Multifunctional Nanoplatform Based on Black Phosphorus Quantum

Jul 3, 2017 - Reactive Oxygen Species Assay Kit was used to verify the intracellular production of ROS. Hep G2 cells were incubated with PEGylated ...
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Multifunctional Nanoplatform Based on Black Phosphorus Quantum Dots for Bioimaging and Photodynamic/Photothermal Synergistic Cancer Therapy Yi Li, Zhiming Liu,* Yuqing Hou, Guangcun Yang, Xixi Fei, Henan Zhao, Yanxian Guo, Chengkang Su, Zhen Wang, Huiqing Zhong, Zhengfei Zhuang, and Zhouyi Guo* MOE Key Laboratory of Laser Life Science & SATCM Third Grade Laboratory of Chinese Medicine and Photonics Technology, College of Biophotonics, South China Normal University, Guangzhou 510631, Guangdong, China S Supporting Information *

ABSTRACT: A multifunctional nanoplatform based on black phosphorus quantum dots (BPQDs) was developed for cancer bioimaging and combined photothermal therapy (PTT) and photodynamic therapy (PDT). BPQDs were functionalized with PEG chains to achieve improved biocompatibility and physiological stability. The as-prepared nanoparticles exhibite prominent near-infrared (NIR) photothermal and red-light-triggered photodynamic properties. The combined therapeutic application of PEGylated BPQDs were then performed in vitro and in vivo. The results demonstrate that the combined phototherapy significantly promote the therapeutic efficacy of cancer treatment in comparison with PTT or PDT alone. BPQDs could also serve as the loading platform for fluorescent molecules, allowing reliable imaging of cancer cells. In addition, the low cytotoxicity and negligible side effects to main organs were observed in toxicity experiments. The theranostic characteristics of PEGylated BPQDs provide an uplifting potential for the future clinical applications. KEYWORDS: black phosphorus quantum dots, bioimaging, photodynamic therapy, photothermal therapy, low cytotoxicity

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INTRODUCTION Given the high incidence of malignant tumors in individuals regardless of sex or age group, a considerable effort has been devoted to the development of effective tumor therapies.1 Traditional cancer treatments include surgical resection, chemotherapy, and radiotherapy,2,3 although these methods remain far from being optimal therapies given their low curative effect and strong systemic side effects.4,5 Photothermal therapy (PTT) and photodynamic therapy (PDT) have attracted increasing interest because of their good therapeutic efficiency and minor injury. PTT employs optical absorbing agents to convert the photo energy to heat under light irradiation, leading to thermal ablation of cancer cells.6−8 Examples of near-infrared (NIR) light-absorbing nanoparticles are gold nanorods, graphene and polypyrrole.9−11 In PDT, the cancer cells can be killed by the cytotoxic reactive oxygen species (ROS), generated by the endocytosed photosensitizer under appropriate irradiation conditions.12−18 However, the efficiency of PDT is limited by low 1O2 production due to the severe hypoxia environment in tumors.19 Combined PDT with PTT has been proposed to overcome this disadvantage, because heat induced by PTT can significantly improve blood flow, which consequently increasing oxygen supply and thus enhancing 1O2 production.20 Moreover, PDT can enhance the sensitivity of tumor cells to PTT by disturbing microenvironmental conditions.21 The combination of PTT with PDT is an © 2017 American Chemical Society

effective therapeutic strategy against superficially located tumors.22 However, the current therapeutic nanoplatforms for combinatorial PDT/PTT often consist of complex components. Two-dimensional (2D) black phosphorus (BP) material has recently attracted considerable attention because of its unique physical structure and excellent optical properties.23−25 This novel 2D material is distinguished from other 2D materials by its layered and puckered honeycomb structure consisting of phosphorus; and each phosphorus atom is linked to three other neighboring phosphorus atoms via a single bond.26 Moreover, BP displays a direct band gap ranging from ∼0.3 eV for bulk to ∼2.0 eV for phosphorene. This layer-number dependent band gap of BP is quite different from that of zero-gap graphene.27,28 BP demonstrates various and impressive performances. However, the applications of BP nanostructures were limited for lack of efficient synthetic strategy and poor ambient stability. It has been reported that mechanically exfoliated BP flakes could be irreversibly degraded to PxOy in exposed environment, and the oxidation rate depend on thickness of BP and oxygen concentration.29,30 Surface passivation modification with suitable strategies have been developed to suppress the Received: April 26, 2017 Accepted: July 3, 2017 Published: July 3, 2017 25098

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

Research Article

ACS Applied Materials & Interfaces

rpm for 60 min to remove NMP. The precipitate was finally resuspended in water for further use. Preparation of PEGylated BPQDs and RdB/PEG-BPQDs. PEGylated BPQDs were prepared through electrostatic bonding. BPQDs (1 mg) were dispersed in 2 mL of mPEG-NH2 solution (Mw = 5000, 1 mg/mL) and sonicated for 20 min. After stirring of 4 h, the mixture was dialyzed using a 100-kDa filter to remove the unreacted PEG-NH2. To prepare RdB/PEG-BPQDs, 10 μL of RdB solution (4 mg/mL) was added into PEGylated BPQDs solution and reacted in the dark for 12 h. Then the mixture solution was centrifuged three times at 9000 rpm for 30 min to remove the unreacted RdB. Evaluation of the Photothermal Effect of PEGylated BPQDs. We investigated the photothermal effect of PEGylated of BPQDs induced by NIR laser irradiation. A 1.5 mL centrifuge tube containing 1 mL of sample was fixed on an iron pedestal and then irradiated with 808 nm laser (2 W/cm2); temperature change in the sample was recorded by an infrared thermal camera (Fluke Ti 200, Fluke Corp, USA). Detection of 1O2. A typical chemical method based on DPBF was used to confirm 1O2 generation. PEGylated BPQDs (240 μg) was added into 4 mL of anhydrous ethanol containing DPBF (20 μg/mL) and stirred for 100 min in dark. The resulting mixture was irradiated with a 625 nm light (80 mW/cm2), and the absorption intensity of DPBF at about 410 nm was recorded on a UV−vis−NIR spectrometer. Evaluation of the 1O2 Quantum Yield. Emission spectra of 1O2 at about 1270 nm was detected by a JY Fluorolog-3 spectrophotometer (Fluorolog-3, USA) with a NIR detector and excited with 530 nm wavelength. The absorption of the standard photosensitizer RB, BPQDs and RdB/PEG-BPQDs at 530 nm were all adjusted to ∼0.2 OD. The 1O2 quantum yields of BPQDs and RdB/PEG-BPQDs were calculated with the followed formula: Φsample = ΦRB (Isample/IRB), where Isample and IRB represent the PL peak areas of 1O2 generated by the obtained nanoparticles and RB, respectively. Confocal Fluorescence Imaging. Human hepatocellular carcinoma (Hep G2) cells and 4T1 murine breast cancer cells were seeded in 20 mm glass bottom dishes (5 × 104 cells/dish) and incubated for 24 h at 37 °C. Then the culture medium was replaced with fresh medium containing 60 μg/mL of RdB/PEG-BPQDs and incubated for 12 h. Cells were finally washed three times with PBS and imaged with the confocal microscope. PDT and PTT Treatments in Vitro. The cytotoxicity of PEGylated BPQDs to Hep G2 cells was investigated using a standard CCK-8 assay. Hep G2 cells were seeded in a 96-well plate (1 × 104 cells/well) in DMEM medium at 37 °C under 5% CO2 for 24 h. Then the cells were incubated with different concentrations of PEGylated BPQDs (0, 5, 10, 25, 50, and 100 μg/mL) for another 24 h. A standard CCK-8 assay was used to determine the relative viability of the cells. For PDT treatment, Hep G2 cells were treated with different concentrations of PEGylated BPQDs (0, 40, 60, and 80 μg/mL) and irradiated with 625 nm light (80 mW/cm2, 10 min) and then incubated for 36 h. For PTT, Hep G2 cells were irradiated with 808 nm laser (2 W/cm2) for 2 min. To investigated the antitumor effect of the combination of PTT and PDT, Hep G2 cells were exposed to 625 nm light (80 mW/cm2) for 10 min and then to 808 nm laser (2 W/ cm2) for 2 min, after 36 h of incubation, standard CCK-8 assay was performed to determine the relative viability of the cells. A classic photosensitizer (Ce6) was used as the positive control. For Hoechst 33258 staining experiment, Hep G2 cells were treated with 60 μg/mL PEGylated BPQDs and then irradiated with the light (808 nm laser: 2 W/cm2, 2 min; 625 nm light: 80 mW/cm2, 10 min). Afterward, the Hep G2 cells were stained with Hoechst 33258 dye for 15 min in dark. Fluorescent images of cells were taken with a fluorescence microscope after the cells were washed with PBS for three times. Reactive Oxygen Species Assay Kit was used to verify the intracellular production of ROS. Hep G2 cells were incubated with PEGylated BPQDs (60 μg/mL) and then incubated for an additional 4 h. The BPQDs were subsequently removed, and 1 mL of basic DMEM medium with DCFH-DA (10 μM) was added and then incubated for

chemical degradation of exfoliated BP sheet, such as fully oxidized BP layer, AlOx overlayers deposition, as well as the covalent aryl diazonium functionalization.31−33 On the other hand, the poor stability of BP nanostructures brings new hope in the biomedical field, especially in tumor therapy, because elemental phosphorus of BP can be degraded into biocompatible phosphorus oxides in vivo.34 Similar to graphene, BP nanosheet possesses ultrahigh surface area and outstanding photothermal feature, which endow it with the capacities for drug delivery and photothermal therapy.35,36 BP sheet can also serves as the biodegradable photosensitizer for the mass generation of 1O2 in tumor photodynamic therapy.34 Recently, given the particles smaller than 10 nm could been effectively eliminated from renal and liver, Yu and co-workers developed BP quantum dots (BPQDs), the ultrasmall derivatives of BP sheets, as novel NIR-absorbing photothermal agents for the high-performance photothermal ablation of cancer cells.37 They then fabricated biodegradation-controlled BPQDs encapsulated in the poly(lactic-co-glycolic acid) nanospheres, allowing more stable PTT effect with inappreciable side effects.38 BPQDs were also applied as the nanotags for photoacoustic or fluorescent imaging of tumor cells.39,40 In this study, water-soluble PEGylated BPQDs smaller than 10 nm were prepared through liquid exfoliation. The asprepared BPQDs display good biocompatibility, excellent NIR photothermal performance, and 1O2 generation capability. For the first time, we integrated the PTT and PDT into a singlecomponent nanoplatform based on BPQDs for cancer treatment. In vitro and in vivo studies verifiy the excellent effectiveness of PEGylated BPQDs for PDT/PTT combination cancer therapy with little toxicity. In vitro fluorescence imaging of cancer cells was also accomplished utilizing PEGylated BPQDs as the loading platform for organic fluorescent dyes.

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EXPERIMENTAL METHODS

Materials. BP crystal powder was purchased from Nanjing XFNANO Materials. Methoxypolyethylene glycol amine (mPEGNH2, Mw = 5000), N-mechyl-2-pyrrolidone (NMP), rhodamine B (RdB), rose bengal (RB), chlorin e6 (Ce6), 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), 2,2,6,6-Tetramethylpiperidine (TEMP), 1,3diphenylisobenzofuran (DPBF) and dimethyl sulfoxide (DMSO) were purchased from Aladdin Reagents (Shanghai, China). Cell Counting Kit-8 (CCK-8) and 2,7-dichloro-dihydro-fluorescien diacetate (DCFH-DA) Reactive Oxygen Species Assay Kit were obtained from Shanghai Beyotime Biotechnology. Dulbecco’s modified Eagle medium (DMEM), RPMI 1640 cell medium, fetal bovine serum, streptomycin and penicillin were purchased from Gibco Invitrogen. Characterization. Transmission electron microscopic (TEM) images of PEGylated BPQDs were taken with a JEM-2010HR transmission electron microscope (JEOL, Japan). Atomic force microscopic (AFM) images were recorded on a FM-Nanoview1000 (FSM-Precision, China). Ultraviolet−visible-near-infrared (UV−vis− NIR) absorption spectra were obtained using a UV−vis−NIR spectrometer (UV-3200S, MAPADA, China). Raman spectra were acquired by a Raman spectrometer (Derbyshire, England) equipped with 785 nm laser as excitation source. Confocal fluorescence microscopic images were obtained using a laser scanning confocal microscopy (Zeiss LSM710, Germany). The electron spin resonance (ESR) spectra were measured by a ESR spectrometer (E-Scan, USA). Synthesis of BPQDs. BPQDs were prepared using the sonication exfoliation technique.37 In brief, 30 mg of milled BP crystals powder was dispersed in 30 mL of NMP; then the mixture was sonicated using an ultrasonic cell disruption system for 6 h (600 W) followed by an ultrasonic cleaning system for 10 h (300 W). The resultant BPQDs solution was centrifuged at 7000 rpm for 20 min to remove the oversized particles. The supernatant was then centrifuged at 18 000 25099

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

Research Article

ACS Applied Materials & Interfaces Scheme 1. Schematic Illustration of PEGylated BPQDs and the Biomedical Applications

Figure 1. (a, b) TEM images of PEGylated BPQDs with different magnifications. (c) AFM image and (d) corresponding height analysis of PEGylated BPQDs. μL) were intratumorally injected to mice. After 24 h of injection, mice in group IV and V were exposed to 625 nm light (80 W/cm2, 20 min) and 808 nm laser (2 W/cm2, 2 min), respectively; mice in group III and VI received 625 nm light and 808 nm laser irradiation. Infrared thermal camera was used to record the change in surface temperature in tumor site. Tumor size and body weight were measured every 2 days using a digital vernier caliper and a electronic scale, respectively. Tumor volumes were estimated using the following formula: (tumor length) × (tumor width)2/2. The normalized tumor volumes were calculated as V/V0, where V0 is the initial tumor volume before treatment. After 16 days, the major organs of the mice in all groups were obtained and stained with hematoxylin and eosin (H&E) for histological analysis.

20 min. The cells were washed three times with PBS and then irradiated with 625 nm light (80 mW/cm2). After 10 min the medium was replaced with PBS, the fluorescence pictures were obtained using a Leica DM-2500 fluorescence microscope. Anticancer Evaluation in Vivo. All female Balb/c mice were purchased from the experimental animal center of Southern Medical University, and performed with protocols approved by South China Normal University Animal Care and Use Committee. 4T1 cells (1 × 106) suspended in 100 μL of PBS were subcutaneously implanted into the flank of 8-week-old Balb/c mice. Two weeks later, when the tumors volume reached 100 mm3, the mice were randomly divided into six treatment groups (five mice per group) and treated as follows: (I) PBS alone, (II) BPQDs alone, (III) PBS + 625 light +808 laser, (IV) 625 light + BPQDs, (V) 808 laser + BPQDs, and (VI) 625 light +808 laser + BPQDs. PBS (100 μL) and BPQDs (500 μg/mL, 100 25100

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

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ACS Applied Materials & Interfaces

Figure 2. (a) UV−vis−NIR absorbance spectra of PEGylated BPQDs at different concentrations. Inset: photos of PEGylated BPQDs solutions. (b) Raman spectra of BPQDs and bulk BP crystal.

Figure 3. (a) Temperature curves of PEGylated BPQDs solutions at different concentrations exposed to 808 nm laser at a power density of 2 W/ cm2. The inset shows the corresponding the infrared thermal images. (b) Temperature variation of PEGylated BPQDs solution under five laser on/ off cycles.

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out-of-plane phonon mode (A1g ) and the in-plane modes (B2g and A2g ), respectively. Compared to the bulk BP, the Raman spectrum of BPQDs shows an obvious red-shift because of their small lateral dimensions and ultrathin thicknesses, consistent with previous findings.42,43 After RdB grafting, a new peak at about 553 nm appears in the UV−vis−NIR absorption spectra of RdB/PEG-BPQDs (Figure S4). The nanocomposites also displays good photoluminescence property (ex 500 nm, em 577 nm; Figure S5), indicating the potential for bioimaging. Photothermal Sensitivity of PEGylated BPQDs. To evaluate the photothermal effect of PEGylated BPQDs, we exposed the aqueous solutions of nanoparticles at different concentrations to 808 nm NIR laser for 10 min (power density: 2W/cm2). Figure 3a shows a concentration-dependent photothermal heating effect of PEGylated BPQDs, the temperature of the solution increases by 30.5 °C after 10 min when the concentration is 70 μg/mL, which is considerably higher than that of water (ΔT ≈ 2 °C). To further evaluate the photothermal stability of PEGylated BPQDs, we exposed the nanoparticles to NIR laser for 5 min (laser on), followed by naturally decreased to room temperature after the 808 nm laser was turned off (laser off); this procedure was repeated five times. As shown in Figure 3b, no significant temperature change is observed during in the five cycles, revealing the excellent photothermal stability of PEGylated BPQDs. Photodynamic Effect of PEGylated BPQDs. We used the electron spin resonance (ESR) spectroscopy to directly detect

RESULTS AND DISCUSSION Characterization of PEGylated BPQDs and RdB/PEGBPQDs. Scheme 1 illustrates the synthetic of PEGylated BPQDs and RdB/PEG-BPQDs and their biomedical applications. The morphologies of nanoparticles are revealed by the TEM images (Figure 1a, b), where the BPQDs are uniform and distributed in 2.5 ± 0.7 nm. The AFM images (Figure 1c, d) show that the average height of BPQDs distributed in 1.3 ± 0.7 nm, corresponding to approximately 1−2 layers of BP. The successful linkage of PEG onto BP nanodot can be proved by the surface charge analysis (Figure S1), in which the zeta potential of BPQDs increases from −27.8 mV to −15.1 mV after mPEG-NH2 modification. The UV−vis absorbance spectrum and Raman spectrum of PEGylated BPQDs (Figure S2a, b) nearly unchanged after 48 h in the dark. In addition, PEGylated BPQDs maintained excellent photothermal conversion performance after 48 h (Figure S2c). These results prove that little degradation of PEGylated BPQDs was taken place. The surface functionalization also improves the physiological stabilities of the nanoparticles (Figure S3). Figure 2a shows the UV−vis-NIR absorbance spectra of the aqueous solution of PEGylated BPQDs at different concentrations, which exhibits broad absorption band across the UV and NIR regions like other 2D layered nanomaterials.41 The Raman spectrum of BPQDs (Figure 2b) shows three prominent peaks at 359, 436, and 463 cm−1, representing the 25101

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

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Figure 4. (a) ESR spectra of PEGylated BPQDs under different conditions, (b) Time-course absorption spectra of DPBF solution when treated with BPQDs and irradiated with 625 nm light at a power density of 80 mW/cm2.

the 1O2 generation and the TEMP was used as 1O2 trapping agent. As illustrated in ESR spectra (Figure 4a), a characteristic signal induced by 1O2, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), is observed under irradiation, suggesting the generation of 1O2. Moreover, DMPO has been used as spin trap agent to exclude the possible generation of other ROS generated by PEGylated BPQDs; no other ROS signals are detected. In addition, we used DPBF as probe molecule to further confirm the generation of 1O2 by PEGylated BPQDs. DPBF can react with 1O2 and then show a reduced absorption intensity at approximately 410 nm. With the increase of irradiation time of 625 nm light, the absorbance intensity of DPBF displays a continuous decrease around 410 nm indicating the photosensitive reaction induced by BPQDs (Figure 4b). No changes are observed in the absorbance spectra in the presence of NaN3 (the scavenger of 1O2) or 808 nm laser irradiation (Figure S6), which proves the generation of 1O2 under the redlight irradiation. Luminescence emission spectra of 1O2 induced by BPQDs and RdB/PEG-BPQDs at about 1270 nm were detected to evaluate the 1O2 quantum yields of them. Herein, RB was selected as the standard photosensitizer (ΦRB = 0.86, in ethanol).34 Figure S7 shows the luminescence emission spectra of 1O2 generated by RB, BPQDs and RdB/PEG-BPQDs. respectively. According to the standard value of RB, the 1O2 quantum yields of BPQDs (ΦBPQDs) and RdB/PEG-BPQDs (ΦRdB/PEG‑BPQDs) are measured to be 0.74 and 0.68, respectively, which are higher than that of most reported photosensitizers.44 Fluorescence Imaging of Cancer Cells. We explored the application of RdB/PEG-BPQDs for bioimaging of cancer cells. As displayed in the confocal microscope images (Figure 5), distinct fluorescence signals are observed in Hep G2 cells and 4T1 cells in the presence of RdB/PEG-BPQDs. It can also be observed that the nanoparticles are located almost exclusively in the cytoplasm. However, no visible fluorescence is observed in the cancer cells without the nanoprobes (Figure S8). These results clearly demonstrate the potential application of PEGylated BPQDs for bioimaging. In Vitro PDT/PTT Treatment. CCK-8 assay was performed to determine the cytotoxicity of PEGylated BPQDs to Hep G2 cells. No significant cytotoxicity is observed even the nanoparticles concentration up to 100 μg/mL (Figure 6a). Figure 6b exhibits the in vitro cancer therapy using BP-based nanomaterials. The viabilities of Hep G2 cells incubated with

Figure 5. Confocal fluorescence images of Hep G2 and 4T1 cells incubated with 60 μg/mL of RdB/PEG-BPQDs (scale bar: 50 μm).

PEGylated BPQDs and subjected to 625 nm light (PDT alone) or 808 nm laser (PTT alone) irradiation shows a downward trend as concentration increases. In comparison, cell death rate in combined PTT/PDT group is significantly higher than that in the separate treatment groups, indicating the synergistic effect of the BPQD-based combination therapy. The inhibition rate of combined therapy reach more than 80% when the nanoparticle concentrate up to 80 μg/mL. In addition, PEGylated BPQDs may be a superior photosensitizer compared to the classic Ce6, because of their low cytotoxicity (Figure S9). Hoechst 33258 staining was performed to visually observe the cell viability. Hoechst 33342 dye can stain nuclear DNA blue after passing through the cell membrane. The fluorescence microscopic images (Figure S10) reflect that nearly all cells are alive in groups I, II, and III. The bright blue fluorescence intensity of the Hep G2 cells in the combination group is clearly stronger than that in other groups, and severe cell death is observed, further demonstrating the superior therapeutic efficacy of BPQDs-based combination therapy. Detection of 1O2 in Cells. A ROS assay kit was used to detect the ROS level produced by BPQDs in living cancer cells, in which DCFH-DA and Rosup reagent were used as the fluorescence probe and ROS positive control reagent, respectively. As shown in Figure 7, the Hep G2 cells incubated with PEGylated BPQDs, and irradiated with 625 nm light 25102

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

Research Article

ACS Applied Materials & Interfaces

Figure 6. In vitro cell experiments. (a) Cell viability of Hep G2 cells incubated with PEGylated BPQDs (0−100 μg/mL) for 24 h. (b) Relative cell viability of Hep G2 cells for 24 h after treatment under different conditions. Data are presented as mean ± SD.

exhibit strong green fluorescence signals, which is consistent with that treated with Rosup reagent. By contrast, nearly no green fluorescence signal is detected in the negative control group and BPQDs alone group. The above results provide direct evidence that PEGylated BPQDs can generate 1O2 in living cancer cells under 625 nm light irradiation. In Vivo Cancer Therapeutic Efficacy. 4T1 tumor-bearing Balb/c mice were used as models to determine the therapeutic efficacy of PEGylated BPQDs in vivo. The infrared thermal camera was used to detect the real-time temperature change of the tumor sites of mice under laser irradiation. As displayed in Figure 8a, the tumor sites in mice treated with PEGylated BPQDs (groups V and VI) exhibit remarkable increase in temperature within 2 min (ΔT ≈ 30 °C) under the NIR irradiation, but not for those only treated with laser (group III), indicating the excellent photothermal efficiency of PEGylated BPQDs. The in vivo therapeutic efficacies of different treatments were evaluated by detecting the tumor sizes using a digital caliper. As shown in the relative tumor volume curves (Figure 8b), the tumors of mice in groups I, II and III experience rapid growth during the 16-day observation period.

Figure 7. ROS levels of cancer cells treated (a) with PBS or BPQDs (b) without and (c) with 625 nm light irradiation, respectively. (d) Rosup reagent is used as the positive control.

Figure 8. (a) Infrared thermal images of 4T1 tumor-bearing mice after 808 nm laser irradiation. (b) Tumor growth curves in different groups subjected with different treatments. Data are presented as mean ± SD. 25103

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

Research Article

ACS Applied Materials & Interfaces Significant tumor growth suppression can be observed in groups IV, V, and VI. Among them, the combined PDT/PTT treatment achieves an optimal therapeutic efficacy, which may be considered as the synergistic effect of between PDT and PTT mediated by PEGylated BPQDs. Representative photos of tumors in mice (Figure S11) also demonstrate the tumor ablation capability of BPQD-based combined therapy; only black small scars are observed in tumor sites. Tumors were collected after the mice sacrificed at day 16 of the treatment. It can be observed that the tumors in groups V and VI are effectively eliminated (Figure 9a, b), especially in

Figure 10. (a) Mean body weight changes in mice after treatment. (b) Micrographs of H&E-stained major organs obtained from different groups (group I, control; group II, BPQDs; group III, 625 nm light +808 nm laser; group IV, 625 nm light + BPQDs; group V, 808 nm laser + BPQDs; group VI, 625 nm light +808 nm laser + BPQDs; scale bar, 200 μm).

Figure 9. (a) Photographs of tumors collected from different groups of mice at the end of treatments (16 day). (b) Average weights of tumors collected from different groups of mice. (c) Representative micrographs of H&E-stained tumor tissue slices obtained from different groups of mice. (group I, control; group II, BPQDs; group III, 625 nm light +808 nm laser; group IV, 625 nm light + BPQDs; group V, 808 nm laser + BPQDs; group VI, 625 nm light +808 nm laser + BPQDs. scale bar, 200 μm).

treatment based on PEGylated BPQDs show no obvious side effects in vivo.

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CONCLUSIONS We have established a multifunctional nanoplatform based on BPQDs by using liquid exfoliation approach for bioimaging and PDT/PTT combination cancer therapy for the first time. The obtained PEGylated BPQDs demonstrated excellent biocompatibility, high water solubility, strong NIR absorbance and the ability to generate cytotoxic 1O2 under light irradiation. The antitumor effect of PDT or PTT therapy alone is significantly improved when these methods are combined, which may be attributed to the synergistic effect of PDT and PTT. Although a considerable amount of work is needed to investigate the systematic long-term toxicity of BPQDs, the low potential toxicity of BPQDs is demonstrated by histological analysis of the major organs of the mice. In addition, bioimaging of cancer cells is achieved in the presence of developed RdB/PEGBPQDs. Our demonstration based on BPQDs provides an effective theranostic strategy for cancer treatment that shows great potential in biomedicine.

group VI, where four out of five tumors totally disappear. Though the volumes of tumors in the PDT alone group decrease obviously, the therapic effect of PDT alone is relative lower than groups V and VI, which may be attributed to the relatively low intensity of 625 nm light. Moreover, H&E staining of tumor slices obtained from each group of mice displays that no significant morphological the tumor cells in control groups, whereas those in the combined PDT/PTT treatment group are severely damaged (Figure 9c). This results are consistent with the date above, probably a consequence of the synergistic effect of PDT and PTT. Generally, weight loss is a criterion used to evaluate the potential toxicity in vivo.45 The body weight of the mice from all groups was measured to determine whether the treatment caused side effects in vivo. No noticeable weight loss is noted in all groups during a 16-day treatment period (Figure 10a). The major organs of the mice in all groups were collected at 16 day after the therapy for histological analysis (Figure 10b). No appreciable organ damage could be found in any group after receiving various treatments, demonstrating that the combined PDT/PTT

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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/acsami.7b05824. 25104

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

Research Article

ACS Applied Materials & Interfaces

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(9) Gou, L. F.; Murphy, C. J. Fine-Tuning the Shape of Gold Nanorods. Chem. Mater. 2005, 17, 3668−3672. (10) Chen, J.; Wang, X.; Chen, T. Facile and Green Reduction of Covalently PEGylated Nanographene Oxide Via a ’Water-Only’ Route for High-efficiency Photothermal Therapy. Nanoscale Res. Lett. 2014, 9, 86. (11) Jin, Y.; Li, Y.; Ma, X.; Zha, Z.; Shi, L.; Tian, J.; Dai, Z. Encapsulating Tantalum Oxide into Polypyrrole Nanoparticles for Xray CT/Photoacoustic Bimodal Imaging-Guided Photothermal Ablation of Cancer. Biomaterials 2014, 35, 5795−5804. (12) Huang, P.; Xu, C.; Lin, J.; Wang, C.; Wang, X.; Zhang, C.; Zhou, X.; Guo, S.; Cui, D. Folic Acid-conjugated Graphene Oxide Loaded with Photosensitizers for Targeting Photodynamic Therapy. Theranostics 2011, 1, 240−250. (13) Tian, B.; Wang, C.; Zhang, S.; Feng, L.; Liu, Z. Photothermally Enhanced Photodynamic Therapy Delivered by Nano-Graphene Oxide. ACS Nano 2011, 5, 7000−7009. (14) Markovic, Z. M.; Ristic, B. Z.; Arsikin, K. M.; Klisic, D. G.; Harhaji-Trajkovic, L. M.; Todorovic-Markovic, B. M.; Kepic, D. P.; Kravic-Stevovic, T. K.; Jovanovic, S. P.; Milenkovic, M. M.; Milivojevic, D. D.; Bumbasirevic, V. Z.; Dramicanin, M. D.; Trajkovic, V. S. Graphene Quantum Dots as Autophagy-Inducing Photodynamic Agents. Biomaterials 2012, 33, 7084−7092. (15) Sun, Y.; Chen, Z. L.; Yang, X. X.; Huang, P.; Zhou, X. P.; Du, X. X. Magnetic Chitosan Nanoparticles as a Drug Delivery System for Targeting Photodynamic Therapy. Nanotechnology 2009, 20, 135102. (16) Seo, S.-H.; Kim, B.-M.; Joe, A.; Han, H.-W.; Chen, X.; Cheng, Z.; Jang, E.-S. NIR-Light-Induced Surface-Enhanced Raman Scattering for Detection and Photothermal/Photodynamic Therapy of Cancer Cells using Methylene Blue-Embedded Gold Nanorod@SiO2 Nanocomposites. Biomaterials 2014, 35, 3309−3318. (17) Bechet, D.; Couleaud, P.; Frochot, C.; Viriot, M. L.; Guillemin, F.; Barberi-Heyob, M. Nanoparticles as Vehicles for Delivery of Photodynamic Therapy Agents. Trends Biotechnol. 2008, 26, 612−621. (18) Han, J.; Park, W.; Park, S. J.; Na, K. Photosensitizer-Conjugated Hyaluronic Acid-Shielded Polydopamine Nanoparticles for Targeted Photomediated Tumor Therapy. ACS Appl. Mater. Interfaces 2016, 8, 7739−7747. (19) Wang, K.; Zhang, Y.; Wang, J.; Yuan, A.; Sun, M.; Wu, J.; Hu, Y. Self-Assembled IR780-Loaded Transferrin Nanoparticles as an Imaging, Targeting and PDT/PTT Agent for Cancer Therapy. Sci. Rep. 2016, 6, 27421. (20) Qiu, J.; Xiao, Q.; Zheng, X.; Zhang, L.; Xing, H.; Ni, D.; Liu, Y.; Zhang, S.; Ren, Q.; Hua, Y.; Zhao, K.; Bu, W. Single W18O49 Nanowires: A Multifunctional Nanoplatform for Computed Tomography Imaging and Photothermal/Photodynamic/Radiation Synergistic Cancer Therapy. Nano Res. 2015, 8, 3580−3590. (21) Waldow, S. M.; Henderson, B. W.; Dougherty, T. J. Potentiation of Photodynamic Therapy by Heat: Effect of Sequence and Time Interval Between Treatments In Vivo. Lasers Surg. Med. 1985, 5, 83− 94. (22) Alvarez-Lorenzo, C.; Bromberg, L.; Concheiro, A. LightSensitive Intelligent Drug Delivery Systems. Photochem. Photobiol. 2009, 85, 848−860. (23) Ç akır, D.; Sevik, C.; Peeters, F. M. Significant Effect of Stacking on the Electronic and Optical Properties of Few-Layer Black Phosphorus. Phys. Rev. B: Condens. Matter Mater. Phys. 2015, 92, 165406. (24) Dahbi, M.; Yabuuchi, N.; Fukunishi, M.; Kubota, K.; Chihara, K.; Tokiwa, K.; Yu, X.-f.; Ushiyama, H.; Yamashita, K.; Son, J.-Y.; Cui, Y.-T.; Oji, H.; Komaba, S. Black Phosphorus as a High-Capacity, HighCapability Negative Electrode for Sodium-Ion Batteries: Investigation of the Electrode/Electrolyte Interface. Chem. Mater. 2016, 28, 1625− 1635. (25) Li, J.; Luo, H.; Zhai, B.; Lu, R.; Guo, Z.; Zhang, H.; Liu, Y. Black Phosphorus: a Two-Dimension Saturable Absorption Material for Mid-Infrared Q-Switched and Mode-Locked Fiber Lasers. Sci. Rep. 2016, 6, 30361.

Zeta potentials, UV−vis, Raman spectra and temperature curves of PEGylated BPQDs, stability test, UV−vis spectra and fluorescence spectra of BPQD-based nanoparticles, absorption spectra of DPBF after various treatments, 1O2 emission spectra induced by BPQDs and RdB/PEG-BPQDs, confocal images of control group, cell viability after incubated with PEGylated BPQDs or Ce6, fluorescence microscopic images, representative photos of tumors in mice (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel/fax: 86-20-85211428. E-mail: [email protected] (Z.M.L). *E-mail: [email protected] (Z.Y.G.). ORCID

Zhouyi Guo: 0000-0003-4518-4862 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (61335011, 61675072, 61275187, 21505047, and 11404116) the Natural Science Foundation of Guangdong Province of China (2014A030310306, S2013040016223, and 2014A030311024), the Science and Technology Project of Guangdong Province of China (2012A080203008 and 2014A020212282), the Science and Technology Innovation Project of the Education Department of Guangdong Province of China (2013KJCX0052).

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REFERENCES

(1) Minelli, C.; Lowe, S. B.; Stevens, M. M. Engineering Nanocomposite Materials for Cancer Therapy. Small 2010, 6, 2336−2357. (2) Hirsch, L. R.; Stafford, R. J.; Bankson, J. A.; Sershen, S. R.; Rivera, B.; Price, R. E.; Hazle, J. D.; Halas, N. J.; West, J. L. Nanoshellmediated Near-infrared Thermal Therapy of Tumors under Magnetic Resonance Guidance. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 13549− 13554. (3) Zhang, W.; Guo, Z.; Huang, D.; Liu, Z.; Guo, X.; Zhong, H. Synergistic Effect of Chemo-photothermal Therapy Using PEGylated Graphene oxide. Biomaterials 2011, 32, 8555−8561. (4) Liu, Z.; Cheng, L.; Zhang, L.; Yang, Z.; Liu, Z.; Fang, J. Sub-100 nm Hollow Au-Ag Alloy Urchin-shaped Nanostructure with Ultrahigh Density of Nanotips for Photothermal Cancer Therapy. Biomaterials 2014, 35, 4099−4107. (5) Chen, Q.; Wang, C.; Cheng, L.; He, W.; Cheng, Z.; Liu, Z. Protein Modified Upconversion Nanoparticles for Imaging-guided Combined Photothermal and Photodynamic Therapy. Biomaterials 2014, 35, 2915−2923. (6) Meng, D.; Yang, S.; Guo, L.; Li, G.; Ge, J.; Huang, Y.; Bielawski, C. W.; Geng, J. The Enhanced Photothermal Effect of Graphene/ conjugated Polymer Composites: Photoinduced Energy Transfer and Applications in Photocontrolled Switches. Chem. Commun. 2014, 50, 14345−14348. (7) Liu, X. J.; Wang, Q.; Li, C.; Zou, R. J.; Li, B.; Song, G. S.; Xu, K. B.; Zheng, Y.; Hu, J. Q. Cu2-xSe@mSiO(2)-PEG Core-Shell Nanoparticles: a Low-Toxic and Efficient Difunctional Nanoplatform for Chemo-photothermal Therapy under Near Infrared Light Radiation With a Safe Power Density. Nanoscale 2014, 6, 4361−4370. (8) Cheng, L.; Wang, C.; Feng, L. Z.; Yang, K.; Liu, Z. Functional Nanomaterials for Phototherapies of Cancer. Chem. Rev. 2014, 114, 10869−10893. 25105

DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106

Research Article

ACS Applied Materials & Interfaces (26) Koenig, S. P.; Doganov, R. A.; Schmidt, H.; Castro Neto, A. H.; Ö zyilmaz, B. Electric Field Effect in Ultrathin Black Phosphorus. Appl. Phys. Lett. 2014, 104, 103106. (27) Castellanos-Gomez, A. Black Phosphorus: Narrow Gap, Wide Applications. J. Phys. Chem. Lett. 2015, 6, 4280−4291. (28) Su, X.; Wang, Y.; Zhang, B.; Zhao, R.; Yang, K.; He, J.; Hu, Q.; Jia, Z.; Tao, X. Femtosecond Solid-State Laser Based on a FewLayered Black Phosphorus Saturable Absorber. Opt. Lett. 2016, 41, 1945−1948. (29) Wan, B.; Yang, B.; Wang, Y.; Zhang, J.; Zeng, Z.; Liu, Z.; Wang, W. Enhanced Stability of Black Phosphorus Field-Effect Transistors with SiO(2) Passivation. Nanotechnology 2015, 26, 435702. (30) Favron, A.; Gaufres, E.; Fossard, F.; Phaneuf-L’Heureux, A. L.; Tang, N. Y.; Levesque, P. L.; Loiseau, A.; Leonelli, R.; Francoeur, S.; Martel, R. Photooxidation and Quantum Confinement Effects in Exfoliated Black Phosphorus. Nat. Mater. 2015, 14, 826−832. (31) Zhou, Q.; Chen, Q.; Tong, Y.; Wang, J. Light-Induced Ambient Degradation of Few-Layer Black Phosphorus: Mechanism and Protection. Angew. Chem., Int. Ed. 2016, 55, 11437−11441. (32) Wood, J. D.; Wells, S. A.; Jariwala, D.; Chen, K. S.; Cho, E.; Sangwan, V. K.; Liu, X.; Lauhon, L. J.; Marks, T. J.; Hersam, M. C. Effective Passivation of Exfoliated Black Phosphorus Transistors against Ambient Degradation. Nano Lett. 2014, 14, 6964−6970. (33) Ryder, C. R.; Wood, J. D.; Wells, S. A.; Yang, Y.; Jariwala, D.; Marks, T. J.; Schatz, G. C.; Hersam, M. C. Covalent Functionalization and Passivation of Exfoliated Black Phosphorus via Aryl Diazonium Chemistry. Nat. Chem. 2016, 8, 597−602. (34) Wang, H.; Yang, X.; Shao, W.; Chen, S.; Xie, J.; Zhang, X.; Wang, J.; Xie, Y. Ultrathin Black Phosphorus Nanosheets for Efficient Singlet Oxygen Generation. J. Am. Chem. Soc. 2015, 137, 11376− 11382. (35) Tao, W.; Zhu, X.; Yu, X.; Zeng, X.; Xiao, Q.; Zhang, X.; Ji, X.; Wang, X.; Shi, J.; Zhang, H.; Mei, L. Black Phosphorus Nanosheets as a Robust Delivery Platform for Cancer Theranostics. Adv. Mater. 2017, 29, 1603276. (36) Chen, W.; Ouyang, J.; Liu, H.; Chen, M.; Zeng, K.; Sheng, J.; Liu, Z.; Han, Y.; Wang, L.; Li, J.; Deng, L.; Liu, Y. N.; Guo, S. Black Phosphorus Nanosheet-Based Drug Delivery System for Synergistic Photodynamic/Photothermal/Chemotherapy of Cancer. Adv. Mater. 2017, 29, 1603864. (37) Sun, Z.; Xie, H.; Tang, S.; Yu, X. F.; Guo, Z.; Shao, J.; Zhang, H.; Huang, H.; Wang, H.; Chu, P. K. Ultrasmall Black Phosphorus Quantum Dots: Synthesis and Use as Photothermal Agents. Angew. Chem., Int. Ed. 2015, 54, 11526−11530. (38) Shao, J.; Xie, H.; Huang, H.; Li, Z.; Sun, Z.; Xu, Y.; Xiao, Q.; Yu, X. F.; Zhao, Y.; Zhang, H.; Wang, H.; Chu, P. K. Biodegradable Black Phosphorus-Based Nanospheres for in vivo Photothermal Cancer Therapy. Nat. Commun. 2016, 7, 12967. (39) Lee, H. U.; Park, S. Y.; Lee, S. C.; Choi, S.; Seo, S.; Kim, H.; Won, J.; Choi, K.; Kang, K. S.; Park, H. G.; Kim, H. S.; An, H. R.; Jeong, K. H.; Lee, Y. C.; Lee, J. Black Phosphorus (BP) Nanodots for Potential Biomedical Applications. Small 2016, 12, 214−219. (40) Sun, C.; Wen, L.; Zeng, J.; Wang, Y.; Sun, Q.; Deng, L.; Zhao, C.; Li, Z. One-Pot Solventless Preparation of PEGylated Black Phosphorus Nanoparticles for Photoacoustic Imaging and Photothermal Therapy of Cancer. Biomaterials 2016, 91, 81−89. (41) Niu, X.; Shu, H.; Li, Y.; Wang, J. Photoabsorption Tolerance of Intrinsic Point Defects and Oxidation in Black Phosphorus Quantum Dots. J. Phys. Chem. Lett. 2017, 8, 161−166. (42) Zhang, X.; Xie, H.; Liu, Z.; Tan, C.; Luo, Z.; Li, H.; Lin, J.; Sun, L.; Chen, W.; Xu, Z.; Xie, L.; Huang, W.; Zhang, H. Black Phosphorus Quantum Dots. Angew. Chem., Int. Ed. 2015, 54, 3653−3657. (43) Niu, X.; Li, Y.; Shu, H.; Wang, J. Anomalous Size Dependence of Optical Properties in Black Phosphorus Quantum Dots. J. Phys. Chem. Lett. 2016, 7, 370−375. (44) Wilkinson, F.; Helman, W. P.; Ross, A. B. Quantum Yields for the Photosensitized Formation of the Lowest Electronically Excited Singlet State of Molecular Oxygen in Solution. J. Phys. Chem. Ref. Data 1993, 22, 113−262.

(45) Yang, H. W.; Lu, Y. J.; Lin, K. J.; Hsu, S. C.; Huang, C. Y.; She, S. H.; Liu, H. L.; Lin, C. W.; Xiao, M. C.; Wey, S. P.; Chen, P. Y.; Yen, T. C.; Wei, K. C.; Ma, C. C. EGRF Conjugated PEGylated Nanographene Oxide for Targeted Chemotherapy and Photothermal Therapy. Biomaterials 2013, 34, 7204−7214.

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DOI: 10.1021/acsami.7b05824 ACS Appl. Mater. Interfaces 2017, 9, 25098−25106