Near-Infrared Light-Mediated Photodynamic Therapy Nanoplatform by

Sep 26, 2016 - (16) In the above-mentioned FRET effect, Yb/Tm codoped with UCNPs absorbs NIR light and emits UV light, which can be absorbed by g-C3N4...
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Near-Infrared Light-Mediated Photodynamic Therapy Nanoplatform by the Electrostatic Assembly of Upconversion Nanoparticles with Graphitic Carbon Nitride Quantum Dots Ming-Hsien Chan,†,‡ Chieh-Wei Chen,† I-Jung Lee,† Yung-Chieh Chan,§ Datao Tu,‡ Michael Hsiao,*,§,∥ Chung-Hsuan Chen,§ Xueyuan Chen,‡ and Ru-Shi Liu*,†,§,⊥ †

Department of Chemistry, National Taiwan University, Taipei 106, Taiwan Key Laboratory of Optoelectronic Materials Chemistry and Physics, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, China § Genomics Research Center, Academia Sinica, Taipei 115, Taiwan ∥ Department of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan ⊥ Department of Mechanical Engineering and Graduate Institute of Manufacturing Technology, National Taipei University of Technology, Taipei 106, Taiwan ‡

S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) is a promising antitumor treatment that is based on photosensitizers. This therapy kills cancer cells by generating reactive oxygen species (ROS) after irradiation with specific laser wavelengths. Being a potential photosensitizer, graphitic carbon nitride (g-C3N4) quantum dots (QDs) are noncytotoxic. Although the use of g-C3N4 QDs is challenged by the limited tissue penetration of UV light, g-C3N4 QDs display excellent ultraviolet (UV) light-triggered cytotoxicity. The g-C3N4 QDs were synthesized using a solid-phase hydrothermal method. The well-distributed hydrophilic gC3N4 can be combined with NaYF4:Yb3+/Tm3+ upconversion nanoparticles via the positive ligand poly(L-lysine) to produce the final nanocomposite, NaYF4:Yb/Tm-PLL@g-C3N4. Upconversion nanoparticles can transfer IR light into UV light and promote g-C3N4 to release blue-to-green visible light to generate different images. Moreover, g-C3N4 is a promising photosensitizer in PDT because g-C3N4 can transfer oxygen into toxic ROS. The singlet oxygen formed by g-C3N4 displays great potential for use in the treatment of cancer. therapy.2,3 In PDT, the core issue is the target of the photosensitizer. Compared with organic reagents, inorganic composite materials are more stable and display a longer duration of action in an organism.4 Thus, graphitic carbon nitride (g-C3N4) has been investigated in the past few years.5 gC3N4 displays a unique band gap (2.7 eV), which can be applied in photocatalysis and in decomposition of water to serve as a medium for electron conduction.6 In fact, g-C3N4 not only can be applied in the energy field but also displays

1. INTRODUCTION Among the current cancer treatments, photodynamic therapy (PDT) has been the focus of medical researchers owing to its noninvasiveness and low side effects. In PDT, a photosensitizer absorbs a specific wavelength, and the absorbed light energy is transferred into an electron, which attacks oxygen to oxidize toxic free radicals,1 also known as reactive oxygen species (ROS). Given its high degree of chemical reactivity, ROS disrupts organelles, such as mitochondrial membranes, upon entry into the cells. On the basis of these reported characteristics, PDT can be used as a better option for cancer treatment because it improves the drawbacks of traditional © XXXX American Chemical Society

Received: June 28, 2016

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DOI: 10.1021/acs.inorgchem.6b01522 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry enormous potential in the biological field.7 The graphene phase in g-C3N4 quantum dots (QDs) can effectively absorb ultraviolet (UV) light bands; in addition, the light energy can transfer into free electrons and generate ROS in the environment.8 The synthesis of different sizes of g-C3N4 allows this photosensitizer to absorb UV light and convert the energy into different fluorescences, ranging from blue to green.9 The brilliant blue and green fluorescences can also be used as a nontoxic bioimaging tool.10 Compared with a conventional organic photosensitizer, g-C3N4 remains stable in a biological environment. Moreover, because g-C3N4 consists of only C and N, its biological toxicity is very low and it can be easily metabolized in organisms.11 On the basis of the advantages described above, g-C3N4 is very suitable as a new light-sensitive reagent.12 However, UV-light emission is a big problem when using g-C3N4 for PDT. In the UV- and visible-light spectra, biological tissues absorb light, dramatically reducing the application of PDT. Furthermore, UV light causes harm to organisms. Approximately 800−1000 nm of the near-infrared (NIR) light can effectively penetrate tissues. To address this problem, researchers used doped lanthanide metal upconversion nanoparticles (UCNPs), which can absorb low-energy light, especially NIR light, and emit high-energy light.13 UCNPs offer practical medical applications.14 Mixing different lanthanide metals causes UCNPs to produce different bands of light; for instance, Tm ions doped in nanoparticles can convert NIR energy into UV and visible light.15 These composites, where UCNPs are bound with photosensitizers, were subsequently developed; when NIR light is irradiated onto the composites, the IR light can be absorbed and is converted into UV light. The photosensitizer can absorb UV light in the fluorescence resonance energy transfer (FRET) mode, resulting in PDT production and release of ROS.16 In the above-mentioned FRET effect, Yb/Tm codoped with UCNPs absorbs NIR light and emits UV light, which can be absorbed by g-C3N4; this mechanism addresses the problem on energy conversion of penetrating UV light, rendering a more effective mechanism for PDT. In addition, given that Yb/Tm codoped with UCNPs emits visible blue light, g-C3N4 can be adjusted from blue to green, and this change in fluorescence can be used as a novel diagnostic system.17 The above-mentioned UCNP bound with g-C3N4 is a new therapeutic and diagnostic nanoplatform and has not yet been reported. We propose a new multifunctional nanopharmaceutical platform that involves IR-light absorption to generate UVlight bands, leading to ROS production and ultimately to PDT. NaYF4 is used as a UCNP host; a high-temperature coprecipitation method was employed to dope Yb3+/Tm3+. The surface ligands were removed by acid pickling, and the oil phase was transformed into an aqueous phase. We subsequently added the amino acid polymer poly(L-lysine) (PLL) to render the UCNP surface positively charged, so that the surface of the nanoparticles becomes entirely positively charged.18 Moreover, urea and sodium citrate were used in the precursor during solid-phase g-C3N4 synthesis. A hydrothermal method involves the negatively charged COO− groups of g-C3N4, and COO− participates in electrostatic binding to form NaYF4:Yb/TmPLL@g-C3N4. NaYF4:Yb/Tm-PLL@g-C3N4 can effectively absorb NIR light and emit UV light for the absorption of gC3N4 and then exerts toxicity on cells via ROS, resulting in apoptosis. This study provides several significant contributions. First is the use of NIR to achieve deeper penetration of the scope of the organization to produce a more significant effect of

PDT. Second is the use of g-C3N4 as a new type of photosensitizer, wherein this nontoxic and metabolically excellent material can effectively produce a single groundstate oxygen, which produces green fluorescence, resulting from increased UV-light absorption. The third one is due to the UPNPs through the surface modification layer on the positively charged PLL, with g-C3N4 electrostatic adsorption negatively charged. g-C3N4 was confined in the PLL layer so that the entire surface remained covered with a weak positive charge. The weak positive surface of NaYF4:Yb/Tm-PLL@g-C3N4 can effectively be endocytosed by cells. NaYF4:Yb/Tm-PLL@gC3N4 is intermediated by IR light and is applied as a nanodrug platform in PDT. NaYF4:Yb/Tm-PLL@g-C3N4 can enter into the cells to produce ROS and damage the mitochondria of cancer cells, leading to cancer cell apoptosis.16

2. EXPERIMENTAL SECTION Materials. Sodium citrate was purchased from J. T. Baker. Y(CH3CO2)3·xH2O (99.9%), Yb(CH3CO2)3·4H2O (99.9%), Tm(CH3 CO2 )3·xH2 O (99.9%), NaOH (98%), NH4 F (98%), 1octadecene (ODE, 90%), oleic acid (OA, 90%), hydrochloric acid (37%), ethanol (99%), urea powder, poly(L-lysine) (PLL), 9,10anthracenediylbis(methylene)dimalonic acid (ABDA), 1,3-diphenylisobenzofuran (DPBF), LysoTracker Red DND-99, 2′,7′-dichlorodihydrofluoroscein diacetate (DCFH2-DA), and JC-1 dye were purchased from Sigma-Aldrich. 4′,6-Diamidino-2-phenylindole (DAPI) and Hoechst 33324 were purchased from Invitrogen. Deionized water was obtained using a Milli-Q SP ultrapure-water purification system from Nihon Millipore Ltd. (Tokyo, Japan). All chemicals were used as received without further purification. g-C3N4 Synthesis. Urea (0.101 g, 1.68 mmol) and sodium citrate (0.041 g, 0.14 mmol) were mixed in an agate mortar (6 cm) and ground into a uniformly sized powder. The mixture was placed in an autoclave and heated to 180 °C. The resultant yellowish mixture was purified by three rounds of alternating washings with ethanol and centrifugation at 12000 rpm and then dialysis against pure water through a dialysis membrane for 24 h. Four species of g-C3N4s with distinct emissions were synthesized by tuning the amount of sodium citrate. Urea (0.101 g, 1.68 mmol) was separately reacted with sodium citrate (0.165 g, 0.56 mmol; 0.081 g, 0.28 mmol; 0.055 g, 0.187 mmol; 0.041 g, 0.14 mmol). By varying the molar ratio of urea to sodium citrate from 3:1 to 6:1, 9:1, and 12:1, we synthesized g-C3N4 displaying different emissions. The as-obtained g-C3N4 particles were completely dissolved in water and emitted bright fluorescence under a UV lamp. The stable aqueous solutions were further characterized by fluorescence and UV−vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Preparation of NaYF4:Yb/Tm Particles. The water-soluble NaYF4:Yb/Tm particles were synthesized using the reported hightemperature coprecipitation method.19 In brief, the precursor of NaYF4:Yb/Tm was synthesized in 6 mL OA and 14 mL ODE solutions. Subsequently, 0.64 mmol of Y(CH3CO2)3, 0.144 mmol of Yb(CH3CO2)3, and 0.016 mmol of Tm(CH3CO2)3 were added, and the mixture was maintained at 120 °C. After the precursor cooled to room temperature, a methanol solution containing 0.1475 g of NH4F and 0.1 g of NaOH was added to the above mixture under vigorous stirring. The reaction mixture was transferred to a 100 mL roundbottomed flask and heated to 290 °C for 2 h. The mixture was cooled to room temperature naturally, and a precipitate was obtained by centrifugation; the precipitate was washed three times with ethanol and cyclohexane. To remove the OA and ODE ligands and transfer NaYF4:Yb/Tm into the water phase, we washed the previous compound three times with alcohol-diluted HCl to obtain watersoluble NaYF4:Yb/Tm. Modification of PLL on the Surface of NaYF4:Yb/Tm (NaYF4:Yb/Tm-PLL). To prepare the PLL-functionalized UPNPs (NaYF4:Yb/Tm-PLL), we first prepared a water solution of PLL and NaYF4:Yb/Tm particles. Different concentrations (2.5, 5, 10, 20, and B

DOI: 10.1021/acs.inorgchem.6b01522 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry 40 mg/mL) of a PLL peptide solution were initially prepared, and the 10 mg/mL particle solution was prepared using the same water solution. A PLL peptide solution (1 mL) was then mixed with 1 mL of the NaYF4:Yb/Tm-PLL solution and then stirred at room temperature for 24 h. Thereafter, the solid product was produced, washed with water, and then centrifuged. The solid products were finally suspended in water and stirred at room temperature. The sample was denoted as NaYF4:Yb/Tm-PLL. g-C3N4-Modified NaYF4:Yb/Tm-Poly(L-lysine) (NaYF4:Yb/TmPLL@g-C3N4). The adsorption isotherm was determined by preparing a series of g-C3N4 solutions of varying concentrations ranging from 40 to 60 μg/mL in water at pH 7.4. NaYF4:Yb/Tm-PLL (1 mg) was then mixed with 500 μL of each g-C3N4 solution in a 1.5 mL centrifuge tube. The mixture was dispersed by vortex for 30 s, and the suspensions were separated by centrifugation at 13000 rpm for 5 min. The difference in the amount of g-C3N4 in solution before and after adsorption was deemed equal to the amount of g-C3N4 adsorbed on the material. After washing three times with water, the precipitate, NaYF4:Yb/Tm-PLL@g-C3N4, was suspended in 0.5 mL water. Characterization of Materials. The morphology and size of NaYF4:Yb/Tm-PLL@g-C3N4 materials were detected using TEM (JEOL, Japan) and high-resolution TEM (HRTEM; JEM-2100F, JEOL, Japan). The DLS diameter and ζ potential were measured by a Malvern Zetasizer-3000 instrument at room temperature. UV-1700 spectrophotometer (Shimadzu, Japan), Fluoromax-3 (Horiba, Japan), and Quantaurus-QY absolute PL quantum yield spectrometer (Hamamatsu, Japen) were used to obtain the absorbance, photoluminescence (PL) spectra, and quantum yield using a 1-cm-wide quartz cell, respectively. PL lifetimes were measured by an Edinburgh Instrument FLS920 spectrometer equipped with continuous-wave (450 W) and microsecond-pulsed xenon lamps. A Bruker D2 PHASER X-ray diffraction (XRD) analyzer with Cu Kα radiation (λ = 1.54178 Å) was utilized to obtain crystallographic information on NaYF4:Yb/Tm and g-C3N4. The Raman spectra of NaYF4:Yb/Tm, gC3N4, and NaYF4:Yb/Tm-PLL@g-C3N4 were elucidated using a Thermo DXR microscope with a 532 nm laser. The transmittance Fourier transform infrared (FTIR) spectra of NaYF4:Yb/Tm, PLL, gC3N4, and NaYF4:Yb/Tm-PLL@g-C3N4 were mixed with KBr powders and detected through a FTIR spectrometer (PerkinElmer, USA). A Leica TCS SP5 confocal microscope was used to obtain confocal microscopy in vitro images. Flow cytometry (FACSAria, BD) was utilized to quantify the fluorescence degree of cells. Measurement of ROS Production in Solution. The amount of ROS generated following in vitro PDT using NaYF4:Yb/Tm-PLL@gC3N4 was measured using ABDA and DPBF bioreagents following the manufacturer’s instructions.20,21 Briefly, NaYF4:Yb/Tm-PLL@g-C3N4 at a concentration of 1 mg/mL in water was sonicated for 20 min, and ABDA dye at a final concentration of 10 μM was added to the suspension solution. The fluorescence of ABDA was measured at 407 nm by PL. The suspension was then irradiated using 980 nm NIR light at a power of 1 W/cm2 for up to 60 min, measuring the fluorescence at every 20 min of irradiation, because the amount of generated ROS is proportional to the fluorescence intensity of ABDA. In addition, the generated ROS is also tested by a DPBF reagent at the same conditions, with the only difference being using a UV−vis spectrophotometer to detect the absoption of DPBF at 450 nm. Cell Viability Test. For cytotoxicity assay, an oral epidermoid carcinoma cell line (OEC-M1) was used as the in vitro model. The OEC-M1 cell line was grown in a RPMI-1640 medium mixed with 10% fetal bovine serum (FBS) and a 1% cocktail of penicillin/ streptomycin/L-glutamine (Gibco, USA). The OEC-M1 cells were incubated in a 5% CO2 incubator at 37 °C. Briefly, an aliquot containing 2000 cells/well was inoculated in a 96-well plate for 12 h. Serially diluted concentrations of a NaYF4:Yb/Tm-PLL@g-C3N4 solution (3, 9, 27, 81, and 250 μg/mL) were treated for another 48 h. After 48 h of incubation, Alamar Blue assay was performed according to the manufacturer’s protocol. Measurements in the Alamar Blue assay were obtained using SpectraMax M2 at an excitation/ emission of 560/590 nm (Molecular Devices, USA). Using the NIR treatment conditions, all cell test experiments will be exposed to

irradiation for a short time, to prevent the culture medium from overheating, with a 980 nm laser (1 W/cm2, 2 min break after 5 min of irradiation). Uptake and Localization of NaYF4:Yb/Tm-PLL@g-C3N4 at in Vivo Conditions. First, the OEC-M1 cell lines were seeded in 6-well plates at a density of 20000 cells/mL and incubated overnight, followed by incubation with NaYF4:Yb/Tm-PLL@g-C3N4 (250 μg/ mL) for 12 h at 37 °C. Next, the cells were washed with phosphatebuffered saline (PBS) to remove particles that were not internalized in cells. After cells were stripped by trypsin, cells would be collected by centrifugation, fixed with glutaraldehyde (2.5%), embedded in pure resin, cut into ultrathin sections, and stained by osmic acid. Finally, the uptake phenomena of NaYF4:Yb/Tm-PLL@g-C3N4 were examined by TEM imaging with a field-emission gun working at 80 kV. In Vitro Confocal Microscopy Image Analysis. The OEC-M1 cell lines (20000 cells/mL) were loaded in 6-well plates with coverslips for 12 h and then incubated with 250 μg/mL NaYF4:Yb/Tm-PLL@gC3N4 at 37 °C and a 5% CO2 atmosphere for another 12 h. After treatment, the culture medium and free NaYF4:Yb/Tm-PLL@g-C3N4 materials were removed and purified by PBS (10 mM, pH 7.4). The cells were then fixed with 4% paraformaldehyde. Subsequently, the nuclei of the cells on the coverslips were stained for 5 min with DAPIcontaining mounting gel. The treated and control cells were finally observed under a confocal microscope. The nuclei were visualized after excitement by a UV laser at 408 nm, and emission was detected at 450−500 nm. In addition, the NaYF4:Yb/Tm-PLL@g-C3N4s materials were excited at 980 nm, and their emission was detected at 450−500 nm. In Vitro ROS Investigation and Mitochondrial Image Analysis. The cell conditions and sample concentrations for ROS investigation and mitochondrial image acquisition are similar to those for cell imaging. The culture medium of OEC-M1 cells was exposed to 250 μL/mL NaYF4:Yb/Tm-PLL@g-C3N4 and labeled through 0.1 μg/ mL DAPI staining for 5 min. Moreover, the treated and control cells were stained with 20 μM DCFH2-DA and then subjected to a photosensitization experiment by irradiation at 1 W/cm2 using a 980 nm laser for 10 and 30 min. The results were obtained through confocal microscopy. DCFH2-DA was excited at 498 nm, and its emission was detected at 530 nm. To evaluate the mitochondrial transmembrane potential, JC-1 staining was made by a sensitive potential probe via a wavelength change of the fluorescence: JC-1 used excitation/emission of 540/570 nm to obtain green fluorescence and excitation/emission of 590/610 nm to obtain red fluorescence. In the experiment, JC-1 dye replaced DCFH2-DA and the other conditions were the same. Flow Cytometry JC-1 Analysis. The OEC-M1 cell lines were seeded at 2 × 105 cells/well into 6-well culture plates and incubated for 24 h. Then the medium was removed, and the cells were treated with a fresh medium containing 250 mg/mL NaYF4:Yb/Tm-PLL@gC3N4 at 37 °C with 5% CO2 for 24 h. After treatment with a 980 nm laser at different times, the cells were washed twice with PBS, then detached using a trypsin−ethylenediaminetetraacetic acid (EDTA) solution, and centrifuged at 2000 rpm for 10 min. The cells were suspended in 2 mL of PBS, and 1 × 104 cell counts were immediately analyzed using a flow cytometer (FACSAria, BD). In order to quantify the degree of JC-1 fluorescence, one filter of polyethylene would receive the red and orange light from gathering JC-1 and the other filter of fluorescein isothiocyanate would obtain the green light from monomer JC-1. Western Blot Analysis. Cells were lysed by a RIPA buffer solution (including 150 nM NaCl, 1% NP-40, 50 mM Tris-Cl at pH 8.0, 2 mM EDTA at pH 8.0, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitors). The primary antibodies were diluted in a 2% BSA/PBS buffer (137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 0.1% Tween 20 at pH 7.4). Cell apoptosis was examined using anticaspase 3 and antucaspase 9, which detected full-length caspase 3 and 9 (32 and 47 kDa), and the cleavage fragment (17 kDa). Anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the control group. Antisecondary antibodies were used to detect anticaspase 3, caspase 9, and anti-GAPDH. C

DOI: 10.1021/acs.inorgchem.6b01522 Inorg. Chem. XXXX, XXX, XXX−XXX

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3. RESULTS AND DISCUSSION This study used the coprecipitation method to dope Tm and Yb in UCNPs to obtain a mixture of blue-fluorescence-emitting material. Various concentrations of PLL were applied to modify the material surface by rendering positive charges. Finally, we synthesized g-C3N4, which emits green light. g-C3N4 was then combined with the PLL-modified UCNPs; the converted energy of the desired nanoparticles can emit UV band energy that is transferred into g-C3N4, which, in turn, produces green light and ROS (Scheme 1). The synthesis methods for triaxle

nanoscale g-C3N4 QDs are presented in ref 22 using the bottom-up approach. The authors proposed changes on the urea and sodium citrate concentrations to adjust the emission region of g-C3N4. The HRTEM image (Figure S1a) revealed that the diameters of g-C3N4 range from 2.6 to 3.5 nm. The inset in Figure S1a shows the representative image of an individual nanoparticle, which demonstrates high crystallinity with a lattice parameter of 0.35 nm. Furthermore, the g-C3N4 surface contributed some electrical charges, as confirmed by the ζ potential. Figure S1b shows that the surface potential of gC3N4 is approximately −34.5 mV; this is because of the g-C3N4 surface with a surface charge of different COO− functional groups. In addition, as previously proposed, adjusting the ratio of urea and sodium citrate of g-C3N4 can change the fluorescence from blue to green. The UV−vis spectra (Figure S2a) display a characteristic absorption peak of g-C3N4 at 344 nm, and a tunable emission of g-C3N4 can be achieved by simply adjusting the molar ratio of these two reactants. Four representative g-C3N4 powders were prepared by tuning the molar ratio of urea to sodium citrate from 3:1 to 6:1, 9:1, and 12:1 (Figure S2b). Figure S2d shows that all four g-C3N4 powders obtained demonstrate good solubility in water. Moreover, all of the obtained g-C3N4 powders dissolved in water emit bright fluorescence under UV irradiation (Figure S2c,d). As measured from the fluorescence spectra (Figure S2b), the fluorescence emission peaks of the four g-C3N4 QDs are located at 450, 467, 536, and 545 nm, further confirming that the tunable emission of g-C3N4 QDs can be achieved by tuning the molar ratio of the two reactants. Table S1 also shows that the quantum yield of the fluorescence emission spectra of the g-C3N4 QDs at excitation wavelengths were approximately 22%. This value is similar to the highest reported quantum yield of the g-C3N4 QD (29%).23 Moreover, NaYF4:Yb/Tm particles exhibiting a dominant hexagonal phase were prepared (Figure S3a). The NaYF4:Yb/Tm particles excited by a 980 nm laser and at emission intensities of 450 and 474 nm were obtained

Scheme 1. Distinct Generation of ROS by NaYF4:Yb/TmPLL@g-C3N4 Hybrids under NIR Illumination and Further Confirmation of the Possibility of Adopting Such Hybrids for PDT

Figure 1. (a) Schematic of the synthetic process of NaYF4:Yb/Tm-PLL@g-C3N4. (b) TEM image of NaYF4:Yb/Tm-PLL. (c) Magnified TEM image displaying a layer of polymer-modified PLL on NaYF4:Yb3+/Tm3+. (d) Modified g-C3N4 on the surface of the PLL layer (inset: magnified position of g-C3N4). D

DOI: 10.1021/acs.inorgchem.6b01522 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 2. Characteristic of NaYF4:Yb/Tm, NaYF4:Yb/Tm-PLL, and NaYF4:Yb/Tm-PLL@g-C3N4: (a) XRD analysis; (b) ζ potential; (c) FTIR spectrum; (d) Raman shift diagram.

were found in the EDS spectrum, indicating the successful conjugation of NaYF4:Yb/Tm with g-C3N4. The amounts of these elements were further quantified (Table S2). The XRD data (Figure 2a) show that, even after modification with PLL and after conjugation with g-C3N4, the hexagonal phase of the upconversion core did not demonstrate any change compared with JCPDS 16-0334. We use three analytical methods to ensure that PLL and g-C3N4 are linked on the NaYF4:Yb/Tm nanoparticles. Furthermore, the ζ potential (Figure 2a) became strongly positive after PLL conjugated on the surface of NaYF4:Yb/Tm nanoparticles. The combination of g-C3N4 with positively charged NaYF4:Yb/Tm-PLL reduced the charge, demonstrating that g-C3N4 was linked on the particle. These results were also proven by FTIR and Raman tests. After NaYF4:Yb/Tm particles combined with PLL, C−N stretching of the amine group was observed at 1180−1360 cm−1. The unique peaks of −COO− (1557 and 1457 cm−1) appeared again after NaYF4:Yb/Tm-PLL combined with g-C3N4 because the latter bears a negative surface that can bind with the positively charged NaYF4:Yb/Tm-PLL particles (Figure 2c). This result is also proven by a Raman test. Figure 2d shows typical Raman scattering. After combination with g-C3N4, spectra were respectively deconvoluted into D and G bands using two Gaussian curves.24 The measured intensity ratios of the D and G peaks (ID/IG) for these films range between 1.2 and 1.4. The obtained NaYF4:Yb/Tm particles exhibiting good dispersibility emitted upconverting light in the UV−vis regions, resulting from NIR laser excitation at 980 nm (Figure 3a). The

for quantification (Figure S3b). The morphology of the particles was observed under TEM (Figure S3c), and good monodispersibility of the particles in aqueous solution was achieved. In addition, fairly uniform spheres with diameters of 22−25 nm were obtained. To ensure the wide biological application of NaYF4 :Yb/Tm, we prepared ligand-free NaYF4:Yb/Tm particles, which were characterized by FTIR analysis. Figure S3d shows the unique peaks of −CH3 (2950− 2858 cm−1) and −COO− (1557 and 1457 cm−1); however, the unique peaks disappeared after ligand removal, and only −OH peaks were observed at 3430 and 1636 cm−1. Synthesis and Characterization of NaYF4:Yb/Tm-PLL@ g-C3N4. The synthetic route for NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposites can be summed up in three steps (Figure 1a). The original NaYF4:Yb/Tm core with a mean diameter of 25 nm (Figure 1b) was synthesized using the thermal decomposition method. After the ligand-free NaYF4:Yb/Tm cores were decorated with PLL (Figure 1c), the g-C3N4 whose surface is negatively charged can combine with the positively charged NaYF4:Yb/Tm-PLL under hydrolysis conditions. Figure 1d shows that the g-C3N4 QDs are attached to the surface of the NaYF4:Yb/Tm-PLL particles, forming NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposites. Moreover, material composition was analyzed by energy-dispersive spectrometry (EDS; Figure S4a,b). First, elemental peaks representing Na, F, Y, and Yb were observed, indicating that these elements constitute NaYF4:Yb/Tm. After NaYF4:Yb/Tm conjugated with a PLL ligand and g-C3N4, novel elemental peaks of N E

DOI: 10.1021/acs.inorgchem.6b01522 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 3. (a) UV−vis absorption (blue line) of the obtained g-C3N4 QDs and PL (black line) spectra of NaYF4:Yb3+/Tm3+ particles. (b) PL spectra of NaYF4:Yb/Tm cores, NaYF4:Yb/Tm-PLL, and NaYF4:Yb/Tm-PLL@g-C3N4. (c) Upconversion emission spectra under different concentrations of PLL-modified NaYF4:Yb/Tm-PLL@g-C3N4 demonstrated under a 980 nm laser. (d) 545 nm green-light emission of NaYF4:Yb/Tm-PLL@gC3N4 excited by a 980 nm laser. Upconversion excited lifetimes at (e) 360 and (f) 474 nm for NaYF4:Yb/Tm-PLL@g-C3N4.

Figure 4. PL changes of ABDA treated with water, g-C3N4, and NaYF4:Yb/Tm by a UV lamp and a NIR laser. (a) g-C3N4 with UV light after 365 nm irradiation for different times and the detection of ROS generation by ABDA. (b) Percentages used to ensure a decrease of the ABDA agent. Different PLL concentrations were used to link NaYF4:Yb/Tm cores and g-C3N4. (c) NaYF4:Yb/Tm-PLL@g-C3N4 modified with PLL (10 mg/mL) under a 980 nm laser. (d) Using percentages of measure of the loss of fluorescence to show the ROS results.

F

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Inorganic Chemistry UV emission peaks of Tm3+ ions can be derived from radioactive transitions of 1I6 → 3F4 (348 nm) and 1D2 → 3H6 (365 nm), and the visible emission can be assigned to 1D2 → 3 F4 (453 nm) and 1G4 → 3H6 (480 nm) transitions of the Tm3+ ions, respectively. Given the suppressed surface quenching of bare NaYF4, the relative emission intensities of NaYF4:Yb/TmPLL@g-C3N4 were approximately 20 times that of the original NaYF4:Yb/Tm core under similar measurement conditions. After modification of g-C3N4 outside the cores with a transferred upconverting emission, all of the emission peaks of the NaYF4:Yb/Tm cores weakened. The emission intensity of the UV region is remarkably diminished, resulting from the absorbance of UV emission light by g-C3N4 (Figure 3b). Moreover, these energy-transfer phenomena can be proven by the generation of new 535 nm green-light emission. This result indicates that part of the energy in the UV region can induce gC3N4 both to facilitate electron transfer and to generate light. To determine the best conditions for NaYF4:Yb/Tm-PLL@gC3N4 nanocomposites, we used different PLL concentrations to obtain improved surface potential and to facilitate combination with g-C3N4. Parts c and d of Figures 3 show that when a greater amount of PLL was added, more g-C3N4 combined with NaYF4:Yb/Tm cores and a greater emission intensity of the UV region was absorbed. However, 545 nm of green light produced the strongest emission at 10 mg/mL PLL. Thus, we choose this PLL concentration to synthesize our subsequent samples. Analysis of the time-dependent PL behavior (or luminescence lifetime) of upconversion emissions is a good way to verify the rate constants (Figure S5). The emission decay times of the NaYF4:Yb/Tm cores and NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposites were measured at 360 and 474 nm. The light source of the lifetime instrument is a pulse excitation source. Using square-wave excitation with a pulse width of 20 ms, a replication rate of 2 Hz (power density 0.12 W cm−2), and a signal gain of 8.7, the UPNPs were tested. Parts e and f of Figures 3 show that, at 360 nm, the PL lifetime demonstrated an obviously decreasing (from 0.18 to 0.07 ms) trend after combining with g-C3N4; however, at 474 nm, the PL lifetime decreased only slightly (from 0.29 to 0.28 ms). This trend in the PL lifetime demonstrated that g-C3N4 can greatly absorb UV light emitted from the NaYF4:Yb/Tm cores, and a small amount of its blue-ray emission was absorbed. ROS Generation of NaYF4:Yb/Tm-PLL@g-C3N4 Nanocomposites via NIR Irradiation. Cytotoxic intracellular ROS can cause damage to DNA, mitochondria, and plasma membranes of live cells, resulting in cell death. Therefore, the ability to generate extracellular and intracellular ROS of the photosensitizer is one of the crucial factors that determine the efficacy of PDT. We initially used a UV lamp to excite g-C3N4, and the chemical probe ABDA was used to detect g-C3N4. ABDA can react with ROS irreversibly and then reduces the intensity of its characteristic emission at around 430 nm (Figure 4a); the intensity of ABDA emission decreased by more than 65% in 1 h. This phenomenon ensured that only g-C3N4 excited by a UV lamp can generate the ROS effect (Figure 4b). Extracellular ROS was generated by NaYF4:Yb/Tm-PLL@gC3N4 upon NIR laser excitation at 980 nm (Figure 4c). In addition, the intensity of ABDA decreased exponentially (an approximately 45% decrease per hour) with increasing NIR illumination time, confirming the efficient generation of extracellular ROS. We also used different PLL concentrations to ensure that more g-C3N4 nanocomposites combine with NaYF4:Yb/Tm cores and thus more ROS effects can be

detected (Figure 4d). To confirm the impact on the experimental conditions under UV, the 407 nm emission of ABDA was decreased under UV light (around 20%) in Figure S6a. However, g-C3N4 produces more decay under UV light because of the generation of ROS. According to Figure S2, gC3N4 has a certain absorption at 407 nm (around 60%). We test different samples irradiated under 407 nm. In Figure S6b,c, compared with g-C3N4 and nanocomposites under UV and NIR excitation, ROS generated at 407 nm is relatively low and the decrease percentages of ABDA fluorescence are 20% and 12%, respectively. Otherwise, we used another chemical probe, DPBF, to prove the ROS effects. When the solution contained ROS, singlet oxygen reacted with DPBF and reduced the absorption compared with that in ABDA. Figure S6d shows an obvious decline in the absorption after NaYF4:Yb/Tm-PLL@gC3N4 was irradiated by a 980 nm laser. In Vitro Cell Viability Test by NaYF4:Yb/Tm-PLL@gC3N4 in NIR-Triggered PDT. To confirm the stability, we conducted the following experiments to analyze changes of the samples in a serum environment. We soaked 1 mg of our major sample, 10 mg/mL PLL-modified NaYF4:Yb/Tm-PLL@gC3N4, in 1 mL of serum for 6, 12, 24, 48, and 72 h, respectively. TEM, DLS, and ζ potential were used to test the samples. The results are shown in Figures S7 and S8. In Figure S7, after treatment with 10% w/w FBS, we used visual imaging and TEM to check whether NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposites were still uniformly dispersed in the water solution. In Figure S8, DLS and ζ potential curves demonstrate that the changes were not obvious after NaYF4:Yb/Tm-PLL@ g-C3N4 was soaked in FBS for long as 72 h. It was confirmed that our materials remained stable in a serum solution. Low cellular toxicity is one of the most important characteristics of g-C3N4. Therefore, we tested the cellular toxicity of NaYF4:Yb/Tm, NaYF4:Yb/Tm-PLL, and NaYF4:Yb/ Tm-PLL@g-C3N4 with different concentrations. This test was performed by Alamar Blue assay (Figure 5a). The results demonstrated that all samples had minimal apparent cytotoxicity. The light-induced anticancer drug clearly demonstrated that NaYF4:Yb/Tm-PLL@g-C3N4 was cytotoxic to OEC-M1 cells. Our experimental design was employed under irradiation for a short time to prevent the culture medium from overheating when irradiated by a NIR laser at 980 nm (1 W/cm2, 2 min break after 5 min of irradiation). Figure 5b showed that the toxicity of NaYF4:Yb/Tm-PLL@g-C3N4 was only observed under 980 nm irradiation. Compared with the two control groups, NaYF4:Yb/Tm and NaYF4:Yb/Tm-PLL have low cytotoxicity after irradiation under 980 nm. To delineate NaYF4:Yb/Tm-PLL@g-C3N4 with or without the effects of cell uptake, NaYF4:Yb/Tm, NaYF4:Yb/Tm-PLL, and NaYF4:Yb/ Tm-PLL@g-C3N4 were incubated with the OEC-M1 cells in Figure 5b. Laser scanning confocal microscopy (CLSM) confirmed the cellular uptake of NaYF4:Yb/Tm, NaYF4:Yb/ Tm-PLL, and NaYF4:Yb/Tm-PLL@g-C3N4. The intensity of intracellular blue fluorescence emitted from Tm3+ directly reflects the amount of internalization of these samples. OECM1 were incubated in different samples (Figure 6), and the results showed that a significantly greater number of NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposites incubated with OEC-M1 cells were taken up compared with NaYF4:Yb/Tm but less than NaYF4:Yb/Tm-PLL. However, linking g-C3N4 onto NaYF4:Yb/Tm-PLL possibly reduced the uptake of materials by the cells. In order to be able to determine the G

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Figure 6. (Left) Confocal microscopy analysis showing the OEC-M1 cell line with (a) blank, (c) NaYF4:Yb/Tm, (e) NaYF4:Yb/Tm-PLL, and (g) NaYF4:Yb/Tm-PLL@g-C3N4, overlapping with a bright field. (Right) CLSM images showing the interactions of OEC-M1 cells with (b) blank, (d) NaYF4:Yb/Tm, (f) NaYF4:Yb/Tm-PLL, and (h) NaYF4:Yb/Tm-PLL@g-C3N4, overlapping with the fluorescence imaging.

Figure 5. (a) Viability and (b) cytotoxicity tests of NaYF4:Yb/Tm, NaYF4:Yb/Tm-PLL, and NaYF4:Yb/Tm-PLL@g-C3N4 to OEC-M1 cancer cells in vitro. For the cell toxicity test, these samples were subjected to a 980 nm NIR laser and measured by Alamar Blue assay.

distribution of nanoparticles in the cellular environment, TEM measurements and costaining experiments with commercial dyes, LysoTracker Red DND-99, were done after 12 h of cell incubation (Figures S9 and S10). Compared with the control group (Figure S9a), NaYF 4 :Yb/Tm nanoparticles and NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposites (Figure S9b,c) can be internalized by cells and then accumulated in the lysosome. In addition, lysosomal staining can also prove that these nanocomposites internalized in lysosome (Figure S10). This in vitro single-cell evidence demonstrates that the nanocomposites could be taken up through an endocytosis pathway.25 In addition, we use z-axis and orthogonal sectioning confocal microscopy imaging to demonstrate a phagocytic situation. As presented in Figure S11a, the NaYF4:Yb/TmPLL@g-C3N4 particles accumulated between the nucleus and membrane. This reveals that the materials have been internalized by the cells and then enter into the cytoplasm. Orthogonal sectioning imaging (Figure S11b,c) detected only a single cell and observed that the particles would be distributed around the nucleus. In conclusion, the different phenomena observed in different samples were possibly caused by differences in the surface charges. Given that plasma membranes bear negative charges, materials carrying positive charges can enhance the subsequent receptor-mediated

endocytosis. In order to further determine the effect of ROS in the cell, we selected the DCF agent to confirm whether ROS is within the cells due to such a light-mediated PDT pathway. Moreover, the intracellular ROS levels in OEC-M1 cells were measured by DCFH2-DA. DCFH2-DA can diffuse into the cell membrane and is hydrolyzed by the enzyme hydrolase within the cell, resulting in nonfluorescent DCFH2. DCFH2-DA can diffuse into the cell membrane and is hydrolyzed by the enzyme hydrolase within the cell, resulting in nonfluorescent DCFH2. When ROS is generated in the cells, ROS can oxidize DCFH2 and cause DCF fluorescence emission of the cells. Thus, the intracellular ROS produced in DCFH2-DA-treated cells may serve as an indicator to detect and quantify the activity of generated oxygen within the cells. Fluorescence of DCFH2-DAtreated cells under different conditions can be excited at 498 nm, and green fluorescence was generated at 530 nm, as signals reveal. Figure 7 shows that adding NaYF4:Yb/Tm- PLL@gC3N4 only led to low ROS concentration in the cells, and the cells did not produce a green DCF fluorescence signal. When NaYF4:Yb/Tm-PLL@g-C3N4 was irradiated under a 980 nm laser for 10 min (1 W/cm2, 2 min break after 5 min of irradiation), ROS production was significantly increased in the cells, and the ROS content can be detected through DCF H

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recorded: death receptor-mediated apoptosis (extrinsic pathway) and mitochondria-mediated apoptosis (intrinsic pathway). Mitochondria are organelles’ means of core survival in most apoptotic pathways. A change in the mitochondrial membrane potential (MMP, ΔΨm) is an early event in apoptosis, and the health of mitochondria can be assessed by JC-1 staining. High ΔΨm indicates healthy mitochondria. JC-1 will accumulate and gather in the mitochondrial matrix, resulting in red fluorescence that can surpass 590 nm to excite the orange fluorescence emission at 610 nm.26 Conversely, if the cells are damaged, resulting in reduced MMP, JC-1 will form a monomer and produce green fluorescence. This green fluorescence can be excited at 540 nm and emit 570 nm green light. Furthermore, the OEC-M1 cells were treated with NaYF4:Yb/Tm-PLL@gC3N4 with or without NIR irradiation, and a 20 μM JC-1 reagent was added into the cell culture medium followed by confocal microscopy (Figure 8). The results when NaYF4:Yb/ Tm-PLL@g-C3N4 alone or NIR-irradiated NaYF4:Yb/TmPLL@g-C3N4 was used greatly varied. Without illumination, the mitochondria emitted red light, revealing that the upper region of the mitochondria was not damaged. When the OECM1 cells were treated with NIR light, the PDT-mediated red fluorescence gradually faded, indicating loss of MMP. The gradual increase in green fluorescence indicated damage in the mitochondrial membrane. To further confirm the above results, we quantify the degree of JC-1 green fluorescence by flow cytometry. The OEC-M1 cells were treated with NaYF4:Yb/ Tm-PLL@g-C3N4 under an irradiating NIR laser, stained with JC-1, followed by flow cytometry (Figure S12). Flow cytometry results showed control cells (Figure S12a), NIR light alone (Figure S12b), and NaYF4:Yb/Tm-PLL@g-C3N4 alone (Figure S12c), and only a few cells were detected in green-emitting

Figure 7. Amount of intracellular ROS evaluated by a ROS-sensitive probe, DCFH2-DA. After treatment with NaYF4:Yb/Tm-PLL@gC3N4 under a 980 nm NIR laser, the level of DCFH2-DA decreased and was transferred to the DCF fluorescent probe.

fluorescence. Moreover, upon irradiation under a 980 nm laser 30 min later, the intensity of green fluorescence surged, confirming that NaYF4:Yb/Tm-PLL@g-C3N4 can produce a significant ROS effect and is a suitable PDT nanocomposite under irradiation with a 980 nm laser. Cell Apoptosis Mechanisms via a Mitochondrial Damage Pathway. Two major apoptotic pathways were

Figure 8. NIR-mediated PDT-induced mitochondrial damage in OEC-M1 cells. Cells treated with NaYF4:Yb/Tm, NaYF4:Yb/Tm-PLL, and NaYF4:Yb/Tm-PLL@g-C3N4 (250 μg/mL) were irradiated under a 980 nm laser (2.8 W/cm2, 30 min, 5 min break after 10 min of irradiation). The nuclei of the cells were stained blue by Hoechst 33324, and the mitochondria were stained with JC-1. A decrease in red fluorescence in the irradiated cells indicates a loss of MMP. I

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ACKNOWLEDGMENTS The authors thank the Ministry of Science and Technology of Taiwan (Contract Nos. MOST 104-2113-M-002-012-MY3, MOST 104-0210-01-09-02, and MOST 105-0210-01-13-01) for financially supporting this research. This work is also supported by the NSFC (Grants U1305244 and 21325104) and the CAS/SAFEA International Partnership Program for Creative Research Teams. Thanks go to C. Y. Chien of the Precious Instrument Center (National Taiwan University) for assistance in the TEM experiments and Li-Wen Lo of The Genomics Research Center, Academia Sinica, for help with confocal microscopy. The Molecular Imaging Center of National Taiwan University, National Center for HighPerformance Computing, Taiwan, and Research Center for Applied Sciences, Academia Sinica, Taiwan, are thanked for their support.

expression (from monomer JC-1), which are localized in the Q2 region of the plot. It reveals no mitochondrial damage. After the OEC-M1 cells were treated with NIR light-mediated PDT, the localization was shifted rightward over different irradiation times (Figure S12d,e), which indicated a loss of MMP and mitochondrial damage. In order to track the apoptotic pathway, we chose caspase 3 and 9 of these two apoptosis markers to make further confirmation by Western blot.27,28 In Figure S13, the left line was added only nanocomposite as the control group, while the right line was added nanocomposite and irradiated with a NIR laser for 10 min as the experimental group. Comparing these two groups, caspase 3 and 9 revealed the high expression of the cleaved forms of caspase 3 and 9 (17 kDa) in the irradiation with a NIR laser. These phenomena referred to NaYF4:Yb/Tm-PLL@gC3N4, confirming the induction of OEC-M1 cell death through the apoptosis pathway, which could be attributed to the toxic ROS, resulting in further damage to the mitochondria.

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ABBREVIATIONS UCNP, upconversion nanoparticles; g-C3N4 QDs, graphitic carbon nitride quantum dots; PDT, phototdynamic therapy

4. CONCLUSIONS In summary, we reported that this photosensitizing platform could, via a NIR laser, generate ROS for PDT based on a NaYF4:Yb/Tm-PLL@g-C3N4 nanocomposite. NaYF4:Yb/TmPLL@g-C3N4 can efficiently produce extracellular and intracellular ROS by converting deeply penetrating NIR light into UV light to excite the g-C3N4 QDs attached on the UCNPs. The results of the investigation on in vitro anticancer activity suggest that induction of apoptosis in NaYF4:Yb/Tm-PLL@gC3N4-treated cells by NIR light-mediated PDT is possibly associated with disruption of the mitochondrial function. We provide an NIR light-mediated strategy for in vivo PDT using NaYF4:Yb/Tm@g-C3N4. Therefore, the NaYF4:Yb/Tm@gC3N4 demonstrates efficient PDT effects and good imaging capability and is a promising multifunctional cancer therapy nanoplatform for simultaneous bioimaging and therapy.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b01522. TEM images of g-C3N4 and UCNP, ζ potential, UV−vis spectrum and PL characterization of g-C3N4, XRD and FTIR characterization of NaYF4:Yb/Tm, proposed ROS reaction of NaYF4:Yb/Tm-PLL@g-C3N4 with DPBF, and a supported in vitro test of NaYF4:Yb/Tm-PLL@gC3N4 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions

All authors have given approval to the final version of the manuscript. M.-H.C. and R.-S.L. designed and performed the experiments and cowrote the manuscript. C.-W.C., I.-J.L., Y.C.C., and D.T. provided technical support and materials. M.H., C.-H.C., X.C., and R.-S.L. discussed the data, and developed the theoretical aspect. Notes

The authors declare no competing financial interest. J

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