A Single 808 nm Near-Infrared Light-Mediated Multiple Imaging and

Nov 12, 2015 - To solve the issue of limited penetration depth and overheating of the excited 980 nm near-infrared (NIR) light, and unstable and insuf...
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A Single 808 nm Near-Infrared Light-Mediated Multiple Imaging and Photodynamic Therapy Based on Titania Coupled Upconversion Nanoparticles Guixin Yang,†,§ Dan Yang,†,§ Piaoping Yang,*,† Ruichan Lv,† Chunxia Li,‡ Chongna Zhong,† Fei He,† Shili Gai,† and Jun Lin*,‡ †

Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin 150001, People’s Republic of China ‡ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130021, People’s Republic of China S Supporting Information *

ABSTRACT: To solve the issue of limited penetration depth and overheating of the excited 980 nm near-infrared (NIR) light, and unstable and insufficient loading amount of photosensitizers (PSs) in photodynamic therapy (PDT), we have constructed a well-defined core−shell structured NaGdF 4 :Yb/Tm@NaGdF 4 :Yb@NaNdF 4 :Yb@NaGdF 4 @ mSiO2@TiO2 (UCNPs@mSiO2@TiO2) nanocomposite by coating a layer of TiO2 PSs/photocatalyst on an effective 808 nm-to-UV/visible upconversion luminescent (UCL) core to achieve simultaneous multiple bioimaging and efficient PDT. The design of quenching-shield layer can eliminate the back energy transfer from activator Tm3+ to sensitized Nd3+, thus significantly improving the UCL emission. The high surface area of mesoporous silica-coated UCNPs facilitates the stable and high loading amount of anatase TiO2. In vivo results indicate that 808 nm NIR light-mediated PDT using UCNPs@mSiO2@ TiO2 as photosensitizers shows much higher antitumor efficacy than those with 980 nm and UV irradiations due to the higher tissue penetration depth. Meanwhile, the platform itself as an imaging nanoprobe endows the sample with multiple imaging (UCL/CT/MRI) properties. Our work makes great progress toward the integrity of diagnosis and PDT induced by a single 808 nm NIR light.

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INTRODUCTION As a minimally invasive and highly selective anticancer therapeutic technique based on the killing effect of cytotoxic reactive oxygen species (ROS) generated from the PSs upon UV or visible light irradiation, PDT has gained considerable interest due to fewer side effects and lower systemic toxicity superior to conventional chemo- and radiation therapy.1−8 However, the limited penetration depth of irradiated UV or visible light greatly hinders the therapy of solid tumors in deep tissue sites. As compared to fluorescent imaging in the visible range that can only penetrate up to 1−2 mm in tissue, the NIR light enhances tissue penetration to several centimeters due to the greatly decreased absorbance in tissue.9−12 Thus, it is highly desirable to shift the wavelength of the irradiation beam to the NIR window (700−1000 nm) located in the optical window of biological tissues. For the traditional organic PDT photosensitizers, Ce6 and ZnPc are facing the problems of easy light bleaching, poor stability, and quick circulation in body, which greatly inhibit their biomedical applications.13−16 Recently, anatase titania (TiO2) has been extensively employed for PDT © XXXX American Chemical Society

irradiated by UV light to generate ROS to kill tumor cells, due to the high activity, excellent stability, good biocompatibility, low cost, and long retention time in the body.17−24 However, the irradiated UV light is harmful to skin and espccially lacks enough penetration depth for deeper therapy. It is well-known that the upconversion process based on rare earth doped particles can convert low energy light like NIR light to UV or visible light,25−38 which has been extensivey explored for biomedical applications due to the low toxicity, minimal autofluorescence, and sharp absorption.39−44 Recently, the UCL coupled TiO2 nanocomposites have been reported for NIR-mediated PDT and MRI-guided PDT.45−51 However, the conjugation/linking mode limited the sufficient amount of PSs attached on UCNPs, which is necessary for efficient ROS production. Particularly, 980 nm NIR light used as an irradiation source for biological applications has an inherent Received: August 13, 2015 Revised: November 10, 2015

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DOI: 10.1021/acs.chemmater.5b03136 Chem. Mater. XXXX, XXX, XXX−XXX

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(CF3COONa), and trifluoroacetic acid (CF3COOH) were purchased from Beijing Chemical Corp. (China). Synthesis of Oleic-Acid-Stabilized β-NaGdF4:Yb/Tm NPs. Oleic-acid-capped β-NaGdF4:Yb/Tm was synthesized according to our previous method.40 In a typical procedure, 1 mmol of RE(oleate)3 (RE = 80% Gd + 19.5% Yb + 0.5% Tm), 12 mmol of NaF, 15 mL of oleic acid, and 15 mL of 1-octadecene (ODE) were added to the reaction vessel, heated to 110 °C under a vacuum, and kept for 30 min to remove residual water and oxygen. After that, the temperature was increased to 305 °C and kept for 1.5 h in N2 atmosphere. After being cooled to room temperature, β-NaGdF4:Yb/Tm NPs were then obtained by washing with acetone and cyclohexane several times. Finally, the obtained NPs were dispersed in 5 mL of cyclohexane for further use. Synthesis of Core−Shell Structured β-NaGdF4:Yb/Tm@βNaGdF4:Yb@β-NaNdF4:Yb@β-NaGdF4 NPs. Briefly, the as-synthesized core NPs dispersed in cyclohexane solution were added into a reaction vessel containing 10 mL of ODE and 10 mL of OA; the solution was then heated to 120 °C in a vacuum with stirring for 1 h in N2 atmosphere. After that, the temperature was raised to 305 °C. Another solution containing 3 mL of ODE/OA (v/v = 1.5/1.5), 0.9 mmol of Gd(CF3COO)3, 0.1 mmol of Yb(CF3COO)3, and 1 mmol of CF3COONa was injected into the former solution directly. After continuous reaction at 305 °C for 1 h in N2 atmosphere, the solution was cooled to room temperature to obtain β-NaGdF4:Yb/Tm@βNaGdF4:Yb NPs. The temperature of the solution then was raised to 305 °C, and another solution with 3 mL of OA/ODE (v/v = 1.5/1.5), Nd(CF3COO)3 (0.9 mmol), Yb(CF3COO)3 (0.1 mmol), and CF3COONa (1 mmol) was added immediately. The temperature was maintained at 305 °C for 1 h in N2 atmosphere, then cooled to room temperature to obtain β-NaGdF4:Yb/Er@β-NaGdF4:Yb@βNaNdF4:Yb NPs. The solution was then heated to 305 °C, and another solution with 3 mL of OA/ODE (v/v = 1.5/1.5), 1 mmol of Gd(CF3COO)3, and 1 mmol of CF3COONa was immediately injected. β-NaGdF 4 :Yb/Er@β-NaGdF 4 :Yb@β-NaNdF 4 :Yb@βNaGdF4 NPs were finally obtained, which were denoted as UCNPs. Synthesis of UCNPs@mSiO2 Nanospheres. First, 2 mL of cyclohexane solution containing UCNPs was mixed with 0.1 g of CTAB and 20 mL of water. The mixture was then stirred vigorously to evaporate the cyclohexane solvent at room temperature, leading to a transparent UCNPs−CTAB water solution. After that, 10 mL of CTAB stabilized UCNPs solution was added to the mixed solution of 20 mL of water, 3 mL of ethanol, and 150 μL of 2 M NaOH solution. The mixture was heated to 70 °C under stirring. When the temperature is stable, 160 μL of TEOS was added dropwise, and the reaction was allowed to proceed for 10 min. The as-synthesized material was centrifuged and washed with enthanol three times. The surfactant CTAB was removed via a fast and efficient ion exchange method, where the as-synthesized UCNPs@mSiO2 (20 mg) was transferred to 50 mL of ethanol containing 0.3 g of NH4NO3 and kept at 60 °C for 2 h. Finally, the obtained UCNPs@mSiO2 NPs were dispersed in ethanol. Synthesis of UCNPs@mSiO2@TiO2 Spheres. The above synthesized UCNPs@mSiO2 NPs were mixed with 20 mL of ethanol under magnetic stirring for 30 min. Next, 4 mL of 0.025 M TiF4 aqueous solution was added dropwise into the above solution under stirring. The as-formed reaction solution was transferred into a 50 mL Teflon-lined autoclave and maintained at 180 °C for 8 h. After being cooled to room temperature, the obtained precipitate was separated by centrifugation and washed with ethanol and deionized water several times. The as-synthesized UCNPs@mSiO2@TiO2 NPs were dispersed in water for further application. For better biocompatibility and higher cell recognition of the final product, PEG500-silane (0.03 mL) was then added to a mixed solution containing 30 mL of ethanol, 6 mL of deionized water, 0.3 mL of NH4OH, and 20 mg of as-synthesized UCNPs@mSiO2@TiO2 NPs. After being stirred for 24 h, the sample was centrifuged to remove the unreacted agents. For facile discussion in the following section, we named the PEG-modified UCNPS@ mSiO2@TiO2 as UCMTs.

drawback of high absorbance of laser light at about 980 nm, leading to restricted penetration depth.52−54 Moreover, the high absorbance can also induce obvious overheating, which may cause serious tissue damage when irradiated for a long time or with high power density. It is therefore necessary to optimize the wavelength of the excited light into an appropriate NIR region within 700−900 nm, which has the minimum absorbance for all biomolecules.54−56 As compared to 980 nm NIR laser, 808 nm NIR light has better penetrability and less overheating.57,58 ROS generated from 808 nm NIR lightirradiated TiO2 is dependent on the spectral overlap between the UV emission from Yb3+/Tm3+ codoped UCNPs and the absorbance of TiO 2 nanoparticles. Thus, high energy converting efficiency from 808 nm NIR light to UV emission is necessary. Currently, Nd3+ has been widely adopted as the sensitizer to realize the UC emission upon 808 nm excitation.53,59−63 To overcome the energy transfer backinduced quenching effect, diverse structures have been designed. Fabricating an inert layer to block the back energy transfer from inner activator Tm3+ to an outer layer containing Nd3+ is one of the most efficient techniques.53,63 In our previous work, we reported an anticancer therapy nanoplatform for 980 nm NIR light-mediated PDT, which was designed by decorating anatase TiO2 on the surface of bare UCNPs.45 However, the low surface area of the bare UCNPs and the linking/conjugation mode of TiO2 make it difficult to load stable and more TiO2 NPs. Moreover, the overheating and the limited tissue penetration depth derived from the used 980 nm NIR laser remarkably hinder the actual clinical application. Here, we designed a multifunctional TiO2 nanoparticlesdecorated and Nd3+-sensitized UCNPs for 808 nm NIR lightmediated and multimodal imaging guided PDT for the first time. The core-trilayer structure of UCNPs can completely eliminate the deleterious cross-relaxation between the activated Tm3+ and sensitized Nd3+ by fabricating an inner quenchingshield layer, thus keeping the UCL emission intensity of the core upon the 808 nm laser excitation. Meanwhile, the outermost inert layer can greatly increase the UCL intensity. More importantly, a stable and high loading amount of anatase TiO2 NPs has been achieved due to the large surface area of mesoporous silica-coated UCNPs, which can produce a significant amount of ROS to effectively kill tumor cells. The in vitro and in vivo biocompatibility and cytotoxicity against tumor cells of this multifunctional nanoplatform for 808 nm NIR-triggered PDT have been systematically investigated, as well as the comparison with 980 nm and UV light-mediated PDT results. Meanwhile, the dopants Tm3+, Gd3+, and Yb3+derived UCL, CT, and MRI imaging properties were also examined to achieve the NIR-induced multiple imaging function, respectively.

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EXPERIMENTAL SECTION

Materials and Synthesis. All of the chemical reagents used in this experiment are of analytical grade without any further purification, including hydrochloric acid (HCl), sodium fluoride (NaF), tetraethoxysilane (TEOS), phosphate buffered saline (PBS), titanium tetrafluoride (TiF4), Gd2O3, Yb2O3, Nd2O3 and Tm2O3 (99.99%) (from Sinopharm Chemical Reagent Co., Ltd.), oleic acide (OA), 1octadecene (ODE), 4′,6-diamidino-2-phenylindole (DAPI), 1,3diphenylisobenzofuran (DPBF), 2,7-dichlorofuorescin diacetate (DCFH-DA), calcein AM, propidium iodide (PI), and 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), which were purchase from Sigma-Aldrich Co. LLC (China). Cetyltrimethylammonium bromide (CTAB), sodium trifluoroacetate B

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Chemistry of Materials Characterization. Power X-ray diffraction (XRD) patterns were obtained from a Rigaku D/max TTR- III diffractometer at a scanning rate of 15° min−1 in the 2θ range from 10° to 80°, with graphite monochromatized Cu Kα radiation (λ = 0.15405 nm). Images were acquired digitally on a transmission electron microscope (TEM, FEI Tenai G2 S-Twin) and with high-resolution transmission electron microscopy (HRTEM). N2 adsorption/desorption isotherms were obtained on a Micromeritrics ASAP Tristar II 3020 apparatus. Pore size distribution was calculated by the Barrett−Joyner−Halenda (BJH) method. UCL emission spectra were obtained on an Edinburgh FLS 980 apparatus using a 980 nm laser diode module (K98D08M-30W) as the irradiation source and recorded from 300 to 800 nm. The UV− vis absorbance spectra of the solutions were measured by a UV-1601 spectrophotometer. Extracellular and Intracellular ROS Detection. The extracellular ROS (1O2) was detected by using a DPBF as a chemical probe and then measuring the absorption via UV−vis spectroscopy. Briefly, 2 mL of ethanol solution containing DPBF (10 mg L−1) was added to 2 mL of UCMTs solution (0.5 mg mL−1) and then transferred into a 5 mL cuvette. The solution was kept in the dark and irradiated by 808 nm NIR laser for 5, 10, 15, and 30 min, respectively. The solution then was centrifuged at 4500 rpm, and the supernatant was collected for UV−vis detection. In a typical procedure for the detection of intracellular ROS detection, HeLa cells were incubated with UCMTs NPs for 6 h in the dark. After that, noninternalization NPs were washed with PBS, and fresh culture media with DCFH-DA (20 μm) was added and incubated for another 20 min in the dark. After irradiation under a 808 nm laser for 30 min (4 W cm−2), the irradiated cells were collected by trypsinization, then rinsed with PBS three times, and filtrated by 35 mm nylon mesh to obtain a single cell suspension. The FACSC caliber flow cytometer was employed to analyze the intracellular ROS using 485 and 525 nm as the respective excitation wavelength and emission wavelength. In Vitro Cytotoxicity and Biocompatibility of UCMTs. Typically, HeLa cells were seeded in a 96-well plate (7000 well−1) and then cultured in 5% CO2 for 12 h at 37 °C to let the cells attach to the bottom of the wells. The ultrasonically treated UCMTs were diluted into concentrations of 7.81, 15.62, 31.25, 62.5, 125, 250, and 500 μg mL−1, and then were added into the wells containing culture to replace the original culture medium for another 24 h incubation at 37 °C. Among them, the first group was with only UCMTs without NIR irradiation to detect the cytotoxicity of UCMTs. The second group with only culture was under 808 nm NIR irradiation, which is used to detect the effect of NIR irradiation to the cells. The third group was added with UCMTs under 365 nm UV irradiation. The fourth group was added with UCMTs under 980 nm NIR irradiation, and the fifth group was added with UCMTs under 808 nm NIR irradiation. Next, 20 μL of the as-prepared MTT solution (5 mg mL−1) was added to each well. The plate was then incubated at 37 °C for another 4 h. At last, 150 μL of DMSO was added to each well and shaken for 10 min to blend DMSO and the formazan completely. The absorbance spectra were measured at 490 nm using a microplate reader. To further assay the cell viability, the cells were costained by calcein AM and propidium iodide (PI) to differentiate live (green) and dead (red) cells, respectively. The in vitro biocompatibility of UCMTs NPs was detected similar to cytoxicity MTT assay by incubating L929 fibroblast cells instead of HeLa cells. Briefly, 200 μL of product per well plated L929 cells with the number of 6000−7000 was put in a 96-well plate with 8 wells left with culture only for blank control, and then incubated for 24 h so as to allow the cells to attach the wells at 37 °C in 5% CO2. Subsequently, the samples were diluted into respective concentrations of 7.8125, 15.625, 31.25, 62.5, 125, 250, and 500 μg mL−1, and then UCMTs were added and incubated for another 24 h. After that, 20 μL of asprepared MTT solution (5 mg mL−1) was introduced into every well, which was further incubated at 37 °C for another 4 h. In the procedure, MTT was reduced into formazan by viable cells, which may be dissolved by DMSO. Finally, 150 μL of DMSO was introduced into each well and shaken for 5 min to blend the solvent and the formazan

completely. The absorbance was measured on a microplate reader at 490 nm wavelength. Optical density that received no drug was used as 100% growth. Hemolysis Assay of UCMTs. To ascertain the in vivo biocompatibility of the as-synthesized sample, the detection of hemolysis is necessary. Typically, the acquired red blood cells were obtained by removing the serum from human blood by washing with 0.9% saline, then centrifuging several times. After that, blood cells were diluted to 1:10 with PBS solution. 0.4 mL of diluted cells suspension was then mixed with 1.6 mL of deionized water as a positive control, 1.6 mL of PBS as a negative control, and 1.6 mL of material suspensions with varying concentrations (15.63, 31.25, 62.5, 125, 250, and 500 μg mL−1). The eight samples were shaken and then kept stable for 2 h at room temperature. Finally, the mixtures were centrifuged, and the absorbance of the upper supernatants was detected by UV−vis spectroscopy. The hemolysis percentage was determined by the following equation:

hemolysis % = (A sample − Acontrol(−))/(Acontrol(+) − Acontrol(−)) where A is the absorbance of the UV−vis spectrum. Cellular Uptake of UCMTs. Cellular uptake by HeLa cancer cells was recorded on a confocal laser scanning microscope (CLSM). HeLa cancer cells were first seeded in a 6-well culture plate and allowed to grow for 12 h to achieve a monolayer, then incubated with assynthesized UCMTs at 37 °C for 30 min, 1 h, and 3 h, respectively. After that, the cells were washed with PBS three times, and fixed with 2.5% glutaraldehyde (1 mL well−1) at 37 °C for 10 min. To carry out nucleus labeling, we stained the nuclei with DAPI solution (20 μg mL−1 in PBS, 1 mL well−1) and FITC solution for 10 min. The cells were then rinsed with PBS three times. The coverslips were placed on a glass microscope slide, and the samples were recorded by CLSM (Leica TCS SP8). UC Luminescence Microscopy (UCLM) Study. HeLa cells (105 well−1) were seeded in a 6-well culture plate, then incubated overnight to get a monolayer. After that, the cells were incubated for different times (0.5, 1, and 3 h) at 37 °C with UCMTs (0.25 mg mL−1), then washed with PBS solution three times, and fixed with 1 mL of 2.5% glutaraldehyde in each well at 37 °C for 10 min. Subsequently, the asprepared cells were washed with PBS three times to remove attached NPs. The UCL imaging was detected on Nicon Ti−S apparatus. For the upconversion luminescence microscopy with different concentrations of UCMTs, the method is similar to the above process, besides that the cells were incubated for 1 h at 37 °C with varying concentrations of 2, 1.5, 1, 0.5, 0.25, and 0.13 mg mL−1. In Vitro and In Vivo X-ray CT Imaging. The in vitro CT imaging experiments were performed on a Philips 64-slice CT scanner at 120 kV. UCMTs were first dispersed in PBS with varying concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 mg mL−1 and then added into a 2 mL tube for the in vitro CT imaging. To carry out in vivo CT imaging, the mice were first anesthetized by injecting 10% chloral hydrate. After that, 120 μL of UCMTs (50 mg mL−1) was intratumorally injected into the tumor-bearing mice in situ. The mice were scanned before and after injection of the NPs. In Vitro and In Vivo MRI. The in vitro MRI was performed on a 0.5 T MRI magnet (Shanghai Niumai Corp. Ration NM120-Analyst). UCMTS nanoparticles were dispersed in water with various Gd concentrations. T1 measurements were carried out using a nonlinear fit to changes in the mean signal intensity within each well versus repetition time (TR) with a Huantong 1.5 T MR scanner. At last, the r1 relaxivity values were calculated by the curve fitting of 1/T1 relaxation time (s−1) as a function of Gd concentration (mM). In Vivo Toxicity. Female Kunming mice (20−25 g) were purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, P. R. China), and all of the mouse experiments were performed in compliance with the criteria of The National Regulation of China for Care and Use of Laboratory Animals. First, the tumors were built up by subcutaneous injection of H22 cells (murine hepatocarcinoma cell lines) into the left axilla of each female mouse. After being grown for 1 week, the tumors reached C

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Figure 1. (A) Schematic illustration for the formation of UCNPs@mSiO2@TiO2. TEM images of (B) NaGdF4:Yb/Tm, (C) NaGdF4:Yb/Tm@ NaGdF4:Yb, (D) NaGdF4:Yb/Tm@NaGdF4:Yb@NaNdF4:Yb, (E) NaGdF4:Yb/Tm@NaGdF4:Yb@NaNdF4:Yb@NaGdF4, (F) UCNPs@mSiO2, and (G) UCNPs@mSiO2@TiO2. (H) EDS and (I) high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and the cross-sectional compositional line profiles of UCNPs@mSiO2@TiO2. the size of about 6−8 mm. The tumor-bearing mice then were randomly divided into six groups (n = 5, each group) and were treated by intratumoral injection with UCMTs, pure 808 nm NIR irradiation, UCMTs with 365 nm UV, UCMTs with 980 nm NIR irradiation, and UCMTs with 808 nm NIR irradiation. The first group was used for blank control without any injection. The injected amount is 80 μL (1 mg mL−1) every 2 days. For the NIR irradiation, the tumor site was irradiated with 808 nm NIR laser for 10 min. The tumor size and body weights were measured every day after treatment. Histology Examination. Histology analysis was carried out at the 14th day after treatment. The typical heart, liver, spleen, lung, and kidney tissues of the mice in the control group and best treatment group were isolated. The organs were dehydrated using buffered formalin, ethanol with different concentrations, and xylene. After that,

they were embedded in liquid paraffin. The sliced organs and tumor tissues (3−5 mm) were stained with hematoxylin and eosin (H&E) and examined on a Leica SP8 microscope.

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RESULTS AND DISCUSSION Formation, Structure, Morphology, Phase, and UCL Properties. A schematic diagram for the synthesis of the UCNPs@mSiO2@TiO2 core−shell−shell nanocomposite is presented in Figure 1A. First, oleic acid capped βNaGdF4:Yb/Tm core was synthesized by a thermal decomposition process from corresponding rare earth oleates. To eliminate the quenching effect, tune the excitation wavelength from 980 to 808 nm, and greatly increase the UCL emission D

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intensity, core−shell structured NaGdF4:Yb/Tm@NaGdF4:Yb@NaNdF4:Yb@NaGdF4 NPs (labeled as UCNPs) have been designed by coating different functional layers on UCL core NPs by a layer-by-layer technique. As shown in Figure 1B, the as-synthesized β-NaGdF4:Yb/Tm core NPs consist of uniform and nearly monodispersed nanoparticles with an average particle size of 22 nm. For the subsequent three layercoated samples, the average diamter increases to 26 nm (Figure 1C), 29 nm (Figure 1D), and 35 nm (Figure 1E), respectively. The HRTEM images of the samples synthesized in each step show the high crystallinity of the samples (Figure S1A−C). For the mesoporous silica-coated sample (UCNPs@mSiO2), we can clearly see that a layer of uniform mesoporous silica with a thickness of 21 nm has been coated on the surface of UCNPs (Figure 1F). Here, TiF4 is served as Ti precursor to estabilish a layer of porous TiO2 shell on the surface of the UCNPs@ mSiO2 NPs. In Figure 1G, the as-synthesized sample still shows uniform particle distribution with size similar to that of UCNPs@mSiO2. Interestingly, the as-synthesized UCNPs@ mSiO2@TiO2 NPs show a clear hollow interior space different from UCNPs@mSiO2, which may be due to the dissolvation of silica by the formed HF derived form the decompostion of TiF4. The EDS (Figure 1H) confirms the existence of Ti, O, F, Na, Gd, Si, Nd, and Tm elements. The cross-sectional compositional line profiles as shown in Figure 1I further

Figure 2. (A) UC emission spectra of UCNPs, UCNPs@mSiO2, and UCNPS@mSiO2@TiO2 excited under 808 nm NIR light, and the corresponding luminescent photographs of (B) UCNPs dispersed in cyclohexane, (C) UCNPs@mSiO2 dispersed in water, and (D) UCNPs@mSiO2@TiO2 dispersed in water.

Figure 3. Extracellular and intracellular ROS generation detection. (A) UV−vis adsorption spectra of DPBF solutions treated with UCMTs under 808 nm laser irradiation versus the treated time. (B) Flow cytometry analysis of HeLa cells incubated with UCMTs under 808 nm irradiation for different time. The level of intracellular ROS was detected using DCFH-DA as the peroxide-sensitive fluorescent probe. (C) CLSM images of HeLa cancer cells after incubation of culture UCMTs with 808 nm MIR laser irradiation deal with DCFH-DA for different times. E

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Figure 4. (A) Inverted fluorescence microscope images of HeLa cells incubated with UCMTs at 37 °C for 0.5, 1, and 3 h. Scale bars for all images are 50 μm. Digital photographs of subcutaneous implantation test of UCMTs on a mouse (B) before and (C) after 808 nm laser light irradiation.

layer (shell 3) can prevent the quenching interactions between the UV-blue upconversion luminescence interior and outer surrounding. As shown in Figure 2A, we can see that assynthesized UCNPs, UCNPs@mSiO2, and UCNPS@mSiO2@ TiO2 samples all emit upconverting emission in UV/visible regions under 808 nm NIR laser excitation, and show obvious blue/bluish color under NIR irradiation (Figure 1B−D). The UV emission peaks of Tm3+ ions can be ascribed to radiative transitions of 1I6 → 3F4 (348 nm) and 1D2 → 3H6 (365 nm), and the visible emissions can be assigned to 1D2 → 3F4 (453 nm) and 1G4 → 3H6 (480 nm) transitions of Tm3+ ions, respectively.64−68 Because of the quenching effect of water molecules, the UCL emission intensity of UCNPs@mSiO2 under 808 nm excitation is decreased.69,70 After coating the TiO2 shell outside the UCNPs@mSiO2 cores, all of the emission peaks of the sample become weaker. Especially, the emission intensity of the UV region has been markedly decreased because of the effcient absorbance of UV light by the TiO2 shell. However, the as-synthesized UCNPS@mSiO2@ TiO2 still shows bright bluish violet emissions irradiated by 808 nm laser (Figure 2D). It is noted that when excited by 980 nm laser, the emission intensities of the samples are not as high as those under the excitation of 808 nm light with the same exciting power (Figure S3).

confirm the multiple core−shell structure. The phase of assynthesized UCNPs@mSiO2@TiO2 NPs was further characterized by X-ray diffraction (XRD) measurement. As shown in Figure S2, two sets of diffraction peaks can be observed. One is indexed to anatase TiO2 phase (JCPDS no. 21-1272), and the other is assigned to hexagonal NaGdF4 phase (JCPDS no. 270699), indicating the formation of anatase TiO2 in the nanocomposite. We have reported that the UV emission derived from the Tm3+ doped UC NPs can activate TiO2 to generate ROS under 980 nm NIR irradiation.45,50 Here, the excitation wavelength of 980 nm has been replaced by 808 nm by coating a layer of NaNdF4:Yb, which can make harsh 808 nm NIR laser light. As shown in Figure S4, as compared to NaGdF4:Yb,Tm@ NaNdF4:Yb, both the UCL emission intensities of NaGdF4:Yb,Tm@NaGdF4:Yb@NaNdF4:Yb and NaGdF4:Yb,Tm@ NaGdF4:Yb@NaNdF4:Yb@NaGdF4 have been markedly enhanced, indicating that the inert NaGdF4:Yb shell-induced interionic distance can efficiently block the energy back-transfer from activator (Tm3+) to sensitizer (Nd3+). Moreover, the enhanced UCL emission intensity of NaGdF4:Yb,Tm@ NaGdF4:Yb@NaNdF4:Yb@NaGdF4 as compared to NaGdF 4 :Yb,Tm@NaGdF 4 :Yb@NaNdF 4 :Yb suggests that the NaGdF4 shell chosen as the luminescence quenching reduction F

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Figure 5. (A) In vitro CT images of UCMTs with different concentrations. (B) CT value of aqueous solution of UCMTs as a function of the particle concentration. CT images of tumor-bearing mice (C,D) before and (E,F) after intratumor injection.

also exhibit obvious green color, confirming the efficient generation of intracellular ROS. UCL/CT/MRI Imaging Properties. Figure 4A gives the respecive inverted UCL images of HeLa cells incubated with UCMTs for 0.5, 1, and 2 h at 37 °C. It is obvious that UCL of UCMTs increases with the enhanced time. Meanwhile, no signal is found outside of the cells, whereas the UCL centers in the intracellular region show that the as-synthesized product has been internalized into the cells rather than merely stained on the membrane surface. The result also suggests that the UCMTs NPs are a promising candidate for in vitro bioimaging with negligible background. Figure S6 gives the respective inverted UCL images of HeLa cells incubated with varying concentrations of UCMTs for 1 h. As shown, when the concentration of the UCMTs is lower than 0.25 mg mL−1, the cells show no signal of UCL. With the increased concentration of as-prepared nanospheres, the emission color of the upconversion fluorescence is enhanced. Therefore, the minimum cell uptake is 0.25 mg mL−1, which allowed for the use of this system for imaging application. More importantly, UCL for the TiO2 loaded sample is needed for antitumor application. Figure 4B and C shows the in vivo UCL imaging of as-synthesized UCMTs, a mouse after hypodermic injection of UCMTs to the subcutaneous tissue of the back and another mouse injected with saline as a control under 808 nm irradiation. It is apparent that the mouse injected with UCMTs exhibits a bright bluish-violet color, while the region of the mouse injected with saline does not show visible color. X-ray CT has been proved an important imaging technique due to the deep tissue penetration and high resolution. The doped Yb3+ and Gd3+ ions endow the sample with CT imaging properties. Figure 5 shows the CT imaging properties of UCMTs in vitro and in vivo. It is clear that CT values and the signal increase with the increased UCMTs concentration (Figure 5A,B), and there is a well-fitted linear relationship with the slope of 11.8 ± 0.13. In vivo CT imaging was studied by intratumorally injecting the solution of UCMTs. In Figure

As mentioned above, the coating of a layer of mesoporous silica can lead to large surface area, which is beneficial for loading more TiO2 nanoparticles. In Figure S5, the N2 adsorption/desorption isotherms of UCNPs@mSiO2 and UCNPS@mSiO2@TiO2 can be classfied as type-IV isotherms, indicating the mesoporous structure. The BET suface areas and average pore sizes of them are calculated to be 797 m2 g−1 and 3.3 nm and 636 m2 g−1 and 3.8 nm, respectively. The high specific surface area and mesoporous pore structure make them a potential for biomedical applications. ROS Detection of UCMTs under 808 nm NIR Irradiation. It has been reported that ROS can bring damage to mitochondria and DNA in cells, leading to the death of tumor cells. Thus, the most important factor to determine the PDT efficacy is the ability to generate intracellular and extracellular ROS. Here, we used 1,3-diphenylisobenzofuran (DPBF) as the chemical probe to detect the generation of extracellular ROS. As a reductive material, DPBF can react with ROS, which can induce the decrease of emission intensities of the characteristic absorbance at 470 nm when excited by UV light under 808 nm NIR light irradiation (Figure 3A). It is found that with the increase of irradition time, the intensity of the absorbance spectra of DPBF gradually decreases, proving the efficient generation of extracellular reactive 1O2. The generation of intracellular ROS was detected by monitoring the fluorescent dichlorofluorescein (DCF) converted from nonfluorescent 2,7-dichlorofluorescein diacetate (DCFH-DA) reacted with ROS irradiated with an 808 nm NIR light after HeLa cells were incubated with UCMTs in the dark. The fluorescent intensity determined by FCM relatively represents the quantitative amount of the generated ROS. As shown in Figure 3B, the fluorescent intensity of DCF increases with the increased irradiation time. DCFH-DA in HeLa cells can be oxidized to DCF by 1O2, which emits green light excited by 552 nm laser that can be detected by CLSM. In Figure 3C, HeLa cancer cells after incubation of UCMTs with 808 nm NIR laser irradiation dealt with DCFH-DA for different times G

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Figure 6. In vitro and in vivo antitumor performance. (A) In vitro cell viability of HeLa cells incubated for 24 h with UCMTs, UMCTS with 365 nm UV irradiation, UMCTS with 980 nm irradiation, and UMCTS with 808 NIR-laser irradiation. (B) CLSM image of HeLa cancer cells after incubation of culture and UCMTs with 808 nm laser irradiation dyed with calcium AM and PI. (C) Normalized body weights and (D) tumor size of H22 tumor in different groups after treatment. (E) Photographs of tumor tissues from different groups obtained after 14 days. (F) H&E stained images of tumor tissues from the control and the best treated groups. (G) Representative photographs of mice after incubation with UCMTs with 980 and 808 nm NIR-laser irradiation.

DAPI is used to mark the nuclei. The overlay of the two channels is also presented. In the first 0.5 h, little green emission is observed, indicating only a few of the UCMTsFITC NPs have been uptaken by HeLa cells. Stronger emission of both green and blue color can be observed in the cells with prolonged time, indicating that more particles have been localized in the cells. The result suggests that the as-synthesized UCMTs can be effectively taken up by tumor cells. In Vitro Cell Viabilities and Hemolysis Assay. Standard MTT assay was employed to detect the short-term viability by using L929 cells. Figure S9A exhibits the viability of the cells after incubation for 24 h with particle concentrations from 7.81 to 500 μg mL−1. It is evident that the cell viabilities decrease gradually with the increased concentration, while the cell viabilities still keep more than 80% even if the concentration is

5C−F, the CT value after injection of the sample is 766.7 HU (Hounsfield units), which is remarkably higher than that (23.53 HU) of the control (injection of the same dose of saline). The in vitro and in vivo results represent that the as-synthesized UCMTs are an effective contrast agent for CT imaging. Figure S7A shows the in vitro T1-weighted MR images of UCMTs versus the Gd concentration. As shown, the signals are positively enhanced with the increased Gd concentration, and the longitudinal relaxivity (R1) value of the sample is 1.588 mM−1 S1− (Figure S5B). Cellular Uptake. Figure S8 shows the CLSM images of HeLa cells incubated with UCMTs-FITC for 0.5, 1, and 3 h at 37 °C to verify the cell uptake process. The green fluorescence from the FITC can be clearly found in confocal images with an excitation wavelength of 552 nm. The blue fluorescence from H

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Figure 7. H&E stained images of heart, liver, spleen, lung, and kidney collected from different groups.

up to 500 μg mL−1, suggesting that UCMTs have no obvious cytotoxicity. The purpose of this work is to prove UCMTs can act as an inorganic PDT agent for 808 nm NIR light triggered photodynamic therapy. To guarantee the safety of the cells, the phototoxicity of the 808 nm NIR laser was also studied using the similar MTT assay. It is found that both the irradiation power intensity and the irradiation time have no obvious cytotoxicity (Figure S9B,C); thus it is inferred that the excited 808 nm light has no toxic effect on the cells. In addition, to prove the potential intravenous administration in vivo, we employed the hemolysis assay on red blood cells. Figure S9D shows the hemolytic result detected by UV−vis absorbance at varying concentrations ranging from 15.6 to 500 μg mL−1 using PBS and deionized water as negative and positive controls, respectively. The red solutions in the tubes (inset of Figure S9D) are ascribed to the presence of released hemoglobin. The result indicates that the sample has excellent blood compatibility. In Vitro and In Vivo Antitumor Properties. To further confirm the PDT efficiency of UCMTs on HeLa cells in vitro, the cytotoxic effects of UCMTs on HeLa cells under 808 nm NIR laser, 980 nm NIR laser, and 365 nm UV light irradiation were investigated, and the results are given in Figure 6A. HeLa cells incubated only with UCMTs without any irradiation act as the control group. The result shows that the UCMTs without any light irradiation have no effect on the cell viability in whole concentration range, also indicating the low toxicity. However, as compared to that only with UCMTs without any irradiation, the cell viability of HeLa cells treated with UCMTs NCs with 980 nm light, 808 nm light irradiation (5 min break and 10 min of irradiation), or 365 nm UV irradiation for 30 min greatly decreased, which suggests the high cancer cells inhibition efficacy resulting from the generated ROS. It is noted that the survival rate of HeLa cell treated with UCMTs irradiated by 365 UV light is slightly higher than those with 980 or 808 nm irradiaton. For NIR irradiated treatments, the HeLa cell death

was controlled by the NIR light triggered PDT effect due to the fluorescence resonance energy transfer (FRET) from UCNP cores to TiO2 shells. The viability of HeLa cells treated with different conditions marked with AM that can mark living cells with green color and PI that can mark dead cells with red color was also measured. In the CLSM image of HeLa cells cultured with UCMTs without NIR laser (top, Figure 6B), almost all of the cells are green, which indicates that UCMTs have no obvious cytotoxicity to cancer cells. However, when HeLa cells were incubated with UCMTs with 808 nm NIR laser irradiation, only red cells can be found (bottom, Figure 6B), indicating that tumor cells are effectively killed due to the efficient PDT effect from UCMTs under 808 nm NIR laser irradiation. To further evaluate the in vivo anticancer efficacy of UCMTs under 808 nm laser irradiation, we chose liver cancer tumor line H22 (murine hepatocarcinoma) as the xenograft model. The tumor-bearing mice were randomized into six groups (n = 5, each group), and the group injected with nothing was used for blank control. Group 2 was only irradiated with 808 nm NIR irradiation for 30 min on the tumor site. Similar to group 2, groups 3, 4, and 5 were intratumorally injected with the UCMTs NPs. Group 4 was exposed to 365 nm UV light for 30 min, group 5 was irradiated by 980 nm NIR laser for 30 min, and group 6 was exposed to 808 nm NIR laser for 30 min. As given in Figure 6C, the body weight of all groups does not decrease with the prolonged time, indicating the little side effect of UCMTs as compared to traditional antitumor drugs. In Figure 6D, it is obvious that the group treated with UCMTs under 808 nm laser irradiation exhibits the best tumor growth inhibition efficacy. The digital photographs of the tumors excised from representative mouse (Figure 6E) also directly show that the tumor size of the mice in group 6 has markedly been decreased as compared to those in other groups. It should be noted that different from the in vitro results, the group under 808 nm NIR light-driven PDT treatment clearly I

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51472058, 51332008), the National Basic Research Program of China (2014CB643803), and NCET in University (NCET12-0622) are greatly acknowledged.

demonstrates better tumor inhibition than that under 365 nm light irradiation due to the deeper tissue penetration depth of 808 nm NIR light than that of UV light. Figure 6F shows the tumor histologic sections of the control group and the group treated with 808 nm NIR light irradiation. It is found that the shape, size, and staining of tumor cells in the control group are at variance, and most of the nuclei have the mitotic phenomena. For the best inhibition group (group 6), the cells seem almost regular with normal cells due to the markedly increased apoptotic and necrotic tumor cells. In Figure 6G, the skin of the mouse after the 808 nm laser irradiation keeps the original appearance, whereas the skin irradiated by the 980 nm laser shows an obvious scar, indicating the apparent photodamage effect on tissues of 980 nm NIR laser light. The representative H&E stained images of the heart, liver, spleen, lung, and kidney organs from the mice in different groups are presented in Figure 7, respectively. Histology analysis of the major organs from mice after injection of UCMT NPs with 808 nm NIR laser irradiation shows that no appreciable damage to organs or noticeable abnormality can be observed, the intercellular gap is ambiguous in the liver organ, and there is remarkable atrophy in the glomerulus. All of the results prove that UCMTs under 808 nm NIR laser-mediated PDT could be a safe and feasible cancer treatment technique with substantial application in biomedical fields.

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CONCLUSIONS In summary, TiO2 nanoparticles with PDT effect were combined with UCNPs for the first time to form a new multifunctional cancer therapy platform with multiple imaging and PDT function upon a single 808 nm irradiation. The large surface area derived from the mesoporous structure of the platform offers more active sites to produce ROS to kill tumor cells. The UCL process in this platform was applied to take advantage of the strength of the PDT effect by the FRET effects. Besides the CT and MRI imaging properties, the nanoparticles also show good UCL imaging effect in vitro and in vivo when excited by a single 808 NIR laser. Therefore, we believe that this new multifunctional cancer therapy platform could be a promising candidate to achieve imaging-guided therapy.

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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/acs.chemmater.5b03136. Figures S1−S9 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

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

G.Y. and D.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC 21271053, 21401032, J

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DOI: 10.1021/acs.chemmater.5b03136 Chem. Mater. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.chemmater.5b03136 Chem. Mater. XXXX, XXX, XXX−XXX