characterization of g-C3N4 QDs. The g-C3N4 nanoparticles with the average size of about 5 nm were synthesized by acid digestion of bulk g-C3N4 as we have previously reported.20 The gC3N4 QDs were well-dispersed in water and remained stable for several months. The X-ray powder diffraction (XRD) was analyzed using a Bruker D8-Advanced X-ray diffractometer with Cu Kα radiation (λ = 1.5418 Å). The luminescent properties were studied by an Edinburgh Instruments FLS 980 steady-state and transient-state fluorescence spectrometer. The morphology was investigated using a high-resolution transmission electron microscope (HRTEM, Fei Tecnai G2F30). Cell Culture. The rat osteosarcoma UMR-106 cell was purchased from the Type Culture Collection of the Chinese Academy of Science. The cells in 25 cm2 tissue culture flasks were cultured in the thermotank with 37 °C and humidified atmosphere of 5% CO2. The cell culture medium was modified Eagle’s minimum essential medium (α-MEM, HyClone, USA) with 4.5 g/L glucose, 10% fetal bovine serum (HyClone), 50 U/mL penicillin, and 50 U/mL streptomycin. In Vitro Blood Compatibility Test. The rabbit’s ear vein blood was used to conduct the hemolysis test in order to evaluate the cytotoxicity of the g-C3N4 QDs as above in vitro. Eight mL blood was taken from the ear of rabbit into the anticoagulant tube, which was diluted to 10 mL with normal saline. According to the ISO10993−1 standard, different concentrations of g-C3N4 QDs (1, 0.8, 0.6, 0.4, 0.2 mg/mL, 1 mL), physiological saline solution (negative control, 1 mL) and double-distilled water (positive control, 1 mL) were incubated in centrifuge tubes at 37 °C for 30 min. Then 0.2 mL diluted blood was added to each tube and the mixtures were kept at 37 °C for 60 min. One hour later, the mixtures were centrifuged at 1000 rpm for 5 min. Finally, the supernatant was transferred to a 96-well plate and the microplate reader was used to measure the optical density (OD) values at 545 nm wavelength. The hemolysis rate (%) was calculated according to the following formula: hemolysis rate (HR) = [OD (test sample) − OD (negative control)]/[OD (positive control) − OD (negative control)] × 100%. All experiments were repeated triplicate. Cellular Uptake Study. UMR-106 osteosarcoma cells were seeded in a 6-well plate at a density of 2 × 106 cells per mL, and 2 mL every well for 24 h. Then different concentrations of g-C3N4 QDs (0.1 mg/ mL, 0.4 mg/mL) were added into the 6-well plate after discarding the medium and then incubated for 24 h. The cells were collected by centrifugation at 1,000 rpm for 5 min. Then, the samples were fixed with 3% glutaraldehyde for 24 h at 4 °C, and washed with phosphate1837

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering the medium was replaced with 100 μL different concentrations of gC3N4 QDs solution (1, 0.4, 01, 0.05, 0.025, 0.0125 mg/mL) diluted in culture medium. For MTT assay, at each of the designated time points, 10 μL MTT (Sigma, USA) solution (5 mg/mL in PBS) was added to 100 μL culture medium and incubated for 4 h at 37 °C to form formazan. After removing the culture medium, dimethyl sulfoxide (DMSO) was used to dissolve the formazan reaction products and the optical density (OD) was measured using a microplate reader (Thermo, Multiskn Go) at 490 nm. Every group was repeated three times and the assay was repeated on three separate days. The data presented in the paper are statistical results from 9 groups of independent data, and the results are presented as the mean ± standard deviation. Cell Viability Test under MW Irradiation. MTT Assay. To evaluate whether the g-C3N4 QDs enhance the effect of microwave ablation, we conducted the MTT assay of cells in vitro as below. Eight mg/mL g-C3N4 QDs solution was diluted into 0.4, 0.1, 0.05 mg/mL with DMEM medium; 100 μL of UMR-106 cell suspension (2 × 104 cells/mL) was seeded into the 96-well plate for 24 h to allow attachment, and then removed from the medium, added to 100 μL of g-C3N4 QDs solution as above. Afterward, the cells were irradiated under microwave at the dose of 3 and 5 W for 5 min and continued to incubate for another 2 h at 37 °C, 5% CO2. MTT assay was performed to evaluate the cell viability after being treated with microwave and the procedure was as shown in the above MTT assay section. Live/Dead Cell Staining Assay. Cell viability under microwave irradiation was also determined by counting the viable and nonviable cells using the live/dead staining. 300 μL of the cell suspension (2 × 104 cells/mL) was irradiated by microwave (3 and 5 W, 2450 MHz) for 5 min. After 2 h, the cells were stained with 300 μL dye (CalceinAM: propiduim iodide: PBS = 2.53 μL: 20 μL: 8.546 mL) (Sigma, USA) for 30 min in dark environment. The MW group was only treated with MW irradiation without g-C3N4 QDs (MW only), and the control group was without any treatment (control). Finally, these samples were observed by fluorescence microscope (Olympus BX51, Japan). In addition, we counted the number of the live (green) and dead (red) cells, and calculated the cell viability for quantitative analysis. Cell Apoptosis. Four hundred microliters of the UMR-106 cell suspension (2 × 104 cells/mL) was seeded into the 24-well plate for 24 h to allow attachment, and then removed the medium, and added different concentrations (0, 0.1, and 0.4 mg/mL) of g-C3N4 QDs solution at 400 μL per well. Then cultured for 24 h, washed three times with PBS, and then resuspended in 100 μL of DMEM with 1% FBS. The positive control was pretreated by coculturing with g-C3N4 QDs for 24 h, and negative control with the cell only. Then each group was irradiated by microwave at the dose of 3 and 5 W for 5 min. The cells were stained in a 96-well microplate with Guava Nexin Reagent (Millipore, Billerica, MA, USA), a premade cocktail containing Annexin V-PE and 7-AAD in a final volume of 200 μL. After 20 min incubation at room temperature, the samples were analyzed using a Guava EasyCyte 5HT flow cytometer (Millipore, USA), and the data were analyzed using Guava Nexin Software v2.2.2. Statistical Analysis. The average values and standard deviation were calculated, and the data were analyzed using SPSS 13.0 software. A one-way ANOVA followed by a Student−Newman−Keuls post hoc test was used to determine the level of significance; p < 0.05 was considered to be significant and p < 0.01 was considered to be highly significant.

inset HRTEM image shows the fringes with lattice spacing of 0.33 nm, in agreement with (002) plane of g-C3N4. Figure 1

Figure 1. Photoluminescence spectra of g-C3N4 QDs excited at 330 and 365 nm. The insets show the g-C3N4 QD aqueous solution (left) and its luminescence excited under a UV lamp (right).

shows the g-C3N4 QDs and their luminescence spectra excited at 330 and 365 nm. As shown inset in Figure 1, g-C3N4 QDs have a strong blue emission at around 440 nm due to the π−π* transition in the aromatic structure.34 3.2. Microwave Activated g-C3N4 QDs on UMR-106 Cells. As shown in Figure 2, when the UMR-106 cells were

Figure 2. Cell viability of UMR-106 cells after being mixed with gC3N4 QDs and treated with microwave for 5 min (*p < 0.05; #p < 0.05).

treated with microwave irradiation (3 and 5 W, respectively) for 5 min without g-C3N4 QDs, only small quantity of tumor cells were killed and the cell viability reached 87% (3 W, 5 min). When UMR-106 cells were treated with g-C3N4 QDs (0.05 mg/mL) under microwave irradiation (3 W, 5 min), many tumor cells were killed and the viability was reduced to 60%. The cell viability is below 40% for the treatment under 5 W MW irradiation with 0.05 mg/mL of g-C3N4 QDs. UMR-106 cells in each group were stained with Calcein AM for live cells and propidium iodide (PI) for dead cells, and the Live/Dead staining fluorescence images of UMR-106 cells after microwave irradiation at the dose of 5 W are shown in Figure 3 (top). In addition, another two trails on two separate days with the images are shown in Figure S3. The corresponding quantitative analysis in Figure 3 (bottom) clearly confirms that the g-C3N4 QDs can promote the death of tumor cells with the help of microwave activation. Apoptosis plays a vital role in the programed cell death. To elucidate the apoptotic potential of microwave-activated g-C3N4 QDs on UMR-106 cells, we investigated apoptosis by flow

3. RESULTS 3.1. Luminescent Property and Morphology of g-C3N4 QDs. To prepare the g-C3N4 quantum dots, melamine was calcined in air at 500 °C for 2 h to obtain a yellow powder.20,33 As shown in Figure S1, the XRD pattern of the powder presents the typical diffraction peaks of the g-C3N4 at 27.4° and 13.1°. The powder was ground, acid-digested, and then dialyzed to get the quantum dots.20 The HRTEM images shown in Figure S2 demonstrate that the particles size is about 5 nm. The 1838

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering

Figure 3. Top: Fluorescence image of UMR-106 cells (×200). (a) cells only; (b) cells under MW irradiation, 5 W, 5 min; (c) cells cultured with 0.4 mg/mL g-C3N4 QDs under MW irradiation; Bottom: The quantitative analysis of live/dead staining fluorescence (*p < 0.05, **p < 0.01).

to 5 min. The temperature of the g-C3N4 QDs solution rose from 27 to 54 °C, whereas the temperature of the control deionized water has a similar raise curve but a slightly higher temperature rise. These results exclude the possibility that gC3N4 QDs may enhance the microwave heating effects. The other possibility is that g-C3N4 QDs may produce ROS under microwave irradiation, similar to what observed in Cu-Cy reported recently by our teams.12 g-C3N4 QDs are efficient photocatalysts24 and it is highly possible that they can produce ROS under microwave irradiation. We used the p-nitrosodimethyl-aniline (RNO)-imidazole (ID) method35 to detect ROS such as singlet oxygen produced by g-C3N4 QDs when irradiated at different doses of MW. RNO is a water-soluble molecule with absorption that can be quenched irreversibly by singlet oxygen in the presence of ID. By comparing the relative quenching of RNO absorption with and without g-C3N4 QDs under MW irradiation, we have observed singlet oxygen generated by the g-C3N4 QDs with different MW doses and irradiation durations. As shown in Figure 5b−d, in the control of RNO-ID, the MW irradiation did not induce any singlet oxygen to quench the RNO absorption. However, with g-C3N4 QDs, the RNO absorption was quenched continuously as a function of MW power or time, indicating a continuous generation of singlet oxygen. The results showed that the RNO absorption was quenched continuously with the increase in the microwave power or time, which means more singlet oxygen was produced as the microwave irradiation power or time increased. These results demonstrate clearly that microwave interaction with g-C3N4 QDs results in ROS including singlet oxygen for tumor cell destruction, and this lays the foundation

cytometry. PI and Annexin V-FITC were used to stain the UMR-106 cells, and then analyzed by fluorescence-activated cell sorting with the results shown in Figure 4. When the UMR106 cells were treated with only g-C3N4 QDs for 24 h, the percentage of apoptotic cells was almost the same as that of the control group. For the treatment with g-C3N4 QDs in combination with microwave, the cell numbers for apoptosis increased from 2% (without MW) to 6% (with MW, 5 W) (Figure 4 bottom). Furthermore, g-C3N4 QDs can enhance the apoptosis efficiency of microwave ablation from 3 to 6%. As the concentration of g-C3N4 QDs increased, the percentage of UMR-106 cells apoptosis is increased. These results confirm further that only the combination of microwave and g-C3N4 QDs may have a fatal effect on UMR-106 cells. Fluorescence image (Figure S4) and apoptosis (Figure S5) of UMR-106 cells under 3 W power microwave irradiation also demonstrate the similar results with those under 5 W power microwave irradiation, further confirming the integrated effect of g-C3N4 QDs and microwave irradiation on cancer cell. 3.3. Killing Mechanism by the Combination of Microwave and g-C3N4 QDS on UMR-106 Cells. Our observations above show clearly that only the combination of microwave and g-C3N4 QDs may have a fatal effect on UMR106 cells. The questions are why and how the combination of microwave and g-C3N4 QDs can destroy tumor cells. First of all, we know that microwave may produce heat for tumor ablation and the combination may enhance the heating effects to increase the killing efficacy. For that, we first measure the microwave heating with and without the g-C3N4 QDs as shown in Figure 5a for the samples irradiated by a 5 W microwave up 1839

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering

Figure 4. Top: Cell apoptosis assays with an Annexin V-FITC apoptosis detection kit. The UMR-106 cells were cocultured with different concentrations of g-C3N4 QDs for 24 h, then they were under microwave irradiation (5W, 5 min). The percentage of Annexin V (+)7-AAD (−) cells revealed apoptosis. (a) 0 mg/mL g-C3N4 QDs; (b) 0.1 mg/mL g-C3N4 QDs; (c) 0.4 mg/mL g-C3N4 QDs; (d) 0 mg/mL g-C3N4 QDs + MW; (e) 0.1 mg/mLg-C3N4 QDs + MW; (f) 0.4 mg/mL g-C3N4 QDs + MW. Bottom: percent of cells undergoing apoptosis (%) in different groups. (*p < 0.05).

Figure 5. Evaluation of the microwave sensitizing properties of the g-C3N4 QDs. (a) MW heating curves of pure water (control) and g-C3N4 QDs (1 mg/mL) under 5 W microwave irradiation. (b) RNO absorption curves of pure water, g-C3N4 QDs (1 mg/mL) under different powers of microwave irradiation for 5 min. (c) RNO absorption curves with the time under 5W microwave irradiation. (d) RNO absorption curves with the time under 10W microwave irradiation.

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DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering

microscope demonstrated that g-C3N4 QDs entered the cells and emitted fluorescence compared Figure 7a, b. When g-C3N4 QDs get into the tumor cells, under microwave irradiation, they may produce ROS to damage the cells. The ROS generated within the cells can be measured with the DCFH-DA method because ROS can oxidize DCFH2 to form DCF with a green fluorescence.37,38 The UMR-106 cells were incubated with g-C3N4 QDs at concentrations of 0 mg/ mL (a, d), 0.1 mg/mL (b, e), 0.4 mg/mL (c, f) for 24 h and exposed to MW (d−f), then the processed UMR-106 cells were observed by the fluorescence microscope. Figure 8 (top) shows that adding g-C3N4 QDs alone led to a little amount of ROS in the cells, and the cells produce very weak green DCF fluorescence. No evident fluorescence was observed in the control or the UMR-106 cells activated with MW alone (a, d), which means almost no ROS were produced in these cells. However, for the UMR-106 cells treated at concentrations of 0.4 mg/mL g-C3N4 QDs under MW irradiation, they show a strong green fluorescence (f), indicating the production of ROS within the cells. Even for the UMR-106 cells treated at a low concentration of g-C3N4 QDs (0.1 mg/mL) under MW, a trace of green fluorescence was also observed, proving the formation of ROS in the cells. The images of another two separate trails are shown in Figure S6. The corresponding quantitative analysis was shown in Figure 8 (bottom), which clearly demonstrates that integrated g-C3N4 QDs and microwave improves the production of ROS. All these observations strongly support that microwave may activate g-C3N4 QDs to produce ROS for cancer cell destruction, which is defined as gC3N4 QDs based microwave induced photodynamic therapy. 3.4. Biocompatibility of g-C3N4 QDs. For cancer treatment by MIPDT, ideally, the nanoparticles should have low to zero dark-toxicity but very high toxicity when induced by microwaves. Because microwaves can be applied locally, using g-C3N4 QDs as photosensitizers can significantly reduce the risks of damage to the surrounding healthy tissue. To illustrate the biocompatibility of g-C3N4 QDs, the hemolysis rates of gC3N4 QDs solutions (0.2, 0.4, 0.6, 0.8, 1 mg/mL) were evaluated using rabbit red blood cells (RBCs) with phosphatebuffered saline (PBS) and deionized water as negative and positive controls, respectively. Figure 9a shows that all hemolysis rates at different concentrations of g-C3N4 QDs are below 5%, indicating that the g-C3N4 QDs have good biocompatibility according to ISO 10993−4:2002. It is found that the g-C3N4 QDs have no destructive effect on erythrocyte membrane. In addition, the potential toxicity of g-C3N4 QDs was also tested with UMR106 cells via the standard 3-(4,5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay. The MTT assay was used to assess the cell proliferation of UMR-106 cells incubated with different concentrations of g-C3N4 QDs (0.125−1.0 mg/mL) for 1, 3, 5 days. Three trails on the three separate days were conducted and the detailed results are as shown in Figure S7. The results of MTT are repeatable from these three trails. The average values of these three independent experiments are shown in Figure 9b. When the cells were cultured with g-C3N4 QDs at concentrations less than 0.4 mg/mL for 24 h, the cell viability is more than 80%. The results indicate that g-C3N4 QDs have low cytotoxicity and are potentially suitable for medical applications

for g-C3N4 QD-based microwave-induced photodynamic therapy. In Figure 5a, the temperature of pure water is higher than 1 mg/mL (only ∼0.1%) QD solution and the reason is as follows. The interaction of microwave with molecules has two effects−the heating effects and the nonheating effects. For the g-C3N4 QD solution, the microwave is used for heating as well as for ROS production, whereas for water, the microwave is mainly for heating. As the QDs need a lot of energy for ROS production, so the heating effect in the QD solution is not so effective as the heating effect in pure water. Cytotoxic intracellular ROS such as singlet oxygen can damage DNA, mitochondria, and plasma membranes of live cells, resulting in cell death.36 g-C3N4 QDs may enter the cells and produce ROS to damage the mitochondria of cancer cells, leading to apoptosis. To ensure whether g-C3N4 QDs entered the cells or not, we observed the UMR-106 cells after incubation with g-C3N4 QDs for 24 h by transmission electron microscope (TEM) as shown in Figure 6. The TEM image

Figure 6. TEM images of g-C3N4 QDs in UMR-106 cells: (a) control; (b) magnification of a; (c) 0.4 mg/mL g-C3N4 QDs; (d) magnification of c.

obviously shows the presence of some aggregated g-C3N4 particles inside the UMR-106 cells. As shown in Figure 7, the UMR-106 cells were incubated with g-C 3 N 4 QDs at concentrations of 0.4 mg/mL for 24 h, and then the processed UMR-106 cells were observed by the fluorescence microscope. The result of the internalization assay by fluorescence

Figure 7. Fluorescence microscope images of g-C3N4 QDs in UMR106 cells (×400). (a) White light imaging of 0.4 mg/mL g-C3N4 QDs. (b) Fluorescence imaging of 0.4 mg/mL g-C3N4 QDs. 1841

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering

Figure 8. Top: Fluorescence image of singlet oxygen in UMR-106 cells, the microwave irradiation is at 5 W for 5 min. Bottom: The quantitative analysis of intracellular ROS (*p < 0.05).

visible light directly and near-infrared (NIR) light indirectly for cancer treatment.20,22,31 Numerous studies have shown that gC3N4 QDs can effectively absorb UV and visible lights to produce free electrons, holes and generate ROS in the environment. g-C3N4 can enter into the cells to produce ROS and damage the mitochondria of cancer cells, leading to cancer cell apoptosis. ROS molecules have unpaired electrons that make them highly reactive when formed in vivo via oxidation−reduction reactions and include free radicals such as hydroxy (HO•) and superoxide radicals (•O2-), and nonradicals including hydrogen peroxide (H2O2) and singlet oxygen (1O2). Therefore, g-C3N4 can work as an agent for ROS-based therapy for cancer treatment. Here our observation that microwave can produce ROS in g-C3N4 will shed a new light on its applications for treating cancers and other defection diseases. Because microwave energy is too low to excite g-C3N4 QDs for ROS production, there must be some other mechanisms for the gC3N4 QD-mediated microwave-induced ROS production. It is well-known that microwave irradiation raises the temperature by stirring up the motion of charged particles and rotation of water molecules.19 As shown in Figure 5, the temperatures in g-C3N4 QD aqueous solutions were even lower than in DI water upon microwave irradiation. This indicates that heating is not the major effect to destroy the cancer cells as observed here. ROS generation by microwave is a common phenomenon.39,40 900 MHz mobile phone radiation can induce ROS formation,41 and a low level of 2.45 GHz microwave irradiation induced oxidative stress not only suppresses implantation but also lead to deformity of the embryo.39 As described above, gC3N4 QDs determine the ROS generation. Without g-C3N4 QDs, almost no ROS or very low concentration of ROS was generated by microwave. This indicated that g-C3N4 QDs catalyze the production of ROS. It is well-known that mitochondria is a major source of ROS by constantly

Figure 9. Biocompatibility of g-C3N4 QDs. (a) Hemolysis rate of red blood cells incubated with g-C3N4 QDs at various concentrations, using deionized water (+) and PBS (−) as positive and negative control, respectively. Inset: Photographs of hemolysis test with different concentrations of g-C3N4 QDs. (b) Cell relative growth rate after culturing with g-C3N4 QDs for 1, 3, 5 days. The error bars represent the standard deviations calculated from three independent experiments.

4. DISCUSSION Graphitic-phase carbon nitride (g-C3N4) quantum dots(QDs) is an excellent photosensitizer that can be activated by UV, 1842

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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ACS Biomaterials Science & Engineering Author Contributions

metabolizing oxygen. One electron transfers to oxygen to form • O2−, which then converts to longer-lasting and membranediffusible H2O2. Peroxidases catalyze reactions involving hydrogen peroxide, resulting in the generation of hypochlorous acid (HOCl) and 1O2, among other species. As a catalyst, gC3N4 QDs may reduce the activation energies required for these reactions and microwave also provides energies for these processes. This is similar to ROS production by microwave activated carbon nanoparticles.42 The released intracellular ROS can cause damage to DNA, mitochondria, and plasma membranes of live cells, resulting in cell death. As one means of tumor ablation, microwave ablation is currently used in the treatment of invasive, malignant bone tumors and metastatic bone tumors by fast heating the tumors. It has some challenges, such as achieving a balance between accurate targeting and minimizing damage to surrounding vital organs and structure. To avoid injury to adjacent organs, the junction sites between lesions and normal tissues must not experience too high a temperature or too long ablation time. Our work showed that g-C3N4 QDs at low power of MW irradiation did not increase the temperature but the combination of g-C3N4 QDs and microwave is very effective for tumor destruction. The cell viability, live/dead and flow cytometry tests endorse that g-C3N4 QDs can enhance the efficiency of microwave ablation to kill the tumor cells. Moreover, because g-C3N4 consists of only C and N, its biological toxicity is very low and it can be easily metabolized in organisms.43 Our in vitro cells cytotoxicity assay and hemolysis tests show that the g-C3N4 QDs has good biocompatibility. All these indicate that g-C3N4 QD-based microwave induced photodynamic therapy is potentially a powerful weapon for cancer treatment.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We appreciate the financial support from the National Natural Science Foundation of China (Grant 81501859, 21620102004, 51472013), Natural Science Foundation of Guangdong Province, China (Grant 2015A030312004), the Science and Technology Planning Project of Guangdong Province (Grant 201604020110), The National Key Research and Development Program of China (2016YFB0700800). We also acknowledge the support from the U.S. Army Medical Research Acquisition Activity (USAMRAA) under Contracts of W81XWH-10-10279 and W81XWH-10-1-0234.

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5. CONCLUSIONS In summary, g-C3N4 QD-based microwave induced photodynamic therapy is reported for the first time. Our observations show that g-C3N4 QDs have good biocompatibility from MTT and hemolysis studies. The addition of g-C3N4 QDs did not increase the microwave heating rate but the combination of gC3N4 QDs and microwave is very effective for tumor destruction because microwave can activate g-C3N4 QDs to produce ROS/singlet oxygen for cancer destruction. The preliminary studies endorse MIPDT as a new modality for cancer treatment and open a new area for g-C3N4 applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00110. XRD pattern of g-C3N4 powder, HRTEM image of the gC3N4 QDs, fluorescence image and apoptosis of UMR106 cells, and cell relative growth rate (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.Y.Z.). *E-mail: [email protected] (W.C.). *E-mail: [email protected] (Y.Z.). ORCID

Junying Zhang: 0000-0002-4860-8774 1843

DOI: 10.1021/acsbiomaterials.7b00110 ACS Biomater. Sci. Eng. 2017, 3, 1836−1844

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