A Versatile Near Infrared Light Triggered Dual-Photosensitizer for

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A Versatile Near Infrared Light Triggered Dual-Photosensitizer for Synchronous Bioimaging and Photodynamic Therapy Lili Feng,† Fei He,*,† Yunlu Dai,† Bin Liu,† Guixin Yang,† Shili Gai,† Na Niu,‡ Ruichan Lv,§ Chunxia Li,⊥ and Piaoping Yang*,† †

Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin 150001, P. R. China ‡ College of Sciences, Northeast Forestry University, Harbin 150050, P. R. China § School of Life Science and Technology, Xidan University, Xi’an 710071, P. R. China ⊥ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) based on Tm3+-activated up-conversion nanoparticles (UCNPs) can effectively eliminate tumor cells by triggering inorganic photosensitizers to generate cytotoxic reactive oxygen species (ROS) upon tissue penetrating near-infrared (NIR) light irradiation. However, the partial use of the emitted lights from UCNPs greatly hinders their application. Here we develop a novel dual-photosensitizer nanoplatform by coating mesoporous graphitic-phase carbon nitride (g-C3N4) layer on UCNPs core, followed by attaching ultrasmall Au25 nanoclusters and PEG molecules (named as UCNPs@gC3N4−Au25-PEG). The ultraviolet−visible (UV−vis) light and the intensive near infrared (NIR) emission from UCNPs can activate g-C3N4 and excite Au25 nanoclusters to produce ROS, respectively, and thus realize the simultaneous activation of two kinds of photosensitizers for enhanced the efficiency of PDT mediated by a single NIR light excitation. A markedly higher PDT efficacy for the dual-photosensitizer system than any single modality has been verified by the enhanced ROS production and in vitro and in vivo results. By combining the inherent multi-imaging properties (up-conversion, CT, and MRI) of UCNPs, an imaging guided therapeutic platform has been built. As the first report of dual-inorganic-photosensitizer PDT agent, our developed system may be of high potential in future NIR light induced PDT application. KEYWORDS: photodynamic therapy, g-C3N4, photosensitizer, up-conversion, imaging, Au25 nanoclusters

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INTRODUCTION

result in unsatisfied therapeutic effect in practical PDT applications.19−21 To date, despite tremendous efforts have been made, it is still a significant challenge and important to develop novel photosensitizer that has the merits of high light conversion efficiency, high stability, and sensitive response to near-infrared (NIR) light. Recently, a novel metal-free semiconductor nanomaterial graphitic-phase carbon nitride (g-C3N4) has emerged, which can be susceptible to ultraviolet and visible light region due to its relatively narrow band gap energy of approximately 2.71 eV. Moreover, g-C3N4 is a thermal and chemical stability semiconductor with excellent

Photodynamic therapy (PDT) has drawn considerable attention in the field of cancer therapy on account of its noninvasion nature, negligible drug resistance, and excellent therapeutic effects.1−12 In a typical PDT process, light with a certain wavelength is used to stimulate photosensitizer drugs to generate reactive oxygen species (ROS) to ablate malignant cells.13−15 Thus, the property of photosensitizer plays an important role in determining the PDT effect. The commonly used photosensitizers containing chlorin derivatives, porphyrin, and ZnPc have been extensively reported, and some of them have been verified for clinical application.16−18 However, these photosensitizers typically have narrow absorption region, premature leakage, and poor hydrophilicity in aqueous media owing to π−π stacking or hydrophobic interactions, which © 2017 American Chemical Society

Received: January 13, 2017 Accepted: April 3, 2017 Published: April 3, 2017 12993

DOI: 10.1021/acsami.7b00651 ACS Appl. Mater. Interfaces 2017, 9, 12993−13008

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ACS Applied Materials & Interfaces

Scheme 1. Schematic Illustration for the Synthesis of UCNPs@g-C3N4−Au25-PEG Nanocomposite and Application as a DualPhotosensitizer for Multimodality Imaging-Guided PDT in Vitro and in Vivo

to solve this question. Recently, thiolate-protected gold nanoclusters (Au25 NCs) have appeared as a new kind of broadly developing prospective photosensitizer, and they exhibit strong absorption in the visible and NIR regions.62−64 The absorption of NIR light is especially conducive to PDT application due to the optimal tissue penetration depth. More importantly, the introduction of gold nanoclusters semiconductor may demonstrate an increased light absorption and effective separation photogenerated electron−hole pairs, which can promote the production of hydroxyl radicals (•OH) and singlet oxygen (1O2), which answer for killing cancer cells.65−68 Hence, exploration of novel NIR-excited dual-photosensitizer drug with high light utilization efficiency and abundant ROS generation for PDT is crucial. For the purpose of abundant ROS production and effective therapeutic response, here we built a novel and multifunctional dual-photosensitizer anticancer nanoplatform for NIR laser mediated and multiple imaging guided PDT. The mesoporous structure, excellent stability, and biocompatibility have been realized by coating mesoporous g-C3N4 shell on UCNPs core, followed by attaching ultrasmall Au25 nanoclusters and PEG molecules (denoted as UCNPs@g-C3N4−Au25-PEG). The emitted UV and visible lights from Tm3+ can activate g-C3N4 to yield ROS, and the intensive NIR emission can excite Au25 NCs, thus realizing the simultaneous activation of two kinds of photosensitizers mediated by a single 980 nm NIR light. In addition, the amusing NIR excitation/emission characteristics integrate the advantages of deep tissue penetration with high sensitivity, minimization of photodamage to biological tissue, and low autofluorescence of NIR light, being anticipated to serve as an optical probe for deep tissue imaging and improve the PDT effect. The physiochemical properties, the ROS production capability, and in vitro and in vivo anticancer performance of this nanoplatform have been investigated in detail.

biocompatibility, nontoxicity, inherent blue light photoluminescence, and high quantum yield.22−26 Nevertheless, up to now, the application report of g-C3N4 in PDT is still relatively rare. The first factor for this situation can be on account of the excitation wavelength of g-C3N4 located in the UV−vis region, which may have unexpected phototoxicity and limited tissue penetration depth.27,28 Especially, the insufficient light absorption and fast electron−hole recombination can greatly decrease the generation rate of ROS under the light irradiation, which directly affects the therapeutic efficiency.29,30 Hence, it is hard to satisfy the condition of clinical PDT application for gC3N4 before these critically technical problems are solved, and more research efforts are still urgent. Up-conversion nanoparticles (UCNPs)-based photosensitizers have been presented as an emerging technology, which can get over several limitations of conventional photosensitizers.31−45 UCNPs can emit high-energy photons (UV− vis and NIR) when irradiated by low-energy NIR light. The utilization of NIR light as an irradiation source has its inherent advantages including the crucial adjective minimization of photodamage to biological tissue, an unexceptionable signal-tonoise ratio, together with enhanced detection sensitivity owing to the inexistence of autofluorescence.46−50 Moreover, the emitted UV and visible lights can activate the photosensitizer to yield ROS through the process of fluorescence resonance energy transfer (FRET) and then avoid impair to spare healthy tissues and poor tissue penetration depth excitation with short wavelength.51−56 Even more remarkably, Tm3+-doped UCNPs attracted particular interest due to the up-converted emission at around 800 nm, which is markedly higher than that of UV−vis emission upon NIR laser excitation.57−61 Although the adsorption band of g-C3N4 has been enhanced from UV to UV−vis region compared with some inorganic photosensitizers,33 and the UV−vis light can also be utilized through the Tm 3+ -induced up-conversion process under NIR light excitation, the dominant NIR emission at about 800 nm of Tm3+ still cannot be used, which leads to low light utilization efficiency. Therefore, combining a photosensitizer on this platform, which can be responsive to NIR light, should be able

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RESULTS AND DISCUSSION Synthesis and Characterization of the Samples. The detailed preparation process and bioapplication of dual12994


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Figure 1. TEM images of (a) NaYF4:Yb/Tm, (b) NaYF4:Yb/Tm@NaGdF4:Yb (UCNPs), (c) UCNPs@mSiO2, (d) UCNPs@mSiO2@g-C3N4, and (e) UCNPs@g-C3N4. (f) Scanning transmission electron microscopy (STEM) image and the corresponding EDS elemental mapping images of UCNPs@g-C3N4−Au25-PEG.

The small size feature of as-prepared negatively charged Au25 NCs with an average particle diameter of 2.36 nm is confirmed by TEM (Figure S2). By electrostatic attraction, the Au25 NCs can be easily adhered on the surface of UCNPs@g-C3N4, which can be attributed to the existence of some uncondensed amine functional groups, which are more stable for Au25 NCs coating in the g-C3N4 shell layer. In the TEM images (Figure 1f), the integrated Au25 NCs do not arise to influence the actual dimension of UCNPs@g-C3N4 because Au25 NCs are too small to be observed. Encouragingly, the corresponding energy dispersive spectrometer (EDS) element mapping gives evidence of the elements, which are distributed uniformly, from which the successful creation of mesoporous shell and the well-dispersed Au25 NCs in UCNPs@g-C3N4−Au25-PEG can also be proved (Figure 1f). Furthermore, the color of asprepared sample changes from white to dark-brown after Au25 NCs loading (inset in Figure S3), which supplementarily verifies the successful loading of Au25 on the UCNPs@g-C3N4 surface. The size of UCNPs@g-C3N4 before and after loading Au25 NCs was also analyzed via dynamic light scattering (DLS) measurement, and the increased size from 100 to 118 nm may be ascribed to the interaction between water molecules and Au25 adsorption on the surface of nanoparticles (Figure S3). In addition, the zeta potential of UCNPs@g-C3N4 nanoparticles changes from the positively charged (+11.6 ± 1.1 mV) to negatively charged (−9.6 ± 2.3 mV) after Au25 NCs decoration (Figure S4), which presents additional evidence for the successful surface modification of Au25 NCs. The introduction of g-C3N4 layer between UCNPs and Au25 NCs not only presents dual-photosensitizer optical properties, but also distinctly inhibits the luminescence quenching effect. The coating amount of g-C3N4 on the surface of UCNPs and the

photosensitizer are illustrated in Scheme 1. Monodispersed core−shell structured UCNPs were prepared and coated with mesoporous silica in accordance with a previously accessible route with simple modification and served as substrates to synthesize dual-photosensitizer.67 First, core−shell structured UCNPs were obtained by a high temperature pyrolysis method to synthesize β-NaYF4:Yb,Tm core, and then external shell (βNaGdF4:Yb) was coated on the surface by sequential epitaxial growth process. Transmission electronic microscopy (TEM) images revealed that the UCNPs are well-dispersed hexagonal nanocrystals with an average particle size changes from 20 to 32 nm after shell growth process (Figure 1a,b). Then the hydrophobic UCNPs were transferred into hydrophilic phase by surface modification with cetyltrimethylammonium bromide (CTAB), which also can be used as surfactant to produce mesoporous silica shell through sol−gel process. The UCNPs@ mSiO2 nanoparticles were achieved after the surfactant was removed. The uniformly dispersed UCNPs@g-C3N4 NPs demonstrated the mean particle size of around 90 nm with outer-shell thickness 27 nm, which were successfully prepared by using monodisperse UCNPs@mSiO2 NPs as template, cyanamide (CY) as nitrogen and carbon sources, and ammonium fluoride as a etchant. Briefly, CY was deposited into the pores of the mesoporous silica shell, subsequently switched into UCNPs@g-C3N4 via the thermal-induced selfcondensation of CY at 550 °C after removal of the silica template with NH4F. The representative TEM images are presented to confirm the significant contrast difference (Figure 1c−e). The as-prepared UCNPs@g-C3N4 possesses huge specific surface area (621 m2 g−1) and mesoporous nanoarchitecture with the average pore size 3.07 nm (Figure S1), which were utilized to load large amount of Au25 NCs. 12995


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Figure 2. (a) XRD patterns of UCNPs, UCNPs@g-C3N4, UCNPs@g-C3N4−Au25-PEG, and the standard JCPDS card 16−0334 of hexagonal NaYF4. (b) FT-IR spectra of UCNPs, UCNPs@g-C3N4, and UCNPs@g-C3N4−Au25-PEG. (c) XPS survey spectra of UCNPs@g-C3N4−Au25-PEG. (d) High resolution C 1s spectra of UCNPs@g-C3N4−Au25-PEG, fitted to two energy components centered at around at 284.6 and 288.3 eV. (e) High resolution N 1s spectra of UCNPs@g-C3N4−Au25-PEG, fitted to three energy components centered at around at 398.9 eV, 399.7 eV, and 401.2 eV. (f) High resolution Au 4f spectra of UCNPs@g-C3N4−Au25-PEG, fitted to two energy components centered at around at 83.6 and 84.5 eV.

loading amount of Au25 NCs are 22% and 1% determined elemental analyzer and ICP-MS, respectively. Afterward, the assynthesized dual-photosensitizer was modified with PEG through Au−S bonds so as to enhance their water solubility and extend blood circulation time in live system. After PEG modification, the zeta potential changes to (+5.8 ± 1.3 mV). PEGylated dual-photosensitizer is light brown in color with a hydrodynamic size about 136 nm and exhibited excellent stability in physiological solution. It should be noted that although the system seems to be complicated in terms of construction, the synthesis methods are mature and facile to make the system be constructed with excellent morphology and component in each step. Accordingly, a novel nanoplatform with all-in-one “smart” functions for simultaneous diagnosis and cancer therapy was successfully designed, where the cancer therapy effect may combine the multimode imaging function to form a photodynamic therapeutic platform and achieve the real time monitoring performance in the process of cancer treatment. Moreover, this designed system allows large specific surface area, high loading amount, almost no leakage of dual-

photosensitizer, high light utilization efficiency, and good interfacial interaction between donor (UCL core) and acceptor (g-C3N4 and Au25), which are favorable for generating a large amount of ROS upon 980 nm NIR light irradiation. The phase composition and structure of the as-synthesize samples in different steps were verified by XRD pattern, which are given in Figure 2a. XRD pattern of as-prepared UCNPs directly matches with hexagonal phase NaYF4 corresponding to the encoding of JCPDS file number 16−0334, the wide peaks reveal the nanosized structure. For UCNPs@g-C3N4, the typical peak at 27.8° is attributed to aromatic segments or the coating of amourphous g-C3N4. In the case of UCNPs@gC3N4−Au25-PEG, three distinct peaks at 38.2°, 44°, and 64.7° could be found, which are consistent with 111, 200, and 220 reflections for Au25 NCs, respectively. FT-IR spectra of the samples synthesized at different steps are demonstrated in Figure 2b. After coating g-C3N4, a broad band at 3200−3500 cm−1 was detected and assigned to NH stretching vibration mode and some hydroxyl groups on the surface of the nanoparticles. The presence of NH stretching vibration mode 12996


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ACS Applied Materials & Interfaces suggests that some uncondensed amine functioned groups still exist in the g-C3N4 shell. For UCNPs@g-C3N4−Au25-PEG, the band at 810−880 cm−1 is assigned to the CN heterocycles of triazine units. The Au−O stretching vibration band at 818 cm−1 is detected. No extra characteristic peak of Au−O vibration is observed due to the overlap of Au−O vibration band with CN heterocycles. In addition, XPS was used to analyze the state of all the elements in UCNPs@g-C3N4−Au25-PEG (Figure 2c). Figure 2d is the high-resolution C 1s spectrum of UCNPs@gC3N4−Au25-PEG, and the two peaks observed at 284.6 and 288.3 eV are attributed to the carbon contamination and N− C−N coordination. It can be observed from Figure 2e that N 1s is divided into three peaks at 398.9, 399.7, and 401.2 eV, respectively. The main peak at 398.9 eV can be attributed to sp2-hybridized nitrogen in triazine rings (CN−C), and the other two peaks at 399.7 and 401.2 eV are given the credit to tertiary nitrogen (N−(C)3) and N−H bonding, respectively. In the Au 4f spectrum (Figure 2f), the peaks at 83.6 and 84.5 eV are derived from Au 4f5/2 and Au 4f7/2 electrons of Au25 NCs. The Au25 NCs loading ratio on the g-C3N4 surface is estimated to be 0.96% by the XPS data. From the high-resolution S 2p spectrum of UCNPs@g-C3N4−Au25-PEG (Figure S5), the two peaks detected at 162.6 and 164.5 eV are assigned to the Au−S and S−H bonds, suggesting the formation of Au−S bond. Therefore, it can be summarized that electrostatic attraction effectively integrated nanosized Au25 NCs with UCNPs@gC3N4, which is also affirmed by above XRD and FT-IR results. Optical Properties and ROS Generation. The upconversion emission spectra of the neat UCNPs in cyclohexane, UCNPs@g-C3N4, and UCNPs@g-C3N4−Au25-PEG in water are given in Figure 3a. Upon irradiation of 980 nm NIR light, the photoluminescence spectra of these three samples are similar. There are five emission bands at 348, 362, 453, 475, and 800 nm, which can be attributed to 1I6 → 3F4, 1D2 → 3H6, 1D2 → 3F4, 1G4 → 3H6, and 3H4 → 3H6 transitions of Tm3+, respectively. After the coating of g-C3N4, the up-conversion emission intensity of UCNPs@g-C3N4 evidently declines in comparison with the initial intensity. This is mainly due to the FRET process from UCNPs to g-C3N4. Moreover, it may also be attributed to an augment in the multiphonon relaxation in aqueous media triggered by high-energy vibration of water molecules. For UCNPs@g-C3N4−Au25-PEG, the emission intensity is further decreased, which is assigned to the migration, transfer of charge carriers, separation and recombination process of the photogenerated electron−hole pairs, and the nonradiative energy transfer from UCNPs@g-C3N4 to Au25 NCs via the stronger quantum size effects. The quantum yield of the final UCNPs@g-C3N4−Au25-PEG upconversion nanomaterials can be measured as 2.11%, which is conducive to obtain fluorescence imaging effect and an excellent PDT effect (Figure S6). The UV−vis absorption of the as-prepared samples was analyzed, as presented in Figure 4. The UV−vis absorption spectrum of the as-synthesized dark-brown Au25 NCs (Figure 4a) consists of four characteristic absorption peaks, which are consistent with the previously reports.69,70 Figure 4c and d demonstrate that the UCNPs@g-C3N4 absorption edge of g-C3N4 is around 500 nm, which corresponds to a band gap of 2.71 eV. Compared with UCNPs@g-C 3 N 4 , the UV−vis absorption spectrum of UCNPs@g-C3N4−Au25-PEG exhibits strong capacity of light absorption in UV, visible, and NIR regions (Figure 4e and f), which can be assigned to the inherent absorption and quantum size effects of Au25 NCs. In addition, the absorption spectrum

Figure 3. (a) UC emission spectra of UCNPs, UCNPs@g-C3N4, and UCNPs@g-C3N4−Au25-PEG under 980 nm laser irradiation. The emission intensity from 300−700 nm is displayed three-times higher than the real value. (b) Decay curves for the 1G4 → 3H6 emission (475 nm) of Tm3+ in UCNPs, UCNPs@g-C3N4, and UCNPs@g-C3N4− Au25-PEG. The same concentrations of UCNPs, UCNPs@g-C3N4, and UCNPs@g-C3N4−Au25-PEG (400 μg mL−1) were used for fluorescence detection.

on the photon energy scale was also measured, and the absorption bands of UCNPs@g-C3N4−Au25-PEG can be detected at about 1.62, 2.21, and 2.54 eV. Compared with the absorption spectrum of Au25 NCs (Figure 4a and b), the visible and NIR absorption bands of the composite are from the loaded Au25 NCs. After loading of Au25 NCs, the separation of electron−hole pair is more effective due to the direct transfer of photoexcited electron from g-C3N4 conduction band to Au25 NCs. Notably, the complete overlay of the absorption and emission spectrum of UCNPs@g-C3N4−Au25-PEG makes it possible to yield more abundant ROS excited by NIR light on account of the FRET process. Therefore, the introduction of Au25 NCs on UCNPs@g-C3N4 significantly enhances the utilization rate of light, which also plays a crucial role in improving the quantum yield of ROS for PDT application. The ROS generation mechanism of UCNPs@g-C3N4−Au25-PEG upon 980 nm NIR laser excitation is schematically illustrated in Scheme 2. To further study the FRET effect of UCNPs@gC3N4−Au25-PEG irradiation with 980 nm laser, we tested the fluorescence decay curves of 1G4 → 3H6 (475 nm) transition of Tm3+ in UCNPs, UCNPs@g-C3N4 and UCNPs@g-C3N4− Au25-PEG, as given in Figure 3b. It is obvious that the fluorescent lifetime becomes shorter after coating the g-C3N4 shell and loading the Au25 NCs on UCNPs, which exhibits an effective FRET process. In spite of the reduced fluorescence intensity and lifetime owing to the FRET effect, the emission intensity of UCNPs@g-C3N4−Au25-PEG can still meet the requirements for in vitro and in vivo upconversion luminescence bioimaging, as discussed in following section. The generation of ROS was examined via the chemical probe, 2,7-dichlorofluorescein-diacetate (DCFH-DA). The 12997


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Figure 4. UV−vis absorption spectra of (a, b) Au25 solution (single crystals redissolved in aqueous solution, inset is the photograph of Au25 NCs in water). UV−vis diffuse reflectance spectra of (c, d) UCNPs@g-C3N4 and (e, f) UCNPs@g-C3N4−Au25-PEG.

Scheme 2. UV−vis Absorption Spectrum (Black) and Emission Spectrum (Red) of UCNPs@g-C3N4−Au25-PEG upon 980 nm NIR Laser Excitation and Schematic Illustration for the ROS Generation Mechanism of UCNPs@g-C3N4−Au25-PEG Irradiated 980 nm NIR Light

C3N4-xAu25−PEG with different content of Au25 NCs irradiated by 980 nm NIR light for 1 and 5 min was measured (Figure S7a−b). As shown, the fluorescence intensity of DCF increases first and then declines with the increasing Au25 concentration but for a certain value, which suggests the optimal amount is 1 wt %. Figure 5a demonstrates the increase in photo-

nonfluorescent DCFH can be oxidized by ROS to form 2,7dichlorofluorescein (DCF), which emits green fluorescence. DCF fluorescence emission intensity at 525 nm would be detected in the presence of ROS. For the ROS generation, herein, different weight percentages of Au25 NCs were loaded on UCNPs@g-C3N4. The ROS production over UCNPs@g12998


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Figure 5. (a) Photoluminescence spectra of DCF as a sensor for detecting the ROS generation from UCNPs@g-C3N4−Au25-PEG under 980 nm NIR laser irradiation for various times. (b) Intracellular ROS generation detected in HeLa cells treated with UCNPs@g-C3N4−Au25-PEG under 980 nm light irradiation. The level of intracellular ROS was measured by FACSCalibur flow cytometry using the peroxide-sensitive fluorescent probe DCFH-DA. (c) CLSM images of HeLa cancer cells after incubation with UCNPs@g-C3N4−Au25-PEG with 980 nm NIR light irradiation dealt with DCFH-DA for 15 and 30 min. All images share the same scale bar of 50 μm.

luminescence intensity of UCNPs@g-C3N4−Au25-PEG at 525 nm illuminated with 980 nm laser for different times. Meanwhile, the ROS generation from UCNPs@mSiO2−Au25PEG, UCNPs@g-C3N4−PEG, and blank contrast (only NIR irradiation) was also measured (Figure S7c−e), and the relative photoluminescence intensity changes at 525 nm with varied illumination time are presented in Figure S7f. Furthermore, the initial reaction rate (Q) of DCFH coexists with ROS as well as UCNPs@mSiO2@Au25−PEG, UCNPs@g-C3N4−PEG, and UCNPs@g-C3N4@Au25−PEG were calculated as in the following equations:62

where the R value is defined as the initial slope (ΔPL/Δtime). PL represents the photoluminescence intensity of these samples at 525 nm (UCNPs@mSiO2@Au25−PEG, UCNPs@g-C3N4− PEG, and UCNPs@g-C3N4@Au25−PEG). The values of Q U C N P s @ m S i O 2 @ A u 2 5 ‑ P E G /Q U C N P s @ g ‑ C 3 N 4 @ A u 2 5 ‑ P E G and QUCNPs@g‑C3N4‑PEG/QUCNPs@g‑C3N4@Au25‑PEG are calculated to be 0.74 and 0.65, indicating a relatively high ROS quantum yield of UCNPs@g-C3N4@Au25−PEG dual-photosensitizer. It is apparent that ROS production efficiencies of UCNPs@mSiO2− Au25-PEG and UCNPs@g-C3N4−PEG are lower in comparison with that of UCNPs@g-C3N4−Au25-PEG. The Au25 evenly dispersed on the surface of UCNPs@g-C3N4 can lead to more effective electron−hole separation and generate a large amount of ROS. The more immediate proof for the ROS production (· OH and 1O2) by UCNPs@g-C3N4−Au25-PEG upon 980 nm NIR laser excitation can be achieved via electron paramagnetic resonance (EPR) measurement (Figure S8). The characteristic peaks of both 1O2 and ·OH can be clearly detected upon 980 nm NIR illumination. In addition, the generation of intracellular ROS was estimated via monitoring the fluorescence intensity of DCF, which is converted from nonfluorescent DCFH-DA when oxidized by ROS, after HeLa cells were incubated with UCNPs@g-C3N4−Au25-PEG NPs shielded from light, and then illuminated by 980 nm laser. The intracellular fluorescence intensity assessed by flow cytometer

Q UCNPs@mSiO2@Au25 − PEG = RUCNPs@mSiO2@Au25 − PEG/PL UCNPs@mSiO2@Au25 − PEG

Q UCNPs@g − C3N4 − PEG = RUCNPs@g − C3N4 − PEG/PL UCNPs@g − C3N4 − PEG Q UCNPs@g − C3N4@Au25 − PEG = RUCNPs@g − C3N4@Au25 − PEG/PL UCNPs@g − C3N4@Au25 − PEG 12999


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UCNPs@g-C3N4−Au25-PEG can be served as an excellent luminescence probe. In addition, the Gd3+-based upconversion nanoparticles are usually designed for T1-weighted MRI. Because of the presence of Gd3+ in UCNPs, our UCNPs@gC3N4−Au25-PEG could serve as a T1 contrast agent for MR imaging. T1-weighted MR images of UCNPs@g-C3N4−Au25PEG solution by a 1.5T MR scanner indicate a concentrationdependent brightening effect, showing a longitudinal (r1) of 0.9195 mM−1 s−1 (Figure S10). After intravenous injection of UCNPs@g-C3N4−Au25-PEG NPs into tumor-bearing mice, obviously brightened signal is examined in the tumor region of mice from in vivo MR imaging. Owing to the better tissue penetration and high-resolution 3D structure, X-ray CT has been widely applied in clinical diagnostics. In previous works, it has been revealed that Yb3+ ions in UCNPs could absorb X-ray and produce strong contrast in CT imaging. On account of these characters, we detected the CT contrast efficacy of UCNPs@g-C3N4−Au25-PEG because the as-synthesized sample contains significant amount of Yb3+. CT images of UCNPs@g-C3N4−Au25-PEG solution demonstrate the linear increase of Hounsfield unit (HU) values with the enhanced concentration of Yb3+, which indicates that contrast effect is hinged on the Yb3+ content (Figure 6). The slope of HU value

(FCM) gives a comparison of relative amount of ROS generated with different illumination time. Figure 5b exhibits the time-dependent fluorescence intensity of DCF. As shown, there are almost no ROS detected without irradiation, and the DCF fluorescence intensity gradually increases with illumination time. Moreover, the CLSM images of HeLa cancer cells after incubation with UCNPs@g-C3N4−Au25-PEG under 980 nm NIR light irradiation dealt with DCFH-DA were also presented to confirm the generation of intracellular ROS (Figure 5c). Analogously, with the increasing of illumination time, brighter green color can be detected in HeLa cells. The above results powerfully proved the generation of extracellular and intracellular ROS and UCNPs@g-C3N4−Au25-PEG, which can act as a potential candidate for NIR laser-induced PDT. Imaging, Uptake, Biocompatibility, and in Vitro Antitumor Properties. Nowadays, imaging diagnostic has been emerging a powerful technology in assisting cancer therapy in terms of early detection and targeting therapy. The UCL with large Stokes shift, sharp emission lines, and superior photostability involves the multiphoton processes of converting the low-energy photons (NIR) into high-energy photons (UV, visible, or NIR). UCL imaging, which relates to the NIR excitation light, possesses high excitation penetration depth in biological tissues and negligible photodamage to living organisms. In addition, Gd-doped UCNPs with unpaired 4f electrons can be acted as T1 contrast agent for MRI, which performs in a noninvasive fashion to gain functional information and high soft-tissue contrast. MRI affords excellent spatial resolution (several tens of micrometers), high penetration depth, and has practice in a clinical setting. UCL and MRI imaging are two complementary modalities that will combine both the advantages of high spatial resolution and sensitivity when integrated into one system, thereby enhancing the quality of bioimaging. CT imaging is clinically diagnostic technique owing to the deep tissue penetration and highresolution 3D structure details. The achievement of multimodality imaging into one single system can make full use of the merits of each modality, thereby enhancing the quality of bioimaging and improving the diagnosis and therapeutic effect. The multiple imaging effects can also combine with the phototherapy effects under NIR light irradiation to achieve realtime imaging-guided cancer therapy. Nowadays, PDT has been used in clinical tumor therapy and shown a good application prospect. Therefore, such a structurally optimized nanomaterial demonstrates interesting properties for clinical PDT application. The time course upconversion luminescence microscopy (UCLM) photographs treated with UCNPs@g-C3N4−Au25PEG nanoparticles for different time (0.5, 1, and 3 h) in the CO2 incubator were performed by an external 980 nm laser as the excitation light source, as presented in Figure S9. A bright blue luminescence of HeLa cells can be found in dark field, which is consistent with the upconversion emission spectrum of UCNPs@g-C3N4−Au25-PEG. It is obvious that a growing number of nanoparticles are internalized into the cells with prolonging incubation time. The stronger fluorescence without any cell autofluorescence can be detected, which demonstrates that the cellular uptake of nanoparticles is time-dependent. Figure S9b and c exhibit the in vivo UCL imaging of asprepared UCNPs@g-C3N4−Au25-PEG. Bright bluish-violet color can be observed after hypodermic injection of UCNPs@g-C3N4−Au25-PEG NPs to the tumor site upon 980 nm light irradiation. These results indicate that the as-prepared

Figure 6. (a) In vitro CT images of UCNPs@g-C3N4−Au25-PEG at different concentrations. (b) HU value of aqueous solution of UCNPs@g-C3N4−Au25-PEG nanoparticles as a function of the concentration. (c) CT imaging and 3D renderings of CT images of a tumor-bearing Balb/c mouse: pre- (top panel) and postinjection (bottom panel) in situ.

of UCNPs@g-C3N4−Au25-PEG was calculated to be 51.68. Then in vivo CT imaging was conducted by intratumoral injection with UCNPs@g-C3N4−Au25-PEG (400 μg mL−1, 100 μL). As presented, the CT value is up to 424.3 HU after injection, which is far higher than that without injection (39.8 HU). The CT imaging results imply that the UCNPs@gC3N4−Au25-PEG produce a great contrast effect for X-ray CT scanning diagnosis. Beyond that, to further investigate cellular uptake process of the nanoparticles, confocal laser scanning microscope (CLSM) photographs of HeLa cells incubated with UCNPs@g-C3N4− Au25-PEG for different time were carried out, as given in Figure S11. In the first 0.5 h, only a few of nanoparticles could be swallowed by HeLa cells. It is obvious that the blue fluorescence attributed to the material itself (g-C3N4) increases near the nucleus with incubation time up to 1 h. We can see that fluorescence intensity monotonically increases with the 13000


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Figure 7. (a) Cell viabilities quantitative assay by standard MTT proliferation test versus incubation concentration (0−800 μg mL−1) of UCNPs@gC3N4−Au25-PEG nanoparticles for 24 h. (b) Hemolysis assay for UCNPs@g-C3N4−Au25-PEG (inset: photographic images for direct observation of hemolysis by UCNPs@g-C3N4−Au25-PEG using PBS as a negative control and water as a positive control (left two tubes), and UCNPs@g-C3N4− Au25-PEG suspensions with different concentrations). (c) Cell viability versus incubated particle concentration (12.5, 25, 50, 100, 200, 400, and 800 μg mL−1) for HeLa cells under different conditions (NIR, UCNPs@g-C3N4−Au25-PEG, UCNPs@g-C3N4−PEG with NIR, UCNPs@mSiO2−Au25PEG with NIR, and UCNPs@g-C3N4−Au25-PEG with NIR). (d) CLSM image of HeLa cancer cells after incubation with culture and UCNPs@gC3N4−Au25-PEG upon 980 nm laser irradiation dyed with calcium AM and PI. Error bars indicate standard deviations, n = 5. All images share the same scale bar of 50 μm.

phototoxicity of 980 nm laser was also investigated. As presented in Figure S12, the cell viability did not decline when irradiation upon 980 nm NIR laser under various laser power for 30 min (5 min break after 5 min irradiation). The short interval irradiation with 980 nm laser could avoid the overheating phenomenon. The above analysis indicated that the 980 nm laser irradiation is safe for the cells. Besides, we utilized cellular staining to estimate the photodynamic toxicity triggered by UCNPs@g-C3N4−Au25-PEG nanoparticles. Calcein-Am/ proiclium iodide staining is a simple and convenient method to distinguish live and dead cells. Under the same culture conditions, the HeLa cells treated with UCNPs@g-C3N4− Au25-PEG irradiated by 980 nm light are almost completely killed, while no dead cells can be observed for the cells incubated with culture medium (Figure 7d), agreeing well with MTT results. To exclude of the influence of heat effect, we performed in vitro and in vivo photothermal experiment by thermal imager (Figure S13). From the experimental results, we can clearly find that the samples do not produce apparent heat effect under NIR laser irradiation for various times. In Vivo Anticancer Efficiency. Encouraged by the remarkable PDT efficiency in vitro and the excellent biocompatibility to the live cells of the as-prepared sample, we therefore performed preliminary phototherapy to confirm the potential of the nanoplatform as an efficient dualphotosensitizer for light-induced PDT in vivo. H22-tumorbearing mice were randomly divided into six groups (five mice per group) in this experiment. The mice were subcutaneously inoculated with H22 tumor cells at left armpit, and the tumor size was allowed to reach about 100 mm3 (5 days after tumor inoculation). Group one injected silane (100 μL) was utilized for blank control. The mice intravenously injected with

incubation time prolonged to 3 h, which also confirms a mass of UCNPs@g-C3N4−Au25-PEG nanoparticles are internalized in the nucleus. The time course CLSM images verify that the as-synthesized nanoparticles can be effectively taken up by HeLa cells since the cytotoxicity of dual-photosensitizer is a vital prerequisite and crucial factor for further biomedical application. Typical methyl thiazolyl tetrazolium (MTT) cell assay was taken on L929 cervical carcinoma cell line to confirm the in vitro cytotoxicity of the UCNPs@g-C3N4−Au25-PEG nanoparticles. As shown in Figure 7a, the cell viabilities are close 100% after incubation with UCNPs@g-C3N4−Au25-PEG nanoparticles for 24 h, revealing no apparent toxic effect in wide concentration range. The excellent biocompatibility may satisfy the demand as potential photosensitive drug for biological applications. In addition, the blood compatibility of the sample was examined to make sure its possible application in vivo. As presented in Figure 7b, no appreciable hemolysis phenomenon can be observed when UCNPs@g-C3N4−Au25PEG NPs were added with different concentrations, indicating the excellent blood compatibility. The cancer cell inhibition effect of UCNPs@g-C3N4−Au25-PEG was performed by a typical MTT assay. Culture mediums containing UCNPs@gC3N4−PEG, UCNPs@mSiO2−Au25-PEG, and UCNPs@gC3N4−Au25-PEG nanoparticles were placed into specific well with different treatment, followed by further incubation for another 24 h. The majority of HeLa cells treated with UCNPs@g-C 3 N 4−Au 25 -PEG are killed after NIR laser irradiation (Figure 7c), while the treated cells in other groups exhibit either negligible or much less cell death after exposure to the laser irradiation. The analysis strongly reveals that the UCNPs@g-C3N4−Au25-PEG is pharmacologically active as a promising dual-photosensitizer than any single modality. The 13001


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Figure 8. (a) In vivo tumor growth curves of tumor-bearing mice in different groups after various treatments. The tumor sizes were measured at the indicated time points. The relative tumor volumes were normalized to their initial volumes before the treatment. (b) Body weight changes of Balb/c mice versus time under different treatments (control, NIR, UCNPs@g-C3N4−Au25-PEG, UCNPs@g-C3N4−PEG with NIR, UCNPs@mSiO2−Au25PEG with NIR, and UCNPs@g-C3N4−Au25-PEG with NIR). Error bars indicate standard deviations (5 mice per group). Error bars indicate standard deviations, n = 5 (∗∗, p < 0.01 as compared with the control group). (c) Digital photographs of tumor-bearing mice after different treatments and corresponding images of excised tumors from representative Balb/c mice after 14 day treatment. (d) H&E-stained tumor sections after 14 day treatment from different groups (control, NIR, UCNPs@g-C3N4−Au25-PEG, UCNPs@g-C3N4−PEG with NIR, UCNPs@mSiO2−Au25-PEG with NIR, and UCNPs@g-C3N4−Au25-PEG with NIR). All images share the same scale bar of 50 μm.

UCNPs@g-C 3 N 4 −Au 25 -PEG, UCNPs@g-C 3 N 4 −PEG, or UCNPs@mSiO2−Au25-PEG are from groups 2−4, respectively. After 48 h, the tumor sites were treated with NIR irradiation last for 30 min (2.5 W cm−2, 5 min break after 5 min irradiation, group 2, 3, 4). Group 5 was injected with UCNPs@ g-C3N4−Au25-PEG nanoparticles in the dark without NIR irradiation, and group 6 was injected with silane with NIR laser illumination (group 6). In each group, the injected amount was 100 μL (400 μg mL−1), and all groups adopted second nanoparticles injection (4 days after first injection) and treatment. In the process of the experiments, no mice died. The average tumor volume and weight change of the tumorbearing mice were estimated every 2 days. As presented in Figure 8a and b, the weight change of the tumor-bearing mice kept steady as time went on, indicating the little adverse side effect of the materials. Yet, the variation of relative tumor volume of different treatment group is complicate. The group treatment with only NIR laser shows slight inhibition of tumor growth than the control group. The group treated with UCNPs@g-C3N4−Au25-PEG in the absence of NIR laser excitation exhibits the similar tumor growth inhibition efficiency. The relatively superior anticancer therapy effects of UCNPs@g-C3N4−PEG and UCNPs@mSiO2−Au25-PEG with NIR irradiation suggest the effect of PDT. Excitingly, the tumor growth in the mice treated with UCNPs@g-C3N4−Au25-PEG

and exposed to NIR irradiation are dramatically inhibited after 14 days treatment. The curves verify that UCNPs@g-C3N4− Au25-PEG achieves better therapeutic effect than other groups. The results again reveal the efficacy of UCNPs@g-C3N4−Au25PEG served as a dual-photosensitizer for PDT. The representative photographs of the tumors ablated from mice in different groups of are presented in Figure 8c. The group treated with UCNPs@g-C3N4−Au25-PEG irradiated by 980 nm NIR laser has a tumor growth inhibition efficacy of 91%, and the tumor size is much smaller than other groups on a certain degree. Accordingly, more necrosis and necrotic tumor cells can be apparently detected in this group (Figure 8d). Little damage can be detected in the tumor tissue sections of the control group, which is consist of tightly circulating tumor cells. The aforementioned results are in accordance with the tumor growth curve, undoubtedly proving UCNPs@g-C3N4−Au25PEG as a dual-photosensitizer has the superior anticancer efficiency than any single modality. We also performed histology analysis of major organs, which include spleen, lung, heart, liver, and kidney from mice after 14 days with various therapeutic methods (Figure 9). No pathological changes can be examined in the main organs after posttreatment, showing a low invasion of the materials to these organs. In addition, ICP-MS was utilized to analyze biodistribution of different nanoparticles (UCNPs@mSiO2@ 13002


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Figure 9. Representative histological H&E stained tissue sections from mice to monitor the histological changes in heart, liver, spleen, lung, and kidney were collected from different groups followed by dissections in 14 days postinjection. All images share the same scale bar of 50 μm.

Au25−PEG, UCNPs@g-C3N4−PEG, and UCNPs@g-C3N4− Au25-PEG) in mice at various intravenous injection points (Figure S14−15) and in vivo blood circulation time of UCNPs@g-C3N4−Au25-PEG. Meanwhile, as the main metabolic products, urine and feces were collected to measure the body clearance. For different nanoparticles injection group, similar experimental results are achieved. Because the spleen is the largest immune organ and lung has abundant blood capillary network with comparatively small diameter, the nanoparticles can accumulate in these organs. In the early stages after the injection (30 min, 1 h, 4 h, and 12 h), the nanoparticles are mainly accumulated in the livers, spleens, and lungs. Meanwhile, the concentrations of nanoparticles are very low in heart and kidney at all time points after postinjection. After 24 h of injection, the Gd concentration reduces in all the organs. We can find that only few samples can be monitored in the liver, spleen, and lung in the seventh day, revealing that the nanoparticles could be excreted from the mice with time prolonged. The pharmacokinetics of blood circulation time curve is exhibited in Figure S16. The longer circulation of UCNPs@g-C3N4−Au25-PEG nanoparticles in blood is conducive to biological diagnosis and cancer therapy. The concentration of Gd in the urine is low at all times from the metabolism of UCNPs@g-C3N4−Au25-PEG experimental results, which reveal that kidney may not be the main metabolic organ. The results of the above analysis could be strong proof to comfirm that Gd3+ cannot separate from UCNPs@g-C3N4− Au25-PEG. Through the analysis on the first day of collecting feces, approximately 60.1% of nanoparticles have been clearanced via the liver and bile (inset in Figure S16). Taken together, the above experimental results suggest the side effects of UCNPs@g-C3N4−Au25-PEG nanoparticles can be negligible after intravenous injection into mice at the current dose and

could be acted as an efficient PDT agent for treatment of malignant cancers.

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CONCLUSIONS In summary, an original and highly efficient single wavelength NIR laser activated dual-photosensitizer with multimodal imaging and PDT function has been designed and fabricated via coating mesoporous g-C3N4 layer on the surface of UCNPs and then attaching Au25 NCs by electrostatic interaction. The dual-photosensitizer exhibits more effective separation electron−hole pairs and markedly higher light utilization efficiency and PDT efficacy than any single modality due to the full use of the emitted light from UCNPs. The cytotoxicity and PDT effect estimated by MTT assay demonstrate that the dualphotosensitizer can obvious impair cancer cells owing to the generation of ROS. The therapeutic efficacy performed through animal experiment and tissue section analysis suggests the tumor can be distinctly inhibited or eradicated and no damage is detected to major organs. Therefore, we predict a promising future of this novel dual-photosensitizer in the diagnosis and treatment of malignant cancers, featured with NIR laserinduced multiple image and PDT function.

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EXPERIMENTAL SECTION

Reagents and Materials. All of the aforementioned chemicals were used without further purification. Re2O3 (99.99%) was all purchased from Sinopharm Chemical Reagent. Oleic acid (OA), 2,7dichlorofluorescein-diacetate (DCFH-DA), 1-octadecene (ODE), and cyanamide were purchased from Sigma-Aldrich. Hydrochloric acid (HCl), cyclohexane, sodium fluoride (NaF), cetyltrimethylammonium bromide (CTAB, ≥ 99%), tetraethylorthosilicate (TEOS), trifluoroacetic acid (CF3COOH), sodium trifluoroacetate (CF3COONa), and 13003


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Preparation of Au 25 (MHA) 18 − Loaded UCNPs@mSiO 2 (UCNPs@g-C3N4−Au25) and UCNPs@g-C3N4 (UCNPs@g-C3N4− Au25). In a feasible procedure, as-obtained UCNPs@mSiO2 solution (8 mg mL−1) was vigorously stirred last 3 h shield from light. Afterward, Au25(MHA)18− solution was appended to the abovementioned solution, then continue to whisk for a further 8 h. The UCNPs@mSiO2-Au25 was achieved by centrifugalization and dried under a vacuum environment. Accordingly, different contents of Au25 loaded nanoparticles were synthesized by the same procedure with varying the quantity of Au25(MHA)18− solution. UCNPs@g-C3N4− Au25 was prepared by the similar procedure except for using UCNPs@ g-C3N4 to replace UCNPs@mSiO2. Preparation of PEG Modified UCNPs@g-C3N4−Au25 (UCNPs@ g-C3N4−Au25-PEG), UCNPs@mSiO2−Au25 (UCNPs@g-C3N4− Au25-PEG), and UCNPs@g-C3N4 (UCNPs@g-C3N4−PEG). In a typical route to synthesize UCNPs@g-C3N4−Au25-PEG, HS-PEGCOOH (10 mg) was placed into deionized water (20 mL) with asacquired UCNPs@g-C3N4−Au25. After being vigorously stirred for 24 h, the nanoparticles were collected by ultrasonication and washed several times with water, which was denoted as UCNPs@g-C3N4− Au25-PEG. UCNPs@mSiO2−Au25-PEG and UCNPs@g-C3N4−PEG were synthesized by the similar procedure. Characterization. Powder X-ray diffraction (XRD) patterns were achieved from a Rigaku D/max TTR- III diffractometer with Cu Kα radiation (λ = 0.15405 nm). Images were carried out on a transmission electron microscope (TEM, FEI Tecnai G2 S-Twin). N2 adsorption/ desorption isotherms were acquired on a Micromeritrics ASAP Tristar II 3020 apparatus. Pore size distribution was estimated via the Barrete−Jonere−Halenda (BJH) method. The surface-charging characteristics of the product were detected using a zetasizer instrument. UCL emission spectra were measured on Edinburgh FLS 980 apparatus using a 980 nm laser as the irradiation source. FTIR spectra were obtained using an AVATAR 360 FT-IR spectrophotometer using a KBr technique. The UV−vis absorbance spectra of the solutions were measured by UV-1601 spectrophotometer. A flow cytometer (FCM, BD Biosciences) with an excitation wavelength of 485 nm was used to quantify the transfection efficiency of each sample. Extracellular and Intracellular ROS Detection. 2,7-Dichlorofluorescein (DCFH) was utilized as a chemical probe for single oxygen measurement. In a feasible detection procedure, the DCFH was formed after a mixture solution containing methanol solution of DCFH-DA (0.5 mL, 1 mmol L−1) and NaOH (2 mL, 0.01 mol L−1) was under continuous stirring for 30 min shielded from light. Moderate sodium phosphate buffer (pH = 7) was placed into the above solution to make it to be neutral. The UCNPs@g-C3N4−Au25PEG nanoparticles (10 μg mL−1) were mixed with DCFH (25 mmol L−1) and then illuminated with 980 nm NIR light for different periods of time. The nonfluorescent DCFH can oxidize by ROS to form 2,7dichlorofluorescein (DCF) carrying green fluorescent rapidly. The quantum yield of ROS was assessed by detecting the fluorescence intensity. For the control experiments, DCF emission was also recorded in the absence of UCNPs@g-C3N4−PEG, UCNPs@mSiO2− Au25-PEG for comparison under the same condition. In a basic experimental procedure for the detection of intracellular ROS detection, after incubate HeLa cells with UCNPs@g-C3N4−Au25PEG nanoparticles for 6 h, PBS was utilized to wash away the noninternalization nanoparticles, and fresh culture media containing DCFH-DA (20 μm) was placed and incubated for another 20 min shielded from light. After irradiated with 980 nm light for different times (2.5 W cm−2), the laser was shut down, and the illuminated cells were collected through a series of operations to obtain single cell suspension. The FACSCalibur flow cytometer was employed to analyze the intracellular ROS using 485 and 525 nm as the respective excitation wavelength and emission wavelength, respectively. In Vitro Cytotoxicity of UCNPs@g-C3N4−Au25-PEG. Typically, HeLa cells were planted in 96-well plate (7000 well−1) and then cultured in 5% CO2 at 37 °C overnight to let the cultured cells grew against the wall of flask. In the first two groups, HeLa cells were incubated with UCNPs@g-C3N4−Au25-PEG for approximately 4 h, and then excitation upon 980 nm laser was performed last 30 min

6-mercaptohexanoic acid (MHA) were obtained from Beijing Chemical Regent. Preparation of Core−Shell Structure β-NaYF4:Yb/Tm@βNaGdF4:Yb. The core−shell structure β-NaYF4:Yb/Tm@β-NaGdF4:Yb nanoparticles has been synthesized through a previous scheme with a little modification. First, for the fabrication of β-NaGdF4:Yb/ Tm, 1 mmol of RE(oleate)3 (RE = 79.5% Y + 20% Yb + 0.5% Tm), 20 mL of oleic acid (OA)/1-octadecene (ODE) (v/v = 1/1), and 12 mmol of NaF component solvent were placed to the flask and temperatured to 110 °C upon a vacuum condition last 30 min to remove of impurities. Afterward, the solution was heated again, and the temperature was kept at 300 °C last 90 min with the protection of nitrogen. The β-NaYF4:Yb/Tm core nanoparticles were achieved through centrifugalization and washed by the ethanol and cyclohexane. For the synthesis of core−shell structure nanoparticles, a template method has been employed. Typically, for the synthesis of βNaYF4:Yb/Tm@β-NaGdF4:Yb sample, 5 mL of cyclohexane stock solution with β-NaYF4:Yb/Tm core sample and 1 mmol CF3COONa, 0.9 mmol Gd(CF3COO)3, and 0.1 mmol Yb(CF3COO)3 were placed into a mixture solution consist of OA (15 mL) and ODE (15 mL) in a vessel. The solution was then stirred last 40 min vigorously, and the temperature was improved to 120 °C under the condition of vacuum for a certain time to remove of impurities. Since then, the solution was temperatured to 310 °C for 60 min in N2. After the temperature down to 25 °C, the β-NaYF4:Yb/Tm@β-NaGdF4:Yb sample was denoted as UCNPs can be obtained by centrifugalization and washed by the ethanol and cyclohexane. They were finally dispersed in cyclohexane solution. Preparation of UCNPs@mSiO2 Nanoparticles. UCNPs@mSiO2 nanoparticles were prepared on the basis of the slightly modified procedure as we previously reported.71 Typically, 2 mL of cyclohexane solution with certain concentration β-NaYF4:Yb/Tm@β-NaGdF4:Yb UCNPs (approximately 4−8 mg mL−1) and 0.1 g of CTAB were added into 20 mL of deionized water. The transparent UCNPs-CTAB mixture solution was acquired after remove cyclohexane by stirred vigorously. Afterward, half of the UCNPs-CTAB solution was mixed with deionized water (20 mL), ethanol (3 mL), and NaOH solutions (150 μL, 2 mol L−1). The mixed solution was heated up to 65 °C with continuous agitation. Next 150 μL of TEOS was dropped into above solution and kept stirring last 12 min. The product can be achieved by centrifugalization. CTAB was removed via an exchanged approach, where the as-obtained UCNPs@mSiO2 (25 mg) was shifted to 40 mL of ethanol consisting of 0.2 g of NH4NO3 and refluxed at 60 °C last 2 h. Ultimately, the UCNPs (β-NaYF4:Yb/Tm@β-NaGdF4:Yb)@mSiO2 nanoparticles with mesoporous shell were achieved. Synthesis of UCNPs@g-C3N4 Nanoparticles. A template method by applying mSiO2 shell as a sacrifice template was applied to obtain g-C3N4 mesoporous shell, and monodisperse UCNPs@ mSiO2 nanoparticles were used as a template to prepare UCNPs@gC3N4. Typically, 1 g of the UCNPs@mSiO2 nanoparticles template was added into 5 g of cyanamide (Alfa Aesar) and kept under ultrasonic vibration and vacuum at 60 °C last 3 h. Afterward, the mixture was put in water bath pot and stirred strictly at 60 °C for 16 h. The product was obtained by centrifugalization and washed by ethanol for three times. The obtained sample was dried, calcined in a crucible at 550 °C (with a ramp rate of 5 °C min−1) for 4 h, and then treated with ammonium fluoride for 8 h to elimination the silica template, and finally the samples were collected for the next step. Synthesis of Monothiolate-Protected Au25 NCs. Aqueous solutions of HAuCl4 (20 mmol L−1) and thiolate ligands (5 mmol L−1) were prepared with ultrapure water. On the basis of the detailed preparation process of MHA-Au25 NCs, aqueous solutions of HAuCl4 and MHA were mixed with moderate deionized water, which resulted in the formation of MHA-Au(I) complexes. An aqueous NaOH solution was then introduced into the above-mentioned mixture, followed by the addition of 0.1 mL of NaBH4 solution (synthesized by dispersing NaBH4 powder in NaOH solution). The Au25(MHA)18− nanoclusters were collected after 3 h for further characterizations and applications.16 13004


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UCNPs@g-C3N4−PEG, and UCNPs@mSiO2−Au25-PEG into the mice from groups 2−4 shielded from light. Group 5 was injected with UCNPs@g-C3N4−Au25-PEG nanoparticles in the dark without NIR irradiation. Analogously, group 6 was only excitation with 980 nm laser for 30 min without any other treatment. The injected amount was 100 μL (400 μg mL−1). After 48 h, the tumor sites were treated with 980 nm NIR irradiation for 30 min (2.5 W cm−2, 5 min break after 5 min irradiation, group 2, 3, and 4). The mice were anesthetized by chloral hydrate solution (5%, 0.1 mL per mouse), and the tumor surface was depilated before irradiation. All the groups adopted second nanocomposite injection (4 days after first injection) and treatment. The average tumor volume and weight change of the tumor-bearing mice were estimated every 2 days, up to 14 days. The diameters of tumors were gauged by a Vernier caliper in two dimensions and estimated according to the formula V = (width2 × length)/2. The average body weight of mice per group was also calculated. Relative tumor volume was estimated as V/V0 (V0 was the initiated volume of the tumor before treatment). In Vitro and Vivo X-ray CT Imaging. A CT scanner (120 kV) was utilized to detect and analyze the in vitro CT imaging quality. For in vitro CT imaging, the UCNPs@g-C3N4−Au25-PEG nanoparticles were dissolved in saline and diluted to different concentrations of 0, 1, 2, 4, 8, and 16 mg mL−1 and then put in 2 mL tubes. First, 10% chloral hydrate anesthetized was injected in the intraperitoneal of tumorbearing mice to execute in vivo CT imaging. After that, 100 μL of UCNPs@g-C3N4−Au25-PEG (400 μg mL−1) was intratumorally injected into the tumor-bearing mice in situ for in vivo CT imaging. In Vitro and In Vivo T1-Weighted MR Imaging. The quality of in vitro MR imaging was operated on a 0.5 T MRI magnet. UCNPs@ g-C3N4−Au25-PEG nanoparticles were dissolved in deionized water with different Gd3+ concentrations. T1 signal values were measured using a nonlinear fit to changes in the mean signal intensity within each well versus repetition time (RT) with a 1.5 T MR scanner. At last, the values of r1 relaxivity were evaluated via the fitted curve of 1/T1 relaxation time (s−1) on the basis of Gd3+ concentrations. First, 10% chloral hydrate anesthetized was injected in the intraperitoneal of tumor-bearing mice to execute in vivo MRI imaging. Afterward, the scanned was performed on tumor-bearing mice before and after 100 μL of UCNPs@g-C3N4−Au25-PEG (400 μg mL−1) was injected. Histology Examination. Histology analysis was carried out after 14 days treatment, immediately. The representative photographs of different tissues (spleen, lung, heart, liver, and kidney) of the tumorbearing mice from different treatment groups were proposed. Then the mouse organs were dehydrated using buffered formalin, different concentrations of ethanol solution, and xylene. Afterward, liquid paraffin was utilized to trap the dehydrated organs. The organs and tumor tissues were sliced and stained with hematoxylin and eosin (H&E) and then observed by Leica TCS SP8. Biodistribution, Circulation, and Metabolism. Balb/c mice were injected with UCNPs@g-C3N4−Au25-PEG (20 mg/kg) intravenously. The mice (seven groups with three mice per group) were then euthanized at various time points (30 min, 1 h, 4 h, 12 h, 1 day, 3 days, and 7 days). The main metabolic products (urine and feces) were collected in different times for further detect and analysis. Then the blood was gained in mice at different points after intravenous injection. The major organs (heart, liver, spleen, lung, and kidney) were collected and treated with HNO3 and HCl (v/v = 1/3), and then heated at 70 °C for 5 min to obtain clear solutions. Afterward, the samples were collected for further detection by ICP-MS.

(group one) and without any irradiation (group two). HeLa cells were treated with UCNPs@g-C3N4−PEG under 980 nm laser for 30 min (group three). As a blank control, the HeLa cells incubated with UCNPs@mSiO2−Au25-PEG were excited upon a 980 nm laser for 30 min (group four). All groups of light conditions showed no difference (2.5 W cm−2, 5 min break after 5 min excitation). Afterward, the cells were continued to incubate for another 10 h to make the samples internalized. Then in each well was placed 20 μL of the as-prepared MTT (5 mg mL−1) and incubated at 37 °C and maintained for another 4 h. At last, to each well was added 150 μL of DMSO and shaken for 10 min to mix DMSO with the formazan completely. Microplate reader was used to detect the absorbance spectra at 490 nm. Further experiments have been performed to prove the cell viability; Calcein AM and propidium iodide (PI) contained assays and were executed to mark dead (red) and live (green) cells, repectively. In Vitro Cell Viability of UCNPs@g-C3N4−Au25-PEG. The in vitro biocompatibility of UCNPs@g-C3N4−Au25-PEG was detected similar to MTT cytotoxicity assay by incubating L929 fibroblast cells. Briefly, 100 μL of product per well was added, eight wells of them were left with culture medium only serve as normal control, and cultured overnight to let the cultured cells grew against the wall of flask at 37 °C in 5% CO2. Subsequently, the samples were diluted into different concentrations of 12.5, 25, 50, 100, 200, 400, and 800 μg mL−1. Afterward, the cells continued to incubate for another 24 h. Then to each well was added 20 μL of the as-synthesized MTT solution (5 mg mL−1) and incubated at 37 °C for another 4 h. At last, to each well was added 150 μL of DMSO and shaken for 10 min to mix DMSO with the formazan completely. Microplate reader was used to estimate the absorbance spectra at 490 nm. Hemolysis Assay of UCNPs@g-C3N4−Au25-PEG. To ascertain the in vivo biocompatibility of as-synthesized sample, the detection of hemolysis was necessary. Typically, the acquired red blood cells were washed with 0.9% saline, then centrifugated for five times. Afterward, blood cells were mixed with 10 mL of PBS buffer solution. Taking 0.3 mL of diluted samples placed into 1.2 mL of deionized water and PBS was considered as a positive and negative control, respectively, and 1.2 mL of material suspensions with a series of concentrations (15.6, 31.25, 62.5, 125, 250, and 500 μg mL−1). The eight centrifuge tubes were agitated and remained stationary last 1 h. Ultimately, the samples were centrifuged, and the upper supernatants were collected to detect the absorbance. Cellular Uptake of UCNPs@g-C3N4−Au25-PEG. The process of cellular uptake by HeLa cells was researched by a confocal laser scanning microscope (CLSM). HeLa cells (105 well−1) were cultivated in a culture plates with six wells, and the cultured cells grew against the wall of flask. Since then, 100 μL of UCNPs@g-C3N4−Au25-PEG nanoparticles (400 μg mL−1) was added to incubate at various intervals. The cells were rinsed with PBS and then immobilized with 1 mL of 2.5% formaldehyde last 10 min and washed with PBS again. Afterward, DAPI was chosen to stain the cells with the purpose of nucleus labeling. Finally, the dyed cells were rinsed with PBS and collected for further characterization. CLSM instrument (Leica TCS SP8) was utilized to assess fluorescence images quality. UCL Microscopy Observation of UCNPs@g-C3N4−Au25-PEG. HeLa cells (105 well−1) were cultivated in a culture plates with six wells, the cultured cells grew against the wall of flask, and 100 μL of UCNPs@g-C3N4−Au25-PEG (400 μg mL−1) was added to incubate at various intervals in the CO2 incubator. Posteriorly, the cells were rinsed with PBS and then immobilized with 1 mL of 2.5% formaldehyde for 10 min, and rinsed several times with PBS. The inverted fluorescence microscopy (Nikon Ti−S) was utilized to assess UCL effect, and meanwhile the samples were irradiated with an external 980 nm NIR light. In Vivo Toxicity Studies. Female Balb/c mice (20−25 g) were purchased from Harbin Veterinary Research Institute. The mice were subcutaneously inoculated with H22 tumor cells at left armpit. The mice with the mean tumor volume of 100 mm3, the tumor-bearing mice, were randomly divided into six groups (five mice per group). Group one did not need any treatment and was utilized for blank control. We intravenously injected UCNPs@g-C3N4−Au25-PEG,

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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.7b00651. N2 adsorption/desorption isotherm and pore size distribution curve; TEM image and particle size distribution; DLS data and zeta potentials; high resolution S 2p spectra; quantum yield of up-conversion 13005


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emission; comparison of ROS generation; inverted fluorescence microscope images; in vitro and in vivo MRI images; CLSM images; in vitro HeLa cell’s relative cell viabilities; in vitro and in vivo infrared thermal photographs; biodistribution of different samples; blood circulation time (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yunlu Dai: 0000-0003-4023-7320 Ruichan Lv: 0000-0002-6360-6478 Piaoping Yang: 0000-0002-9555-1803 Author Contributions

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC 21401032, 51472058, 21401019, 51422209, 51602072, and 51572258), China Postdoctoral Science Foundation (2014M560248, 2016T90269, 2015M581430, and 2015T80321), Outstanding Youth Foundation of Heilongjiang Province (JC2015003), Heilongjiang Postdoctoral Fund (LBH-Z14052, LBH-Z14070, and LBHTZ0607), and the Fundamental Research funds for the Central Universities is greatly acknowledged.

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