Multifunctional Anticancer Platform for Multimodal Imaging and Visible

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A Multifunctional Anticancer Platform for Multimodal Imaging and Visible Light Driven Photodynamic/Photothermal Therapy Ruichan Lv, Chongna Zhong, Rumin Li, Piaoping Yang, Fei He, Shili Gai, Zhiyao Hou, Guixin Yang, and Jun Lin Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/cm504566f • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Chemistry of Materials

A Multifunctional Anticancer Platform for Multimodal Imaging and Visible Light Driven Photodynamic/Photothermal Therapy Ruichan Lv,†,§ Chongna Zhong,†,§ Rumin Li,† Piaoping Yang,†,* Fei He,† Shili Gai,† Zhiyao Hou,‡ Guixin Yang,† and Jun Lin‡,* †

Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Science and Engineering, Harbin Engineering University, Harbin 150001, P. R. China ‡ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130021, P. R. China §

These authors contributed equally to this work.

KEYWORDS. Y2Ti2O7, Photodynamic, photothermal, bio-imaging, up-conversion ABSTRACT: As a potential photosensitizer for photodynamic therapy (PDT), pure titanium dioxide has the drawbacks of low tissue penetration and the possible damage to skin due to the triggered UV light. To realize near-infrared (NIR) laser induced multimodal imaging guided therapy, we constructed a multifunctional core-shell structure (TiO2@Y2Ti2O7@YOF:Yb,Tm) by a facile co-precipitation route, followed by an annealing process. Under a single NIR laser irradiation, the highly cytotoxic reactive oxygen species (ROS) required for PDT can be generated due to the energy transfer from YOF:Yb,Tm to the Y2Ti2O7 photocatalyst which is responsive to blue emission (visible light), and the thermal effect can be simultaneously produced due to the non-radiative transition and the recombination of electron-hole pairs. The NIR light induced PDT and photothermal therapy (PTT) can efficiently suppress tumor growth, which was evidenced by both in vitro and in vivo results. Moreover, the rare earth ions in the composite make the material have good up-conversion luminescence (UCL) imaging and CT imaging properties, thus achieving the target of PDT and PTT synergistic therapy under the multimodal imaging guidance.

INTRODUCTION Nowadays, the traditional anti-cancer therapies, such as chemotherapy and the ionizing radiotherapy, usually bring about serious side effects, drug resistance, and inevitably induce lesions on irradiated tissues.1 As mild and noninvasive techniques based on the photochemical reactions of photosensitizers (PSs) and thermal effect, photodynamic therapy (PDT) and photodynamic therapy (PTT) have become booming powerful tools to apply in the biological and medical field.2 The conventional organic PDT agents, such as ZnPc and Ce6 photosensitizers, suffer from poor water-solubility, easy premature leaking, low bio-stability, and other unclear security problems.3 Alternatively, titanium dioxide (TiO2), which can absorb ultraviolet (UV) light to generate reactive oxygen species (ROS) to kill tumor cells, has motivated intensive experimental and theoretical studies, due to its excellent biocompatibility, high photo-catalytic properties, low cost, and high chemical stability.4 However, in the field of titanium dioxide based PDT agents, there are serious problems which inhibit the biomedical application. Firstly, most of the TiO2 crystals can only be irradiated by UV light to generate ROS.5 To improve the separation probability and increase photo-induced electron-hole pairs, it is necessary to transfer the absorption peaks of TiO2 from UV to visible regions. Some modification strategies have been employed to achieve this target, such as decoration with metal complexes.6 In particular, doping is a powerful strategy to modify electronic structure and construct hetero-atomic surface structures, which allows high photo-catalysis efficiency

under visible light irradiation.7 However, the required UV or visible light still have the drawbacks of low tissue penetration and possible damage to skin. Thus it is of special importance if the exciting laser locates at the longer wavelength, such as 980 nm NIR, which has the high penetration to kill the inner tumor cells to achieve improved therapy efficacy.8 It is well known that lanthanide-doped nanocrystals possess a unique ability to convert excited NIR light to visible emissions, which can be used as the imaging agent on one side.9 On the other side, the UCL emissions in visible regions can be utilized as the donors for transferring energy to PDT agents.8 Meanwhile, the NIR laser which locates within the optical transmission window of biological specimens has the merits of high penetration depth and low tissue absorbance.10 Furthermore, if referred to imaging-guided PDT, two separate lights with different wavelengths were often adopted to realize diagnosis and therapy, which makes it difficult for real-time monitoring and assessing efficacy.11 Therefore, developing a multifunctional platform combining the UCL and TiO2-based materials which can achieve the imaging-guided PDT therapy triggered by a single NIR light is highly desirable. In the UCL field, lanthanide oxyfluorides (LnOF) have been considered as one of the most excellent luminescent hosts for achieving efficient UCL, which are even better than lanthanide fluoride.12 Among all the commonly used co-dopants, only Yb3+/Tm3+ ions can emit the strong blue emissions used for the bio-imaging and optical carrier. Meanwhile, Y2Ti2O7, as one of the classical titanium dioxide-based compound, has attracted considerable attention recently as a possible candidate for

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photo-catalysis due to its unique structural characteristics of which the electron acceptor of Y3+ ions may attract the electron to improve the photocatalysis efficiency.13 It is thus meaningful if the good UCL host and PSs (Y2Ti2O7) are integrated. In this contribution, a core/shell/shell structured TiO2@ Y2Ti2O7@YOF:Yb,Tm (denoted as TYY) platform has been synthesized through a facile and mild co-precipitation method, followed by a calcination process. The phase, composition, and structure of the composites were tuned by adjusting the molar ratio of TiO2/Ln3+. Core/shell/shell structured TYY UCNPs have been optimized for the biomedical experiments. The as-synthesized TYY up-conversion nanoparticles (UCNPs) were employed for MTT assay using HeLa cells and hemolysis experiment using human red blood cells to detect the in vitro and in vivo biocompatibility. Meanwhile, the cytotoxicity against tumor cells was systematically studied by MTT assay, trypan blue and AM/PI marked HeLa cells in vitro. The photo-thermal imaging, the UCL imaging and CT imaging properties were carried out on tumor-bearing mice in vivo simultaneously. The animal experiment and the histologic section assay were performed to demonstrate the potential clinical application of this anti-cancer nanotheranostic.

EXPERIMENTAL SECTION Reagents and Materials. All the chemical reagents are of analytical grade and used without any further purification, including HNO3, urea, KF, Y2O3, Yb2O3, and Tm2O3 (99.99%), glutaraldehyde (from Sinopharm Chemical Reagent Co., Ltd.), 1-(3-dimethy laminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), folic acid (FA), doxorubicin (DOX), hydrophilic titanium dioxide (TiO2), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), 4’,6-diamidino-2-phenylindole (DAPI), dimethyl sulfoxide (DMSO), trypan blue, calcein AM, propidium iodide (PI), 1,3-diphenylisobenzofuran (DPBF) (from sigma-Aldrich Co. LLC.). Synthesis of TYY UCNPs. 1 M Ln(NO3)3 (Ln = Y, Yb, Tm) was firstly prepared by dissolving corresponding Ln2O3 into dilute HNO3 with gradual heating. Meanwhile, hydrophilic TiO2 were dispersed in 20 mL deionized water by ultrasonic wave. In a typical process for the synthesis of TYY UCNPs (TiO2/Ln3+ molar ratio of 1:1), 1 mL of 1 M Ln(NO3)3, 3 g of urea, and 0.1 g of KF were dissolved in 15 mL deionized water mixed with 15 mL ethanol with continuous stirring, and then the previously dispersed TiO2 nanoparticles were added. Another 15 mL ethanol was added to the solution. After continuously stirring for 10 min, the mixture was heated to 90 °C and kept for 3 h. The resulting mixture was collected by centrifugation at 6000 rpm for 4 min, and was repeated three times. After dried at 60 °C for 12 h, the obtained powder was calcinated at 600 °C for 3 h. The final product was TiO2@Y2Ti2O7@YOF:Yb,Tm (simply labeled as TYY). TiO2@YOF:Yb,Tm and Y2Ti2O7@YOF-YOF:Yb,Tm were synthesized by the similar procedure except for adding TiO2/Ln3+ with molar ratio of 1:2 and 2:1, respectively. Surface Modification of TYY UCNPs with FA. FA molecules were conjugated onto the surface of TiO2@Y2Ti2O7@YOF:Yb,Tm by coupling effect of NHS and EDC. Briefly, 6 mg of EDC, 2 mg of NHS, and 1 mg of FA were added into 20 mL of deionized water and stirred in dark

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for 2 h. Then, the as-synthesized TYY UCNPs dispersed in 20 mL deionized water were added and stirred in dark overnight. The product was collected by centrifugation and washed with deionized water and ethanol several times to remove the free FA. The as-prepared TYY UCNPs modified with FA was stored for subsequently biological test. Singlet Oxygen Detection of UCNPs. DPBF was employed as a chemical probe to determine singlet oxygen by measuring the absorption via UV-vis spectroscopy. Typically, 2 mL of ethanol solution containing DPBF (10 mmol/L) was added to 2 mL of TYY UCNPs solution and then transferred into a 5 mL cuvette. The solution was kept in dark and irradiated by a 980 nm NIR laser for 1 min, 2 min, 3 min, and 5 min, respectively. Then, the solution was centrifuged at 6000 rpm and the supernatant was collected for UV-vis detection. The other two samples obtained with TiO2/Ln3+ ratio of 2:1 and 1:2 were also detected by the same process. In Vitro Cytotoxicity and Biocompatibility of TYY UCPCs. Briefly, HeLa tumor cells were seeded in a 96-well plate with the number of 7000 per well, then cultured at 37 °C in 5% CO2 for 12 h. After that, pure culture media were added into the first and the third group, and TYY UCNPs were added to the second and the fifth group, pure DOX was added into the fourth group, and then incubated with cells at 37 °C in 5% CO2 for another 24 h. Among them, the first group was the control group; the second group with only added UCNPs was without NIR irradiation in order to detect the cytotoxicity of the UCNPs; the third group with only culture was under NIR irradiation, which is used to detect the effect of the NIR irradiation to the cells; the fourth group was with pure DOX added; and the fifth group added with UCNPs was under NIR irradiation which is to detect the anti-cancer efficiency with integrated PDT/PTT. The second and fifth group were compared to evaluate the cytotoxicity of the photodynamic effect of the assynthesized UCNPs, and the results of fifth group compared with the conventional pure DOX (the third group) to evaluate the therapeutic effect of the as-synthesized UCNPs. The UCNPs samples were diluted into respective concentration of 15.63, 31.25, 62.5, 125, 250, and 500 µg mL–1, and DOX solution was diluted into different concentration of 3.13, 6.25, 12.5, 25, 50, and 100 µg mL–1. Then, 20 µL of as-prepared MTT solution (5 mg/mL) was added to each well. The plate was then incubated at 37 °C for another 4 h. Finally, 150 µL of DMSO was added to each well and shaken for 10 min to blend the DMSO solvent and the formazan completely. The absorbance was measured at 490 nm using a micro-plate reader. Optical density which received no drugs was regarded as 100% growth. For the trypan blue measurement, three different plates were used for each cell sample set, the first plate of HeLa cells was incubated with culture only with and without NIR irradiation, the second plate was incubated with TYY UCNPs with and without NIR irradiation, and the third plate was incubated with pure DOX. The cells on the bottom of the wells in one plate were stained with 1 mL of 0.4% trypan blue dye to determine the level of cell damage, and the dead cells were marked with blue color while the live cells were not dyed. The three plates could be divided to five groups (with and without NIR irradiation) corresponding to the MTT cytotoxicity assay.

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The in vitro biocompatibility of TYY UCNPs was detected similar to the cytotoxicity MTT assay by incubation of L929 fibroblast cells instead of HeLa cells. Hemolysis assay of TYY UCNPs was used to determine the in vivo biocompatibility. Briefly, red cells were obtained by removing the serum from human blood after washing with 0.9% saline, and centrifugated at 4000 rpm for five times to make the supernatant clear. Subsequently, blood cells were diluted with PBS solution. 0.3 mL of diluted cells suspension was then mixed with 1.2 mL of PBS (as a negative control), 1.2 mL of deionized water (as a positive control), and 1.2 mL of UCNPs suspensions with varying concentration of 15.63, 31.25, 62.5, 125, 250, and 500 µg mL–1. The eight samples were shaken for several minutes, and then kept steady for 2 h. Finally, the mixtures were centrifuged at 4000 rpm and the absorbance of the upper supernatants was measured by UV-vis spectroscopy. The hemolysis percentage was calculated as follows: Hemolysis (%) = (Asample - Acontrol(-)) / (A control(+) - Acontrol(-)), where A is the absorbance. Cellular Uptake of TYY UCNPs. The cellular uptake was examined by confocal laser scanning microscope (CLSM) incubated with HeLa cells. Typically, HeLa cancer cells were cultured in a 6-well plate and grew overnight to obtain a monolayer. Then, they were incubated with the as-synthesized TYY UCNPs at 37 °C for 0.5 h, 1 h, and 3 h, respectively. After that, the cells were rinsed with PBS solution several times, fixed for 10 min with 2.5% glutaraldehyde (1 mL well– 1 ), and further rinsed with PBS three times. Subsequently, the nuclei were stained for 10 min with DAPI solution (20 µg mL– 1 in PBS, 1 mL well–1) in order to perform nucleus labeling. Finally, the cells were rinsed with PBS three times. The coverslips were placed on a glass microscope slide, and the samples were measured by CLSM (Leica TCS SP8). UC Luminescence Microscopy (UCLM) Measurement. To obtain a monolayer, HeLa cells (5 × 104 well–1) were firstly seeded in 6-well culture plates. Then, TYY UCNPs were introduced into the 6-well culture. After that, these cells with UCNPs were incubated at 37 °C for different times (0.5 h, 1 h, and 3 h), respectively. At each incubated time, the cells in each well were washed with PBS solution and fixed with 2.5% formaldehyde (1 mL) at 37 °C for 10 min. Subsequently, the as-prepared cells were washed with PBS several times to remove the attached UCNPs. In Vivo Biodistribution and Circulation of TYY UCNPs. Female Kunming mice (20-25 g) were purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China), and all the mouse experiments were performed in compliance with the criterions of The National Regulation of China for Care and Use of Laboratory Animals. Balb/c mice were intravenously injected with 100 µL of TYY UCNPs (20 mg/mL). The mice were euthanized at different time (10 min, 1 h, 4 h, 12 h, 1 day, 3 days, 7 days, and 14 days). The tumor, blood, and major organs of kidney, lung, spleen, liver, heart were collected and dissolved in 5 mL of HNO3 and HCl (v/v = 1:3), and then heated at 70 °C for several minutes to obtain clear solutions. Then, the solutions were centrifuged and the supernatant were collected for ICPOES detection. For blood circulation assay, more dense time points (10 min, 30 min, 1 h, 2 h, and 4 h) were acquired. In Vivo Toxicity of TYY UCNPs. The tumors were founded by subcutaneous injection of H22 cells (murine hepatocar-

cinoma cell lines) in the left axilla of each female Kunming mouse (about 20 g). After grown for one week, the tumor sizes reached about 5-8 mm in size with the body weight of 26-29 g. The tumor-bearing mice were randomized into five groups (n = 5, each group) and were treated by intravenous injection with different treatment. Group 1: with none treatment, Group 2: with TYY UCNPs only without irradiation, Group 3: under NIR irradiation only, Group 4: with pure DOX, and Group 5: with TYY UCNPs under NIR irradiation. The first group was made for blank control. The injected TYY UCNPs amount is 100 µL (1 mg/mL) every two days, and the injected pure DOX is 100 µL (1 mg/mL) every two days. For the NIR irradiation process, the tumor site was irradiated with 980 nm laser for 10 min after injecting different amount of materials for 2 h. The body weights and tumor sizes were measured every two days after treatment. Histology Examination of TYY UCNPs. Histology analysis was studied at the 14th day after treatment. The typical heart, liver, spleen, lung, kidney, and tumor tissues of the mice in the five different treatment group were isolated. After that, the organs were dehydrated by buffered formalin, ethanol with different concentrations, and xylene, then embedded in liquid paraffin. After that, the organs and tumor tissues were sliced to 3-5 mm and stained with Hematoxylin and Eosin (H&E) and examined by CLSM (Leica TCS SP8). Characterization. Powder X-ray diffraction (XRD) patterns were measured on a Rigaku D/max TTR-III diffractometer at a scanning rate of 15°/min in the 2θ range from 20° to 80°, using graphite monochromatized Cu Kα radiation (λ = 0.15405 nm). The morphology and structure were studied on scanning electron microscope (SEM, JSM-6480A) and transmission electron microscopy (TEM, FEI Tecnai G2 S-Twin). UCL emission spectra were acquired using 980 nm laser diode (LD) Module (K98D08M-30W, China) as the irradiation source and detected by R955 (HAMAMATSU) from 400–800 nm. DOX concentration was detected by UV-1601 UV-vis spectrophotometer at the wavelength of 480 nm. Inductively coupled plasma (ICP) was carried out on a Thermo Electron X Series II ICP instrument.

RESULTS AND DISCUSSION Phase, Structure, Morphology and UCL Properties. Schematic diagram of the anti-cancer therapy mechanism of TiO2@Y2Ti2O7@YOF:Yb,Tm under a single NIR irradiation is presented in Scheme 1, which indicates the as-prepared UCNPs may have synergistic PDT/PTT effect to cancer cell. As shown, the blue emissions could be obtained due to the upconversion energy transfer process of YOF:Yb,Tm (the outer shell of the TYY UCNPs) under the 980 nm laser irradiation. After that, the Y2Ti2O7 (the middle of the TYY UCNPs) as the photocatalyst responsive to the blue light plays an important role in generating the ROS with high level due to the decreased distance between the valence band and conduction band. During the energy transfer process, the thermal effect could be also acquired due to the non-radiative process and the recombination of electron and hole pairs. The generated ROS and thermal effect may result in the nuclei apoptosis and kill the cancer cells. Figure S1 shows TEM images of hydrophilic TiO2 precursor. The precursor is well-dispersed with the size of 20-25 nm. In Figure S2, when the TiO2/Ln3+ molar ratio is

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2:1, high-dispersed microspheres with an average size of 140 nm are obtained which can be due to the uniform coating of Y(OH)CO3F:Yb,Tm shell on the surface of TiO2 cores. When the molar ratio is altered to 1:1, uniform nanospheres with an average size of 70 nm are generated. During this coprecipitation process, the extra Y3+ ions react with electronnegative TiO2, to form a layer of Y2Ti2O7 shell between TiO2 inner core and Y(OH)CO3F:Yb,Tm outer shell, leading to a core/shell/shell structure.14 When the molar ratio is adjusted to 1:2, because more Y3+ ions exist in the solution, the inner survived TiO2 crystals completely transfer to Y2Ti2O7 (100 nm). Meanwhile, the excessive Y3+ ions further react with F– ions to form YOF particles (200 nm).15 After calcination, Y(OH)CO3F transform to YOF phase, while the size and dispersibility (Figure S3) are kept similar to those of the precursors. The phases and compositions of the samples synthesized with different TiO2/Ln3+ molar ratios can also be confirmed by the XRD results, as shown in Figure S4. For the sample synthesized with molar ratio of 2:1 (TiO2@YOF:Yb,Tm), two sets of diffraction peaks can be found. One is assigned to the tetragonal TiO2 phase (JCPDS No. 21-1272), and the other to tetragonal YOF phase (JCPDS No. 06-0347). For TiO2@Y2Ti2O7@YOF:Yb,Tm, three sets of diffraction peaks can be well indexed to tetragonal TiO2, tetragonal YOF and cubic Y2Ti2O7 phase (JCPDS No. 42-0413). In the XRD pattern of the sample synthesized with the TiO2/Ln3+ molar ratios of 1:2, only tetragonal YOF and cubic Y2Ti2O7 are found, while no TiO2 phase is detected. The schematic diagram for the synthesis process of TYY UCNPs is depicted in Figure 1a. The SEM image (Figure 1a) and TEM images (Figure 1b-d) reveal that the final TYY UCNPs are well-dispersed with an average size of 70 nm. The HRTEM image (Figure 1e) shows different crystal lattices which indicates the product is composited of several phases. The corresponding FFT image of the e1 region shows a typical tetragonal crystal. The respective interplanar spacing of 0.35 nm, 0.19 nm, and 0.17 nm corresponds to the (101), (200), and (211) planes of tetragonal TiO2 (JCPDS No. 21-1272). In the FFT image of the e2 region, the typical parallel lattices with the distances of 0.58 nm, 0.29 nm, and 0.15 nm are well consistent with the (111), (222), and (444) planes of cubic Y2Ti2O7 (JCPDS No. 42-0413). In the e3 region, typical parallel lattices of 0.31 nm and 0.16 nm match well with the (101) and (202) planes of tetragonal YOF (JCPDS No. 06-0347). The results clearly reveal the TYY UCNPs are composited with TiO2, Y2Ti2O7, and YOF phases. Meanwhile, the TEM images with the corresponding selected area electron diffraction (SAED) images of different areas for TYY UCNPs are given in Figure S5. The SAED lattice rings of single nanoparticle (Figure S5a, b) and many nanoparticles (Figure S5c, d) are well consistent except for a slight difference of the densities, indicating the particles are composited with the same phases. The high-angle annular dark-field scanning TEM (HAADF-STEM) image shows the core-shell structure (Figure 1f). EDS analysis (Figure 1g) indicates there are Y, Yb, Ti, F, C and O elements. The cross-section compositional profile lines (inset) indicate the product has core-shell structure with YOF outside and TiO2@Y2Ti2O7 inside. The elemental mapping images (Figure 1h) further shows the F element distributes in the thick shell and Y, Yb, and Ti distribute in the

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particles uniformly, which further prove the core-shell structure. It is well known the luminescence intensity and energy transfer efficiency between donor and accepter are influenced by the crystal sizes, the morphologies, the distances between donor and accepter. Usually, in the UV-vis diffuse reflection spectra of TiO2, there are two major peaks which locate at around 370-470 nm. The wide peak at lower than 388 nm is attributed to the band gap recombination of TiO2, and the peaks at 450-480 nm are related with transition from localized surface states to the valence band of TiO2.13a Researches show that the absorbance peaks of Y2Ti2O7 at around 470 nm are well crossed with the max luminescence peaks of Tm3+ ions.13a,16 In order to utilize the cross to generate ROS under NIR irradiation, the influence factors (phase, size, and structure) which play prominent roles in the energy transfer efficiency should be considered. Figure 2a-c shows the SEM images with schematic morphology (insets) of the samples synthesized with different TiO2/Ln3+ molar ratios. The respective average size of TiO2@YOF:Yb,Tm and TYY is 140 nm and 70 nm, while Y2Ti2O7@YOF-YOF:Yb,Tm has two size distributions (100 nm and 200 nm). Figure 2d-f shows the emission spectra of the three samples excited at 980 nm for 0.5 min and 3 min, respectively. The same emission peaks at 476 nm and 803 nm are assigned to 1G4 → 3H6 and 3H4 → 3H6 transitions of Tm3+ ions.17 Figure S6A shows the UCL emission spectra of different samples at 980 nm laser excitation. The TYY UCNPs almost have the same intensity as the pure YOF:Yb,Tm synthesized by co-precipitation method, while the other two samples have much weaker emission intensities. The different UCL intensities should be attributed to the diverse compositions, surface defects, sizes, and shapes. Figure S6B shows that the intensity of TYY UCNPs decreases in the whole regions (blue and NIR emissions) with the prolonged time due to the nonradiative transitions. The two main peaks (476 nm and 803 nm) ratio can indicate the energy transfer efficiency because only the blue emission regions have the cross with the Y2Ti2O7 photocatalyst. As shown in Figure 2d-f and Figure S6B, the decreased blue emissions may transfer the energy to the photocatalyst which can generate ROS and thus the two weak ratio is decreased. For TYY with the strongest UC emission intensity, the ratio decreases from 4.38 to 1.42 when the irradiation time is adjusted from 0.5 min to 3 min, and the emission colour changes from blue to purple. This emission transformation is also proved by the CIE chromaticity (Figure S7) and the in vitro and in vivo experiment (Figure S8), while there is a slight change for TiO2@YOF:Yb,Tm (3.68 to 3.3) and Y2Ti2O7@YOF-YOF:Yb,Tm (2.94 to 2.68). The low peak ratio of Y2Ti2O7@YOF-YOF:Yb,Tm may be due to the fast energy transfer process between YOF and Y2Ti2O7 within 0.5 min. Figure 2g-i presents the UC emission stability at 476 nm (1G4 → 3H6 of Tm3+ ions) of the three samples with irradiation time interval of 5 min. We can see the intensity of TiO2@YOF:Yb,Tm doesn’t change with the prolonged irradiation time, while the intensity of TYY and Y2Ti2O7@YOFYOF:Yb,Tm both decreases obviously. The lifetime at 476 nm of TYY UCNPs (Figure S9B) was detected compared with pure YOF:Yb,Tm (Figure S9A) to further prove the energy transfer process. As shown, the lifetime of TYY at 476 nm is 0.30 ms, which is much shorter than that of pure YOF:Yb,Tm

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(0.76 ms). Figure 2j-l gives the absorbance spectra of the solutions of three samples with DPBF solution added with different irradiated times. It is apparent that the absorbance of DPBF decreases with prolonged time due to the generation of ROS. Especially, the TYY UCNPs have obviously superior photodynamic property. In a sum, the energy transfer from the YOF:Yb,Tm to Y2Ti2O7 in the blue region may produce the photodynamic effect, while the decrease of energy in the whole wavelength may generate the photothermal effect. Thus, the as-synthesized TYY UCNPs should have potential application in the anti-cancer therapy field. Figure 3a,b present the UV-Vis diffuse reflection absorption spectra and Tauc-plots (for indirect band gap transition) calculated from the absorption spectra for band gap energy calculations of different samples: pure TiO2 and the three samples, which are well consistent with the UCL result. Figure 3a shows the absorbance of the Kubelka-Munk function (F(R)) as a function of wavelength (λ). There is an obvious difference between pure TiO2 and the samples synthesized with different TiO2/Ln3+ molar ratio. Furthermore, in Figure 3b for the Tauc-plots (for indirect band gap transition) calculated from the absorption spectra for band gap energy calculations, there is a relationship between the absorption coefficient and the band gap energy: α = A × (hν - Eg)n/(hν), where α is absorption coefficient which is proportional to F(R), A is a constant, ν is the frequency, n = 2 for the indirect transitions, and Eg means the band gap energy which could be changed through different co-doped ions. Thus, the Eg can be calculated using intercept at (F(R) × hν)1/2 = 0 with the x axis of hν.13c Table S1 summarizes the calculated band gap energy of Eg using the Slope-Intercept form of linear equation and the corresponding absorbance edge λ. Compared with pure TiO2, the TYY UCNPs has an obvious red shift of the absorption edge (from 387.5 nm to 484.1 nm). Interestingly, compared with the other three curves, the curve of TYY UCNPs has two different slopes which are well composited to the wavelength of 484.1 nm. This result may be due to the composition of TYY UCNPs: two different photocatalysts of TiO2 and Y2Ti2O7. It indicates the co-dopant of Y decreases the conducting band of the TiO2 to Y2Ti2O7 and narrows the band gap, which is favorable to the cross of the phosphor and the photo-catalyst. Figure 3c schematically shows the generation of ROS and the thermal effect caused by energy transfer process of TYY UCNPs. Firstly, blue emissions could be generated due to the two-/three-photon UCL process under NIR irradiation. The absorption of pump photons from the NIR irradiation only populates photons from the ground state 2F7/2 to 2F5/2 level of Yb3+. Then, through successive energy transfer process, the excited photons transfer from Yb3+ ions to Tm3+ ions and populate the 1G4, 1D2, and 3H4 levels of Tm3+ ions. After all these process, the excited Tm3+ ions will fall to lower energy levels: 1 D2 → 3F4 (449 nm), 1G4 → 3H6 (476 nm), 1G4 → 3F4 (646 nm), and 3H4 → 3H6 (803 nm). Secondly, ROS are generated from the Y2Ti2O7 photocatalyst, which is excited by the blue emissions generated from Yb3+/Tm3+. During the following process, there are three steps: (a) as shown in the absorbance spectra in Figure 3a, the Y2Ti2O7 photocatalyst is sensitive to the blue light. Then, Y2Ti2O7 will produce holes and electrons in the valence band (VB) and conduction band (CB), respectively. (b) The excited holes and electrons separate and migrate to the surface of Y2Ti2O7. Note that the recombination of

electron and hole pairs may decrease the catalytic ability, which is affected by the phase, structure, and size of the Y2Ti2O7. (c) The photo-generated electrons and pairs will migrate from the inner region to the surfaces of Y2Ti2O7, resulting in the formation of high ROS: OH', O2·, H2O2, and 1O2. It should be noted that this efficient transfer process is benefited from the close attachment of YOF and Y2Ti2O7 due to the master design of TYY UCNPs. As shown in TEM images (Figure 1d,f), the TYY UCNPs surfaces are not smooth but have channels and pores which may be caused by the released CO2 by the decomposition of the precursor. That means, the water and oxygen could be exchanged between the nanoparticles and the surrounding environment. Additionally, as shown, the thermal effect generated under NIR irradiation can be due to two reasons. First, during the second energy transfer process from the two different crystals of YOF to Y2Ti2O7, photons in all the energy levels of Tm3+ ions will relax nonradiatively and the decreased UCL in the whole region may result in thermal effect. Second, the recombination of the holes and electrons may also lead to thermal effect. Through designing this catalytic core and the luminescent shell, two advantages are obtained: the UCL material as the shell is beneficial to emit the visible light for tracking and diagnosis, while the ROS and thermal effect generated from the catalytic substance inside could cross the channels to kill the tumor cells. Cell Viability, Drug Release and MTT Cytotoxicity Assay. Standard MTT assay was studied on L929 cells to detect the short-term viability. Figure 4a demonstrates the cell viability with particle concentrations from 15.63 to 500 µg mL–1 incubated for 24 h. The biocompatibility of the sample in all dosages is 101.9-118.0%. Even at the highest concentration of 500 µg mL–1, the cell viability keeps as high as 106.5%, suggesting the good biocompatibility in vitro. Meanwhile, the hemolysis assay on red blood cells was employed to guarantee the potential intravenous administration in vivo. Figure 4b shows the hemolytic result detected by UV-vis absorbance, and the photograph of the red solution in the tube (inset) is taken to show the presence of hemoglobin. There is no visually red colour in the solution with added UCNPs, indicating the negligible hemolysis in the controlled tubes. The highest hemolytic efficiency is 0.12% with particle concentration from 15.63 to 500 µg mL–1, indicating almost no hemolysis occurs. In conclusion, the MTT assay and the blood compatibility of TYY UCNPs are excellent which is almost nontoxic to live cells. Cellular cytotoxicity and dead cells marked by trypan blue were utilized to evaluate the potential application of anticancer therapy. The cytotoxicity of HeLa cells incubated with TYY UCNPs, with pure DOX, with TYY UCNPs under NIR irradiation, and irradiated with NIR only is given in Figure 4c. When the culture was exposed to NIR irradiation, the pump power was adjusted to 0.72 W cm–2 and kept for 5 min. As shown in Figure 4c, when only TYY UCNPs is added, more than 96.0% of HeLa cells are viable under a varying concentration range from 15.63 to 500 µg mL–1. The results indicate that TYY UCNPs have no cytotoxicity to cancer cells. When the cells are incubated with culture only under the NIR irradiation, the cell viability is lower (73.0%-83.5%) than that without NIR irradiation, which may be due to the slight absorbance of culture to NIR laser. When incubated with the anticancer drug of DOX, the cells viability is 34.5%-53.4% with DOX

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concentration of 3.13-100 µg mL–1. The calculated IC50 value of pure DOX to HeLa cells is 4.47 µg mL–1. In the last group, the viability of the HeLa cells incubated with UCNPs with concentrations from 15.63 to 500 µg mL–1 under NIR irradiation is 28.1%-38.4%, which is lower than that of the cells incubated with pure DOX. The calculated IC50 value of UCNPs with NIR irradiation is 5.06 µg mL–1, which is almost as low as the value of pure DOX. The viability of HeLa cells with different treatments marked with trypan blue was also detected. The dead cells can be dyed with blue colour. As shown in Figure 4d, the cells almost survive completely when only incubated with UCNPs without NIR irradiation, while most of the cells are dead when incubated with UCNPs under NIR irradiation, which is well coincident with above cytotoxicity MTT assay. The confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with added UCNPs with and without NIR irradiation dyed by AM and PI (Figure S10) further confirm the PDT/PTT effect of UCNPs triggered by NIR light. The results indicate that the as-synthesized TYY UCNPs should be an efficient anticancer candidate triggered by a single NIR laser. The cell up-taken property was also studied. HeLa cells were incubated with TYY UCNPs for 1 h, then dyed by DAPI and detected by CLSM. As discussed above, the emission wavelength of TYY UCNPs is 476 nm of blue region and 803 nm of NIR region. We utilized 488 nm as the excitation wavelength, and red emission can be interestingly detected. Thus, there are blue emissions emitted by DAPI in the nuclei and red emissions emitted by the UCNPs. Figure 5a reveals the TYY UCNPs are evident in the intracellular region, and no superfluous luminescent signals outside is found. The result indicates the as-synthesized UCNPs are internalized into the cells rather than merely stained in the membrane surface. Furthermore, Figure S11 indicates that stronger red fluorescence emission of UCNPs is observed in the cytoplasm with the enhanced incubation time, suggesting that more naoparticles are localized in the cells. Particularly, it is apparent that the UC luminescence microscopy (UCLM) images (Figure 5b) of HeLa cells incubated with TYY UCNPs presents strong blue emissions, and there are stronger emissions and more internalized nanoparticles inside of HeLa cells with prolonged times from 0.5 h to 3 h (Figure S12). The results further confirm that TYY UCNPs can be effectively taken up by tumor cells and emit blue light under 980 nm irradiation. As discussed above, the photothermal effect is generated due to the energy transfer process between YOF:Yb,Tm and Y2Ti2O7 and the recombination of hole-electron pairs in Y2Ti2O7. The in vivo infrared thermal images of tumor-bearing mice after injection of saline and TYY UCNPs versus irradiation time with the pump power of 0.72 W cm–2 are displayed in Figure 5c, and the temperature curves as a function of irradiation time is depicted in Figure 5d. It is obvious that the second group with UCNPs injected has the obviously increased temperature (up to 49.7 °C) than that (42.1 °C) injected with saline, which proves the photothermal effect caused by the TYY UCNPs. The controllable temperature is beneficial to therapy which can supply various choices and has a synergistic therapy with PDT. Figure 5e presents the digital photograph of a mouse subcutaneously injected with TYY UCNPs under 980 nm NIR irradiation, and apparent purple light can

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be found. With respect to potential clinical application, the detection of the bio-distribution and blood circulation of the particles with different intravenous injection times is necessary. As shown in Figure 5f, in the early stages (10 min, 1 h, 4 h, and 12 h after the injection), the nanoparticles accumulate in the livers, spleens, lungs, and targeted tumors. In the whole detection time, Y concentrations still keep low in kidney and heart. After three days of post-injection, the Y concentrations reduce obviously in all the organs. In the 7th and 14th day after injection, the nanoparticles in the liver, spleen, and lung almost disappear. The results reveal that the injected TYY UCNPs can be excreted from the mice with prolonged times. Figure 5g shows the pharmacokinetics of blood circulation curve. The fitted curve presents a two-compartment model with the first and second phase blood circulation half-lives at 0.018± 0.013 day (about 0.432 h) and 0.437 ± 0.069 day (about 10.5 h), respectively. CT imaging has been proved an effective diagnostic technique owing to the high resolution and deep tissue penetration. Because rare earth ions doped products can be used as contrast agents for CT imaging, we measured the imaging properties of TYY UCNPs in vitro and in vivo, which are presented in Figure 6. It is obvious that the signal and CT value increases with the increase of UCNPs concentration (Figure 6a, b), and there is a well-fitted linear relationship between them with the slope of 10.66 ± 0.61. In vivo CT imaging was further measured by intratumoral injection of tumor-bearing Balb/c mice (Figure 6c-f). As shown, the CT value after injection is 959.5 HU (Hounsfield units), which is markedly higher than that (39.8 HU) of the control (without injection). The in vitro and in vivo results show the as-synthesized TYY UCNPs are effective contrast agent for CT imaging. The good biocompatibility and synergistic anti-cancer effect in vitro stimulate us to study the in vivo anti-tumor effect. As a proof of principle experiments, the H22 tumor-bearing mice were treated with different conditions: without treatment as the control group, with TYY UCNPs intravenously injected only, with NIR irradiation on the tumor site, with pure DOX, and with TYY UCNPs under NIR irradiation. Here, the NIR irradiation is carried for 10 min with the pump power of 0.72 W cm–2. The mice were under treatment when the tumor size grew to 4-8 mm in size (one week). After different treatments of the five groups for two weeks, the result of each group is given in Figure 7a and b. It is obvious that the tumor size is very large (about 27 mm) without any treatment. When UCNPs are injected only, the tumor size keeps similar to the control group (24 mm). The tumor is inhibited a little with NIR irradiation with the tumor size of 19 nm, which may be due to the photothermal effect absorbed by the tumor cells. The pure DOX was also injected intravenously with 100 µL (1 mg/mL) every two days, and the tumor size is 15 mm. It is noted that the mouse of this group has the thinner body which may be caused by the side fact of the anticancer drug. For the last group injected with UCNPs under NIR irradiation, the tumor size (9 mm) is obviously smaller than that of the other four groups. Figure 7c shows the body weights of all of the five groups increase with prolonged time, and the result also indicates the weights are almost caused by the increased tumor volumes (Figure 7b). Notably, the body weight of the best treated group increases slightly, which indicates the tumor can be efficiently inhibited due to the simultaneous PDT and PTT

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effect. Figure 7d presents the tumor histologic section acquired from different groups. There are obvious degenerative changes of coagulative necrosis with karyorrhectic debris and marked karyolysis in control group, and the conditions become better from the left to the right. For the best inhibition group, due to the markedly increased apoptotic and necrotic tumor cells, the cells in this group seems to be almost regular with normal cells. In order to evaluate the nanotoxicity of the as-synthesized material, the H&E stained images of heart, liver, spleen, lung, and kidney from different groups are given in Figure 8. In the control group, necrosis is found in the histological samples, and there are symptoms of inflammation and glomerulus atrophy. Compared with the control group, the organs with best treatment show the following result: no pulmonary fibrosis is detected in all the lung samples. Hepatocytes in the liver samples are found normal, and the glomerulus structure in the kidney section is observed clearly. The results clearly demonstrate the potential clinical applicability of the as-synthesized TYY UCNPs as anti-tumor system.

CONCLUSIONS In summary, uniform core-shell structured TiO2@Y2Ti2O7 @YOF with UCL and photothermal/ photodynamic properties under NIR irradiation were synthesized by a facile coprecipitation method. Different composites were obtained by simply adjusting the TiO2/Ln3+ ratio. The reactive oxygen species (ROS) and thermal effect could be generated under NIR irradiation due to the energy transfer from YOF:Yb,Tm to the photocatalyst of Y2Ti2O7. The results reveal that the assynthesized TYY UCNPs have good biocompatibility. The composite exhibit obvious cytotoxicity to HeLa cancer cells, which were evidenced by MTT assay and CLSM images of HeLa cells dyed by trypan blue and AM/PI. In particular, the product also shows clear photo-thermal imaging, UCL imaging and CT imaging in vivo. The final animal experiment and the histologic section assay also prove the anti-cancer therapy efficiency of the as-synthesized product.

ASSOCIATED CONTENT Figure S1-S12 and Table S1 can be seen from the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected] (P. Y.) [email protected] (J. L.)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial supports from the National Natural Science Foundation of China (NSFC 21271053, 21401032, 51472058, 51332008) are greatly acknowledged.

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Scheme 1. Schematic diagram of the anti-cancer therapy mechanism for TYY UCNPs under a single NIR irradiation .

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Figure 1. (a) Schematic diagram of the synthetic process, (b) SEM image, (c, d) TEM images, (e) HRTEM image, (e1-e3) the corresponding FFT images, (f) HAADF-STEM image, (g) EDS with crosssection compositional line profiles inset, and (h) elemental mapping images of TYY UCNPs. 10


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Figure


2.

(a-c)

SEM

images

of

TiO2@YOF:Yb,Tm,

TiO2@Y2Ti2O7@YOF:Yb,Tm

and

Y2Ti2O7@YOF-YOF:Yb,Tm. Insets are the corresponding structure sketches. (d-f) The UCL emission spectra of the samples under 980 nm irradiation for 0.5 min and 3 min. (g-i) The UC emission stability of Tm3+ at 476 nm under 980 nm excitation within 10 min with each re-irradiation for 5 min. (j-l) The absorbance spectra of TiO2@YOF:Yb,Tm, TiO2@Y2Ti2O7@YOF:Yb,Tm and Y2Ti2O7@YOFYOF:Yb,Tm with DPBF solution added under 980 nm irradiation.

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Figure 3. (a) UV-vis diffuse reflection absorption spectra and (b) Tauc-plots (for indirect band gap transition) calculated from the absorption spectra for band gap energy calculations of pure TiO2 and the three samples synthesized with different TiO2/Ln3+ molar ratio. (c) Schematic diagram of the energy transfer process of TYY UCNPs between the Yb/Tm ions and the Y2Ti2O7 photo-catalyst.

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Figure 4. (a) L929 fibroblast cell viability incubated with TYY UCNPs with different concentrations for 24 h. (b) The hemolysis property of TYY UCNPs to human red blood cells. (c) In vitro viability of HeLa cells after various treatments: incubated with TYY UCPCs with different concentration for 24 h, with NIR irradiation only, incubated with pure DOX, and incubated with UCNPs with different concentration under NIR irradiation. (d) In vitro CLSM images of HeLa cells treated under different conditions marked by trypan blue.

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Figure 5. (a) CLSM images and (b) UCLM images of HeLa cells incubated with TYY UCNPs for 1 h. (c) In vivo infrared thermal images of tumor-bearing mice after injection of saline, and TYY UCNPs versus irradiation time under 980 nm NIR irradiation with the pump power of 0.72 W cm–2. (d) Temperature profiles of the mice after intravenous injection with saline and UCNPs as a function of irradiation time. (e) UCL photograph of a mouse after injection of TYY UCNPs under 980 nm irradiation with the pump power of 0.72 W cm–2. (f) The biodistribution and (g) blood circulation of TYY UCNPs in H22 tumor-bearing mice. 14


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Figure 6. (a) In vitro CT images of TYY UCNPs with different concentrations. (b) CT value of aqueous solution of TYY UCNPs as a function of the particle concentration. CT imaging of tumor-bearing Balb/c mouse (c, d) before intratumor injection and (e, f) after injection.

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Figure 7. (a) Representative photographs of tumor-bearing mice after various treatments: without any treatment as the control group, injected with TYY UCNPs only, with NIR irradiation only, injected with pure DOX, and injected with UCNPs under NIR irradiation, all of the materials were injected intravenously; digital photographs of tumor tissues after different treatments for 14 days. (b) The normalized tumor size, and (c) body weight of H22 tumor in different groups after different treatments. (d) H&E stained images of tumors from the different groups. 16


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Figure 8. H&E stained images of heart, liver, spleen, lung, and kidney from different groups at 14th day.

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Table of Contents (ToC) Graphic

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Multifunctional Anticancer Platform for Multimodal Imaging and Visible

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