Engineered g-C3N4 Quantum Dots for Tunable Two-Photon Imaging

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Engineered g-C3N4 Quantum Dots for Tunable Two-Photon Imaging and Photodynamic Therapy Xiaoxia Wu, Lingyan Yang, Liang Luo, Guohua Shi, Xunbin Wei, and Fu Wang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00055 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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ACS Applied Bio Materials

Engineered g-C3N4 Quantum Dots for Tunable Two-Photon Imaging and Photodynamic Therapy

Xiaoxia Wu1, 2, Lingyan Yang2, Liang Luo2, Guohua Shi*,3, Xunbin Wei*,4, and Fu Wang*,1

1School

of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai

200240, China 2Laboratory

of Environmental Sciences and Technology, Xinjiang Technical Institute of Physics &

Chemistry, Chinese Academy of Sciences, Urumqi, Xinjiang 830011, China 3Jiangsu

Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and

Technology, Chinese Academy of Sciences, Suzhou 215263, China 4Med-X

Research Institue and School of Biomedical Engineering, Shanghai Jiao Tong University,

Shanghai 200030, China

*Corresponding Authors: Guohua Shi (E-mail: [email protected]); Xunbin Wei (E-mail: [email protected]) and Fu Wang (E-mail: [email protected])

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ABSTRACT Near infrared (NIR) light triggered diagnostic and therapeutic systems normally are the integration of several components, whose complex structures and low reproducibility restrict their further applications. Herein, we proposed a single component of defect graphitic-phase carbon nitride quantum dots (g-C3N4 QDs) as dual-functional nanoplatform, which could synchronously achieve two-photon imaging (TPI) and two-photon excited photodynamic therapy (TPE-PDT) under 800-nm NIR laser. In order to regulate the competitive capability between TPI and PDT, three kinds of engineered g-C3N4 QDs (CN, CN-DPT and CN-THDT QDs) with different two-photon catalytic capabilities to generate reactive oxygen species (ROS), were prepared through copolymerization melamine with selected monomers. In the evaluation of in vitro cytotoxicity, three g-C3N4 QDs possessed very good biocompatibilities which could be delivered to tumor cells for imaging and therapy. Under 800-nm NIR light, CN-DPT QDs exhibited bright TPI effect and produced efficient ROS to achieve TPE-PDT, thus suggesting the single component of CN-DPT QDs could perform as an appropriate dual-functional TPI and TPE-PDT probe triggered by 800-nm NIR light. The engineered g-C3N4 QDs with high stability and excellent biocompatibility have contributed to the method of TPI and TPE-PDT, which may lead to g-C3N4 based nanomaterials as novel imaging and therapeutic agents in cancer treatment.

KEYWORDS: graphitic-phase carbon nitride quantum dots (g-C3N4 QDs), defects, near infrared (NIR) light, two-photon imaging (TPI), two-photon excited photodynamic therapy (TPE-PDT)

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1. INTRODUCTION Over the past several decades, engineered multifunctional delivery systems have been implemented for cancer therapy and made remarkable progresses to treat cancer more effectively.1-4 Among numerous diagnosis and therapeutic systems, near infrared (NIR) light triggered fluorescence imaging and photodynamic therapy (PDT) have attracted widespread attentions due to deep tissue penetration, minor auto-fluorescence background, low photo-bleaching, and no damage to normal tissue.5-8 In 2017, Xu et al. reported a nanoplatform of IR-808 dye sensitized upconversion composites with chlorin e6 (Ce6) for PDT and NIR light imaging.9 However, the complicated multifunctional nanoplatform, which has integrated a number of separated components, need skilled and timeconsuming procedure to prepare.10-14 Therefore, developing a multifunctional system with single component and straightforward method is of sovereign importance.15 Gu et al. recently utilized an organic photosensitizer of aggregation-induced fluorescence emission for two-photon imaging (TPI) and PDT in cancer cells.16 Under the NIR light source, effective TPI and PDT were achieved with single organic photosensitizer. According to the relaxation routes of excited electron, the reactive oxygen species (ROS) generation for PDT and two-photon fluorescence for imaging are two competitive processes.17 Therefore, the generation of TPI and PDT from the organic photosensitizer, which originated from two-photon absorption of its intrinsic molecular structure, was difficult to be modified to adjust TPI and PDT abilities.18 Moreover, the organic photosensitizer had low stability and biocompatibility that limited the applications in treatment.19 In order to achieve the balanced imaging and therapeutic capabilities for cancers, it is highly desired to propose a stable and single component system with tunable PDT and TPI abilities. The graphitic-phase carbon nitride quantum dots (g-C3N4 QDs), a semiconductor nanomaterial, has been presented as an inorganic agent of TPI or PDT because of high stability, excellent 3



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biocompatibility, and especially extremely low cost compared with organic photosensitizer.20-22 However, the generation of ROS for g-C3N4 normally needs UV or blue light, which might result in unexpected phototoxicity and low tissue penetration depth.23 Inspired by the mechanism of TPI, gC3N4 QDs also can simultaneously absorb two photons to achieve charge-transfer transition and generate ROS to achieve two-photon excited PDT (TPE-PDT) with NIR light.24,25 Most importantly, it could adjust the photon-generated ROS through producing active sites in the framework of g-C3N4, thus give the opportunity to regulate the competitive relationship of TPI and PDT, implying that the g-C3N4 QDs could act as two-photon photosensitizer and fluorescent probe for tunable deep tumor TPE-PDT and TPI. According to previous reports of introducing active sites of disordered structure or defects to pristine g-C3N4 by simply copolymerizing melamine (MA) with other monomers,26 we prepared three kinds of g-C3N4 QDs as dual-functional nanoplatforms for two-photon guided TPI and TPE-PDT. Those g-C3N4 QDs include pure g-C3N4 QDs (CN QDs), g-C3N4 QDs modified by monomer with phenyl ending (CN-DPT QDs) and g-C3N4 QDs modified by monomer with rich nitrogen element (CN-THDT QDs). All samples were synthesized through a common calcination approach and a subsequent simple quenching step. The as-obtained nanoplatforms preserved similar size, simple structure, diverse defects, high repeatability and excellent stability. Interestingly, the capability of three g-C3N4 QDs for TPI and PDT could be regulated under 800-nm NIR light. In comparison, CNTHDT QDs possessed better ability for TPI and produced adequate ROS to interfere mitochondrial function for TPE-PDT. This g-C3N4 nanomaterials provided an alternative way to DPT QDs system as a dual functional nanoplatform, which performed great TPE-PDT and effective TPI induced by 800-nm NIR light, suggesting the single component of CN-DPT QDs could be an alternative choice for dual-functional TPI and TPE-PDT triggered by NIR light. 4


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2. EXPERIMENTAL SECTION 2.1. Materials. Melamine (MA), sodium hydroxide (NaOH) and cyanuric chloride (CC) were purchased from Tianjin Chemical Regent Co., Ltd. (Tianjin, China). Dimethyl sulfoxide (DMSO) and 4-diamino-6-phenyl-1,3,5-triazine (DPT) were purchased from Macklin Scientific Co., Ltd. (Shanghai, China). 2,7-dichlorofluorescein-diacetate (DCFH-DA) was purchased from SigmaAldrich. 2.2. Preparation of CN, CN-DPT and CN-THDT QDs. The CN-DPT QDs were synthesized by copolymerization of melamine with DPT.26 Experimentally, 3.0 g of MA and 0.3 g of DPT were grinded in an agate mortar for 20 min, then transferred to ceramic crucible and calcined at 520 °C for 2 h in muffle furnace. Before temperature decreased from 520 °C, the resulted CN-DPT was rapidly added to 30 mL pure water and disrupted by ultrasonic for 10 min. Then the obtained CN-DPT QDs were centrifuged (5000 rpm, 10 min) and collected supernatant for further use. As a comparison, the CN QDs was prepared by direct treatment of MA (3.0 g) under the same conditions. CN-THDT QDs were synthetized by two steps. Firstly, the monomer of 2,4,6-trihydrazino-1,3,5triazine (THDT) was synthetized by a modified method.27 5 g of CC was mixed with 10 mL of ethanol in a three flask. Then 40 mL of 80% hydrazine was added by drop by drop with a constant temperature oil bath of 130 ℃ and refluxed for 3 h. After cooling down to room temperature, the products were collected by centrifugation (3000 rpm, 10 min) and dried at 60 ℃. Secondly, 3.0 g THDT was weighed to prepare CN-THDT QDs by a similar process of calcination and quenching under the same conditions of CN-DPT QDs. 2.3. Characterizations. Transmission electron microscope (TEM) images were analysed by Tecnai G20 (FEI, USA). Particle size and zeta potential were obtained by dynamic light scattering (DLS, Nano-ZS, Malvern, UK). X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), 5



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electron paramagnetic resonance (EPR) and Fourier transform infrared spectra (FTIR) were analysed according to previous reports.26 Two-photon spectra and imaging were taken by two-photon imaging system constructed by our group.28,29 2.4. Extracellular and intracellular ROS detection. To evaluate extracellular ROS generation of three g-C3N4 QDs, DCFH-DA was utilized as a fluorescence probe to detect the generated ROS.22,30 In a typical procedure, DCFH-DA in methanol (0.5 mL, 1 mM) was mixed with NaOH (2 mL, 0.01 M) and kept stirring rigorously for 30 min in dark at room temperature to produce DCFH. Then 10 mL of PBS (pH = 7.4) was added to adjust the pH of above solution till neutral. Then 1 mL of asprepared DCFH solution was mixed with 1 mL of g-C3N4 QDs solutions (20 μg/mL) and transferred into a quartz cell. The mixing solution was irradiated by 800-nm NIR femtosecond laser (MAI TAI, Spectra-Physics, USA) for different time (1.0 W/cm2, 0–20 min) in dark. The photoluminescence spectra of the dispersions were measured every 2 min, and DCFH solution without g-C3N4 QDs was used as a control. For intracellular ROS detection of three g-C3N4 QDs under 800-nm NIR laser, human breast cancer cell line (MCF-7) cells were studied as cell model and cultured at 37 ℃ in 5% of CO2. MCF cells were incubated with three g-C3N4 QDs (1 ug/mL) for 4 h, washed away the free g-C3N4 QDs by PBS, and culture media containing DCFH-DA (20 μM) was placed and incubated for another 30 min in dark. The cells were washed three times with PBS to remove the free DCFH-DA. After irradiated with 800-nm lights for different times (1.0 W/cm2, 0–15 min), fluorescence images of g-C3N4 QDs and DCF were collected using a total internal reflection fluorescence microscopy (TIRFM, Nikon Eclipse Ti, Japan). 2.5. In vitro cytotoxicity of three g-C3N4 QDs. MTT assays were used to evaluate the cytotoxicity in vitro. The details can be found in our previous work,31 and the concentration of three g-C3N4 QDs 6


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were 0–80 μg/mL in culture medium. Three kinds of g-C3N4 QDs were nanomaterials without exact molar mass, so we used the quality instead to do the following experiments. 2.6. Two-photon and one-photon fluorescence imaging. For TPI, 2 mL of MCF-7 cells (1×105 cells/mL) were seeded in a culture dish for 24 h. Then, 2 mL of fresh culture containing three kinds of g-C3N4 QDs (1 μg/mL) were added and cultured for another 4 h. The MCF-7 cells were washed with PBS. Three kinds of g-C3N4 QDs were excited by 800-nm NIR laser, and the two-photon fluorescence images were collected using a TPI system constructed by our group. Similar method was used to study the one-photon fluorescence imaging. In additionally, the MCF cells were treated with propidium iodide (PI, 5 μg/mL) at room temperature to stain the nucleus. The fluorescence images of PI and three kinds of g-C3N4 QDs were obtained using a TIRFM. 2.7. In vitro TPE-PDT. In vitro TPE-PDT of g-C3N4 QDs for MCF-7 cells was evaluated by cell viability assay,32 and the details can be found in our previous work.31 The concentration of three gC3N4 QDs were 0–50 μg/mL, and 800-nm NIR laser (1.0 W/cm2, 20 min) was used for TPE-PDT. The absorbance was recorded to calculate the cell viability. The calculation formula is as follows, which ODNo refers to absorbance without the irradiation of 800-nm laser, and ODX means the absorbance of MCF-7 cells incubated with CN, CN-DPT or CN-THDT QDs under the 800-nm laser in different concentration of 0–50 μg/mL. Cell viability = 𝑂𝐷𝑋 𝑂𝐷𝑁𝑜

3. RESULTS AND DISCUSSION The preparation and application of dual functional nanoplatform guided by 800-nm pulsed laser for cancer TPE-PDT and TPI were illustrated in Scheme 1. Specifically, the modified g-C3N4 were prepared by calcination at 520 °C to form graphene-like structures.21 The g-C3N4 QDs were produced by immediate ultrasonic inspired from the metal quenching when the C-N bonds were instable and 7



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easy to be breached at high temperature.33 Under the excitation of two photons, the resulted g-C3N4 QDs could emit bright fluorescence and catalyze the generation of ROS simultaneously, indicating both processes were well preserved. To further regulate the capabilities for PDT and TPI, we then prepared three kinds of g-C3N4 QDs with different active sites by the copolymerization of MA with other monomers of DPT and THDT. The incorporation of precursors (DPT and THDT) increased the disordered structure and lattice defects in the framework of g-C3N4, which can generate the defect energy level between the conduction band and valence band of semiconductor materials to reduce the recombination of photogenerated carriers and improve the ability of ROS generation.26 The possible ROS formed in the reaction include hydroxyl radicals (·OH), superoxide radicals (O2-), hydrogen peroxide (H2O2) and other ROS according to previous reports.34,35 Therefore, the increase of defects can effectively improve the effect of PDT. Subsequently, g-C3N4 QDs with different abilities to generate ROS and fluorescence were applied for cancer therapy and imaging. 3.1. Characterization of the dual-functional nanoplatform. Figure 1a-d present TEM images of CN QDs (a, b), CN-DPT QDs (c) and CN-THDT QDs (d). The g-C3N4 QDs were ~30 nm in size, but are slight agglomeration in TEM images of Figure S1 (Supporting Information). The HRTEM images of three g-C3N4 QDs are shown in Figure 1b and Figure S2, the clear lattice stripes with the interplanar distance are 0.32, 0.34 and 0.33 nm for CN, CN-DPT and CN-THDT QDs respectively, which are apparently graphene like.36 The size distribution and zeta potential of g-C3N4 QDs dispersed in water are shown in Figure 1e-f. The average size of CN, CN-DPT or CN-THDT QDs are 246 nm, 213 nm and 133 nm respectively, which are larger than particle sizes in TEM results, attributing to the agglomeration of g-C3N4 QDs in aqueous dispersion. The storage stabilities of three kinds of engineered g-C3N4 QDs were measured by DLS and shown in Table S1 and Table S2. It can be seen that the sizes of three g-C3N4 QDs increased with the increase of storage time, indicating that 8


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agglomeration occurred in the water for three kinds of g-C3N4 QDs. Because they were synthetized via the top-down method which were easy to agglomerate into the larger particles in high concentration. Nevertheless, three g-C3N4 QDs have different size distributions, their sizes are all about 200 nm which could passively target to tumor cells by enhanced permeability and retention effect.37 Furthermore, the zeta potential of three g-C3N4 QDs had no significant changes after three days from Table S2. From the Figure 1f, it could be found that the zeta potential of three g-C3N4 QDs are slightly lower than their corresponding compounds. Because C-N bonds are instable and easy to be breached with immediate ultrasonic at high temperature in the preparation of three g-C3N4 QDs, so that tertiary amine (R3-N) in frameworks become secondary amine (R2-NH) and primary amine (R-NH2). More R2-NH and R-NH2 groups resulted in the increase of zeta potential for three g-C3N4 QDs.31 The crystal structures of prepared g-C3N4 QDs were further characterized by XRD analysis. As shown in Figure 2a, the dominant peaks at 27.4° and 13.0° are the characteristic peaks of g-C3N4,26 indicating that the ordered lattice structure is formed by calcination. Compared to CN QDs, the diffraction peaks of CN-DPT QDs and CN-THDT QDs are slightly shifted to low angle, which might result from the incorporation of functional monomers and following generation of disorders in the layer structure.38 From the XRD spectra, the interplanar distance at 27.6° is 0.32 nm corresponding to (0 0 2) plane, which is consistent with the HRTEM result for three g-C3N4 QDs.39 The SAED pattern of CN, CN-DPT and CN-THDT QDs are shown in Figure S3 (Support Information). The diffraction ring of CN QDs is clearly visible, and the interplanar distance is calculated to be 0.312 nm corresponding to the lattice plane of (0 0 2) from the XRD results. Similar to CN QDs, the interplanar distances of CN-DPT and CN-THDT QDs are 0.330 nm and 0.329 nm, respectively, will matched with the lattice plane of (0 0 2) from the XRD results.40,41 9



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Supported by FTIR analysis in Supporting Information (Figure S4), there are no notable changes in functional groups and structures after quenching, suggesting that those g-C3N4 QDs possess similar structure as g-C3N4. Chemical composition and state of g-C3N4 QDs were also analyzed by XPS in Figure S5, confirming that incorporation with DPT and THDT could change the chemical composition and state of g-C3N4, and consequently change the catalytic performance. Figure 2b exhibits the UV-vis spectra of g-C3N4 QDs. The wide absorption peaks are the characteristic peaks of g-C3N4.20 The UV-Vis spectra are the absorption of three g-C3N4 QDs under one-photon lights which does not mean that the absorption at 800 nm are low under 800-nm femtosecond laser in Figure 2b. The two-photon absorption (TPA) cross section is an important factor for evaluation of TPI and TPE-PDT. According to previous reports, g-C3N4 QDs have high TPA cross section at 800 nm.38For the evaluation of photothermal conversion of three g-C3N4 QDs, all temperatures of three g-C3N4 QDs aqueous dispersion (0.2 mg/mL) increase to 35.9, 36.7 and 37.6 ℃ from room temperature after 6 min irradiation of 800-nm laser (1 W/cm2) which are similar with control group of pure water (35.4 ℃), indicating that three g-C3N4 QDs have low hurt from heat for cells. One-photon and two-photon fluorescence spectra of three g-C3N4 QDs under the excitation of 400 nm and 800 nm are shown in Figure 2c-d. The emission peaks of CN QDs and CN-DPT QDs are at 433 nm and 435 nm under excitation wavelength of 400 nm, the emission peak shifts to 450 nm under 800 nm excitation, which is similar with previous report.42 The one photon and two photon fluorescence of CN-THDT QDs are different with other two g-C3N4 QDs (CN QDs and CN-DPT QDs) due to the structure difference in framework.26 In order to estimate the abilities of three g-C3N4 QDs for PDT, the generated ROS under 800-nm NIR femtosecond laser are exhibited in Figure 2e. DCFH-DA was used as a fluorescence probe for ROS detection.22 Under the NIR irradiation, the amounts of generated ROS for three kinds of g-C3N4 10


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QDs increase with the irradiation time, suggesting that all g-C3N4 QDs possess efficient carrier generation activities triggered by two-photon in contrast to the one-photon excitation.23 The observed distinct ROS generation abilities for g-C3N4 QDs might be due to the different defects and disordered structures in the framework.26 Therefore, the defects of the synthesized g-C3N4 were characterized by EPR. As shown in Figure 2f, the EPR signal indicates the unpaired electron of π-conjugated aromatic rings trapped on defects, while the intensity of signal increase significantly, indicating that the ability of trapping unpaired electron on defects to produce photo-generated carriers was stronger.26 The unpaired electron influence the photocatalytic ability of semiconductors, indicating that g-C3N4 QDs with higher EPR signal resulting in higher ability of ROS formation.33 Obviously, CN-THDT QDs showed the strongest catalytic activity and lowest fluorescence synchronously,43 which are consistent with the results of ROS generation and fluorescence emission spectra guided by two-photon excitation. Furthermore, the intracellular ROS generation was also investigated in MCF-7 cells incubated with CN, CN-DPT and CN-THDT QDs (1 μg/mL) as shown in Figure S6. With the increase of irradiation time, the fluorescence intensity of DCF increase, especially for CN-DPT and CN-THDT QDs, indicating the generated ROS rose significantly. The performances of high amount of generated ROS for CN-DPT and CN-THDT QDs further confirm that the increase of defects in framework of g-C3N4 could improve the PDT in cancer cells. Therefore, the dual-functional system of g-C3N4 QDs could realize high effective TPE-PDT with the increase in active sites under the irradiation by 800-nm NIR laser. 3.2. In vitro cytotoxicity of three g-C3N4 QDs. The biocompatibility is an indispensable factor for all nanoplatforms in delivery system.44 The cytotoxicity of three kinds of g-C3N4 QDs was evaluated via the MTT assay shown in Figure 3.45 The viability of MCF-7 cells is about 100% for all g-C3N4 QDs in the concentration of 10–80 μg/mL, indicating that the low cytotoxicity of the as11



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prepared all g-C3N4 QDs. The slightly better biocompatibilities of CN-THDT and CN-DPT QDs than CN QDs resulted from the modification of g-C3N4 and the smaller size distributions of CN-THDT and CN-DPT QDs.46 From results of MTT assay, all g-C3N4 QDs possess very good biocompatibilities and could be delivered to tumor cells for imaging and therapy. 3.3. In vitro TPI of g-C3N4 QDs. Fluorescence imaging is a powerful method in biology, while two-photon fluorescence technology with a NIR laser offers much deeper penetration and lower background signal compared to visible light excitation.47,48 As previously reported, g-C3N4 QDs are more stable and biocompatible than organic dyes and other semiconductor quantum dots, which have great potential capability in cancer imaging.21,42 To investigate the TPI performance of three probes, MCF-7 cells were incubated with CN, CNDPT and CN-THDT QDs. As shown in Figure 4, the fluorescence signals were collected in the range of 400–500 nm under the excitation of 800-nm NIR laser. CN-THDT QDs possess very low twophoton fluorescence in the same concentration from Figure 2c-d, so that the collected emission wavelengths of 400–500 nm has no significant influence in the imaging of CN-THDT QDs. All gC3N4 QDs have apparent TPI abilities for MCF-7 cells compared with the control in the absence of probes. Among three g-C3N4 QDs, the two-photon fluorescence intensity of CN-DPT QDs is the highest, while the signal of CN-THDT QDs is lowest. This was probably because the higher catalytic activity of CN-THDT QDs whose two-photon fluorescence is lower as the conservation of energy under the same excitation energy.26 In contrast to TPI, TIRFM was used to collect one-photon fluorescence images of three g-C3N4 QDs (Figure 5). All g-C3N4 QDs emit blue light under the excitation of 405 nm. Nevertheless, the background of images is very obvious due to the autofluorescence of cells under the excitation of blue light.49 3.4. In vitro TPE-PDT of three g-C3N4 QDs. From the assessment of catalytic performance for g12


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C3N4 QDs, they all have high efficiency to generate ROS, which could interfere mitochondrial function and trigger cell apoptosis and necrosis.50,51 Moreover, upon 800-nm NIR laser irradiation, it could achieve noninvasive therapy and low side effects in deep tissues for TPE-PDT.52 Therefore, we evaluated the performance of g-C3N4 QDs for PDT in Figure 6. Under 800-nm laser (1.0 W/cm2, 20 min), MCF-7 cells incubated with g-C3N4 QDs showed fairly low viability of 37.2%, 32.3% and 20.6%, respectively. Compared with the same conditions without laser, the viability of the MCF-7 cells incubated with g-C3N4 QDs all are about 100%, indicating the very low cytotoxicity of g-C3N4 QDs without 800-nm light. Compared with groups of g-C3N4 QDs with or without 800-nm irradiation, the differences are statistically significant (**P = 0.001). Among those g-C3N4 QDs, CNTHDT QDs have the best effect of PDT due to more ROS generation guided by two-photon, while the PDT effects of CN-DPT and CN QDs are weaker, which is consistent with previous results of high ROS generation in corresponding g-C3N4 QDs.42 The above findings indicate that the TPE-PDT of g-C3N4 QDs, especially for CN-THDT QDs, have significantly low cytotoxicity and high therapeutic efficacy against cancer cells in contrast to other therapeutic system,53 with the TPI effect of CN-THDT QDs being the lowest from above TPI results in MCF-7 cells imaging. According to the requirement of imaging and therapeutic system, the nanoplantform should simultaneously possess efficient TPI and PDT abilities.27 Therefore, for the as-prepared dual-functional system of g-C3N4 QDs, CN-DPT QDs exhibit the best ability of TPI and a better PDT effect, which could successfully achieve its TPI and TPE-PDT effect for deep tumor cells simultaneously.

4. CONCLUSIONS We have developed the dual-functional TPI and TPE-PDT nanoplatform based on single g-C3N4 component guided by 800-nm NIR light. In order to balance the catalytic activity and TPI ability of g-C3N4 QDs, we copolymerized MA with other monomers to make different disordered structure and 13



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defects in framework, which will induce different photocatalytic abilities. The prepared g-C3N4 QDs of CN, CN-DPT and CN-THDT QDs with different ROS generation capabilities can interfere mitochondrial function to achieve TPE-PDT, consequently exhibit different TPI effects for cancer cells under 800-nm NIR laser. In order to achieve effective TPI and PDT abilities at the same time, the single CN-DPT QDs. which present balanced abilities for TPI and TPE-PDT, demonstrate efficient TPI and TPE-PDT effects under 800-nm NIR light, providing a new avenue to fabricate infrared light stimulated imaging and therapeutic probe based on single photocatalyst dots.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available. Details of the characterization of TEM, HRTEM, SAED, FT-IR spectra, XPS spectra and intracellular ROS detection. AUTHOR INFORMATION Corresponding Authors Fu Wang: E-mail, [email protected] Xunbin Wei: E-mail, [email protected] Guohua Shi: E-mail, [email protected] ORCID Fu Wang: 0000-0001-7423-078X Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by National Key Research and Development Program of China, (Grant No. 2017YFC0110202) and National Natural Science Foundation of China (Grant No. 21503271).

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Inorganic Photosensitizer Coupled Gd-Based Upconversion Luminescent Nanocomposites for in Vivo Magnetic Resonance Imaging and Near-Infrared-Responsive Photodynamic Therapy in Cancers. Biomaterials 2015, 44, 82-90.

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FIGURES AND FIGURE LEGENDS Scheme 1. The illustration of the preparation of three g-C3N4 QDs as dual-functional nanoplatforms for TPI and TPE-PDT in breast cancer.

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Figure 1. Characterization of three g-C3N4 QDs. (a, b) TEM images of CN QDs (a) and the high resolution images of CN QDs (b); (c, d) TEM images of CN-DPT QDs and CN-THDT QDs; (e, f) Size distributions and zeta potential of the CN, CN-DPT and CN-THDT QDs in pure water.

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Figure 2. Physicochemical performance characterization of the as-prepared nanoparticles of the dual functional nanoplatforms. (a) XRD patterns of CN, CN-DPT and CN-THDT QDs; (b) UV-Vis spectra of the CN, CN-DPT and CN-THDT QDs dispersed in pure water; (c) The emission spectra of CN, CN-DPT and CN-THDT QDs which the excitation wavelength is at 400±2.5 nm; (d) The two-photon emission spectra of CN, CN-DPT and CN-THDT QDs with the excitation wavelength at 800 nm; (e) The ROS detection of CN, CN-DPT and CN-THDT QDs by fluorescent probe of DCFH-DA under NIR irradiation (800 nm, 1.0 W/ cm2) for different time in PBS buffer; (f) EPR spectra of the CN, CN-DPT and CN-THDT QDs.

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Figure 3. In vitro biocompatibility of CN, CN-DPT and CN-THDT QDs incubated with MCF-7 cells.

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Figure 4. Two-photon fluorescence images of MCF-7 incubated with CN, CN-DPT and CN-THDT QDs (1 μg/mL) for 4 h. MCF-7 cells without three g-C3N4 QDs were used as a control. The g-C3N4 QDs was excited at the wavelength of 800 nm, and the two-photon fluorescence images were recorded at emission wavelengths of 400–500 nm. Scale bar: 20 µm.

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Figure 5. Total internal reflection fluorescence microscopy images of MCF-7 incubated with CN, CN-DPT and CN-THDT QDs (1 μg/mL) for 4 h. MCF-7 cells without g-C3N4 QDs were used as a control. The MCF-7 cells were treated with PI (1 μg/mL, EM 550–660 nm; EX 543 nm) at room temperature to stain the nuclei and cytoskeletons. Three g-C3N4 QDs was excited at the wavelength of 405 nm, and the fluorescence images were recorded at emission wavelengths of 430–490 nm. Scale bar: 50 µm.

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Figure 6. The viabilities of MCF-7 cells incubated with CN, CN-DPT and CN-THDT QDs for 24 h without or with laser irradiation (800 nm, 1.0 W/cm2, 20 min). Statistical analysis was determined by a one-way ANOVA followed by Student-Newman-Keuls test using SPSS. The significance level was fixed as P < 0.05. **P = 0.001.

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Graph Abstract

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Engineered g-C3N4 Quantum Dots for Tunable Two-Photon Imaging

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