Highly Erbium-Doped Nanoplatform with Enhanced Red Emission for

Nov 16, 2018 - Thereafter, the enhanced red emission transfers energy to the conjugated photodynamic therapy (PDT) agent for tumor inhibition efficacy...
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Highly Erbium-Doped Nanoplatform with Enhanced Red Emission for Dual-Modal Optical-Imaging-Guided Photodynamic Therapy Miao Feng,† Ruichan Lv,*,† Liyang Xiao,† Bo Hu,† Shouping Zhu,† Fei He,‡ Piaoping Yang,*,‡ and Jie Tian*,†,§

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†

Engineering Research Center of Molecular and Neuro Imaging, Ministry of Education, School of Life Science and Technology, Xidian University, Xi’an, Shaanxi 710071, China ‡ Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical Engineering, Harbin Engineering University, Harbin 150001, PR China § Key Laboratory of Molecular Imaging of Chinese Academy of Sciences, Institute of Automation, Chinese Academy of Sciences, Beijing 100190, China S Supporting Information *

ABSTRACT: Generally, luminescence quenching at high doping concentrations typically limits the concentration of doped ions in the lanthanide material to less than 0.05−20 mol %, and this is still a major hindrance in designing nanoplatforms with improved brightness. In this research, a nanoplatform capable of dual-modal imaging and synergetic antitumor cells therapy was designed. NaYF4:x%Er@NaXF4 (x = 5, 25, 50, and 100; X = Lu and Y) core@shell nanoparticles with Er3+ ion concentration up to 100 mol % were synthesized, and the luminescence properties under near-infrared (NIR) excitation were detected. The results show the strong coupled of surface and concentration quenching effects in upconversion nanoparticles (UCNP). Upconversion luminescence (UCL) and NIR-II emission intensity increased with negligible concentration quenching effect under 980 and 800 nm NIR lasers because of the growth of epitaxial shells. Therefore, the enhanced red luminescence transfers energy to photosensitizer ZnPc as the photodynamic therapy (PDT) agent for tumor inhibition efficacy.

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INTRODUCTION In recent decades, the molecular imaging has been applied to display specific molecules at cellular/subcellular levels through imaging techniques, reflect changes of living organisms, and study biological behavior qualitatively/quantitatively using imaging methods. Optical imaging was one of the most important imaging tool in early disease diagnosis, as it enables noninvasive real-time feedback using nonharmful, nonionizing radiation with high spatial resolution and sensitivity.1−6 Lanthanide-doped nanoparticles generated wide applications from optical displays and photovoltaics to photoactivated bioapplications,7−9 and selective photophysical processes including downshifting/ upconversion processes can be implemented in such materials.10−13 Upconversion nanoparticles (UCNP) are a cluster of lanthanide-doped nanoparticles that could transfer the long-wavelength near-infrared (NIR) light excitation to shortwavelength ultraviolet or visible light. Although recent synthesis © XXXX American Chemical Society

advances have led to the precise control of the crystal phase, structure, morphology/shape, and luminescence intensity/color, it is still hard to achieve strong upconversion luminescence (UCL).14−17 Conventional lanthanide-based UCNP under NIR excitations were typically codoped with Yb3+ (generally under 980 nm irradiation) or Nd3+ (generally under 808 nm irradiation) sensitizer ions and Er3+, Ho3+, or Tm3+ activator ions.18−20 The most used hosts of NaYF4 were usually doped with Yb3+/Er3+, thus emitting stronger green light and weaker red light under 980 nm laser excitation.21,22 One decisive restriction to the lanthanide-doped nanoparticles application was that the concentration of sensitizers is generally limited to a low doped concentration, and the concentration of the activators is low in Received: August 9, 2018

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DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry range of 0.05−20%.23−27 The luminescence intensity reduces with increasing doped activator or sensitizer ion concentrations, referred to as “concentration quenching”. There are two prevalently accepted explanations for concentration quenching: (a) cross-relaxation between adjacent ions and (b) enhanced energy transfer through resonant energy transfer to surface defects.28−31 Although the origin of concentration quenching in lanthanide-doped nanoparticles was still under discussion, the directions and the optimal concentrations for doped ions were commonly used in lanthanide nanoparticles.32,33 Attempts to solve the “concentration quenching” problem included coating nanoparticles with epitaxial shells to minimize surface quenching or using precious metal nanostructures to enhance the surface plasmon energy transfer rate.34−37 In the photodynamic therapy (PDT) field, upconversion emission could have excellent potential with nanoparticles, because the NIR excitation laser falls into the optical window of biological tissues that could penetrate tissue deeply.38−41 Recent efforts have focused on Nd3+-doped UCNP that allow for excitation at 808 nm with a dramatically reduced heating effect.42−45 Meanwhile, it should be noted that the red UCL emission is beneficial for PDT therapy since most of the photosensitizers selectively absorb red light and then transfer energy to surrounding oxygen molecules to generate 1O2.46−50 For a safe and efficient antitumor method, good biocompatibility is essential. Several approaches were proposed to transfer hydrophobic nanoparticles into hydrophilic ones for biological application, such as ligand exchange or modifying by amphiphilic polymer. However, silica coating with suitable porous structures is still the most widely accepted due to easy surface modification, large loading capacity, and high stability under aqueous conditions.51,52 In this work, we proposed that the 5 nm epitaxial shell would couple the surface quenching and concentration quenching with a high concentration of doped Er3+ in the core. A series of core@shell nanoparticles with an inert shell composed of a core of different Er3+ doped NaYF4 nanoparticles were synthesized, and these particles could be used for dual-mode imaging with both upconversion fluorescence and NIR-II emission under 808 nm laser irradiation. Through increasing the Er3+ concentration in the core, the visible red emission enhanced and became stronger than green emission. The designed highly erbium-doped nanoparticles is novel and meaningful and emits higher red luminescence than that of traditional NaYbF4:2% Er@NaYF4 nanoparticles (Figure S1). Finally, the PDT agent of ZnPc was carried after mesoporous silica-coating which could absorb red emission light and produce reactive oxygen species (ROS).

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dissolving the corresponding rare-earth oxides in trifluoroacetic acid at 90 °C. Synthesis of Core Nanoparticles. NaYF4:x%Er (x = 5, 25, 50, and 100) core nanoparticles were prepared with the traditional method. Typically, 1 mmol of RECl3 (RE = Y and Er) (molar ratio of 95:5, 75:25, and 50:50) was mixed with 9 mL of oleic acid (OA) and 15 mL of octadecene (ODE) in a three-necked bottle. The mixture was heated to 160 °C for 45 min under an argon atmosphere to dissolve RECl3 and then cooled to 40 °C naturally. Then, 10 mL of methanol solution mixed with 0.148 g of NH4F and 0.100 g of NaOH was added to the mixture drop by drop, and the mixture was stirred for 1 h. Subsequently, the solution was slowly heated to remove methanol, degassed at 100 °C for 30 min, and the mixture quickly heated and maintained at 300 °C under an argon atmosphere for 1 h. The solution was cooled to room temperature, and the nanoparticles were centrifuged and washed with 20 mL of ethanol for 2 times. Finally, the OA-stabilized NaYF4:x%Er (x = 5, 25, 50, and 100) nanoparticles were dispersed in 10 mL of cyclohexane for further use. Synthesis of Core@Shell Nanoparticles. First, 1 mmol of Lu(CF3COO)3 and 1 mmol of sodium trifluoroacetate (CF3COONa) were mixed with NaYF4:x%Er(x = 5, 25, 50, and 100) in 9 mL of OA and 15 mL of ODE. After stirring at 120 °C under an argon atmosphere for 30 min, the clarified solution was heated and maintained at 310 °C for 30 min. Finally, the cooled solution was centrifuged and washed with 20 mL of ethanol 3 times. OA-stabilized NaYF4:x%Er@ NaLuF4(x = 5, 25, 50, and 100) was obtained and dissolved in cyclohexane. The Yttrium epitaxial shells were synthesized in the same way. The core@shell nanoparticles are named UCNP. Phase Transfer of UCNP@mSiO2. The silica coating process was carried out by a modified reported procedure. Typically, a beaker containing 30 mL of deionized water and 0.12 g of CTAB to obtain a transparent solution, and 3 mL of cyclohexane containing UCNP (20 mg mL−1) was added. The mixture was then stirred vigorously for 12 h to obtain a homogeneous UCNP-CTAB solution. After that, the UCNP-CTAB aqueous solution, 6 mL of ethanol, and 300 μL of NaOH (2 M) were added to deionized water. Then, the mixture was heated and stirred at 60 °C, and 300 μL of TEOS was added slowly into the solution and stirred vigorously for 10 min. The precipitation was centrifuged and washed with ethanol several times. The CTAB template was extracted by ion exchange process. Typically, UCNP@mSiO2 was added into 40 mL of ethanol solution together with 0.18 g of NH4NO3. The solution was heated and kept at 60 °C for 2 h. Finally, the UCNP with mesoporous silica coating (named @mSiO2) were obtained. Preparation of UCNP@mSiO2−ZnPc. The as-prepared UCNP@ mSiO2 was dispersed in deionized water. Subsequently, 100 μL of APTES was dropped into the solution and then heated and stirred at 45 °C for 6 h; the nanoparticles were collected by centrifugation and washed with ethanol several times. Then, 1 mL of ZnPc (1 mg mL−1) was dropped into the amine-conjugated samples and stirred overnight. UCNP@mSiO2−ZnPc was obtained after centrifugation. Characterization. Transmission electron microscopy (TEM) was obtained and detected on a JEOL-JEM 2100 field emission apparatus operated at an acceleration voltage of 200 kV. Upconversion luminescence spectra and luminescence decays were measured by an Edinburgh FLS980 spectrometer equipped with either a 980 or 808 nm continuous-wave (CW) diode lasers as the excitation source. Energy dispersive spectroscopy (EDS) tests were carried out on a HITACHI SU8220 with a field emission gun operating at 3.0 kV. In Vitro Viability of UCNP@mSiO2−ZnPc. The activity measured using MTT was observed in a 96-well plate. Typically, the MCF-7 cells were cultured to obtain monolayers cell for 12 h, and different concentrations of UCNP@mSiO2−ZnPc nanoparticles (500, 250, 125, 62.5, 31.3, 15.6, and 7.8 μg mL−1) were then added to the three groups. The cells were cultured with nanoparticles under 5% CO2 atmosphere at 37 °C for another 24 or 48 h. Then, 20 μL of MTT solution (5 mg mL−1) was added to each well, and the entire plate was incubated for an additional 4 h. Then, the solution in each well was carefully aspirated, and 100 μL of DMSO was added. Finally,

MATERIALS AND EXPERIMENTS

Chemicals. YCl3 (99.95%), ErCl3 (99.9%), LuCl3 (99.9%), sodium trifluoroacetate (CF3COONa, 98%), oleic acid (OA, 90%), 1-octadecene(ODE, 90%), trifluoroacetic acid (CF3COOH, 99%), Calcein AM (96.0%), propidium iodide (PI, 94%), hexadecyltrimethylammonium bromide(CTAB, ≥99.0%), tetraethyl orthosilicate (TEOS, 99.99%),(3-aminopropyl) triethoxysilane (APTES, 98%), 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT), and PBS buffer were purchased from Aladdin. Zincphthalocyanine (ZnPc, 97%) was purchased from Sigma-Aldrich. Ammonium fluoride (NH4F), hydrochloric acid (HCl), sodium hydroxide (NaOH), methanol, ethanol, dimethyl sulfoxide (DMSO), and cyclohexane were obtained from Sinopharm Chemical Reagent Co., Ltd. Ammonium nitrate (NH4NO3) from Tianjin Kermel Chemical Co., Ltd. Re(CF3COO)3 was prepared by B

DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Scheme 1. UCNP@mSiO2−ZnPc Design and Characterizationa

carried out as control. Finally, the cells were stained with Calcein AM and PI at 37 °C and 5% CO2 for 15 min, washed with PBS several times, and then imaged by using a Leica DMI 4000 B instrument. In Vivo X-ray CT Imaging. The in vivo CT imaging experiments were performed on a XLCT system at a voltage of 80 kV. Female BALB/c mice (∼20 g) were purchased from Shaanxi Experimental Animal Center, Xi’an Jiaotong University (Xi’an, China). 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. The mice were first narcotized with 10% chloral hydrate by intraperitoneal injection. Then, 100 μL (10 mg mL−1) of UCNP@ mSiO2−ZnPc solution were injected subcutaneously for scanning. In Vivo Toxicity of UCNP@mSiO2−ZnPc. Female mice were separated arbitrarily into two groups: Mice were injected with PBS as control group, and mice were injected intravenously with 100 μg mL−1 UCNP@mSiO2−ZnPc as the treated group. After treatment for 14 days, the organs (heart, liver, spleen, lung, and kidney) were frozen and stained with H&E, and the microscopy images were taken on the optical microscope. In Vivo Photodynamic Therapy Effect of UCNP@mSiO2− ZnPc. H22 (murine hepatocarcinoma) cells were injected in the left axilla of each female mouse by subcutaneous injection of cell line. After 7 days of growth, the tumor size was about 6−8 mm. Mice were divided into four groups (n = 5 in each group) and injected as follows: with PBS as the control group, with UCNP@mSiO2−ZnPc in the dark, with NIR irradiation, and UCNP@mSiO2−ZnPc under NIR irradiation. Mice injected with 100 μL (1 mg mL−1) UCNP@mSiO2− ZnPc every 2 days. The tumor site was irradiated with 808 nm laser for 10 min each time (pump power was 0.76 W cm−2) and sustained irradiated for 14 days.

(a) Schematic diagram of the design of UCNP@mSiO2−ZnPc. (b) Upconversion emission photos of colloidal dispersion of coreshell nanoparticles (λexc: 980 nm). (c) Representative TEM image of NaErF4@NaLuF4@mSiO2−ZnPc nanoparticles. a

the plate was transcribed with a microplate reader at a wavelength of 560 nm. Live/dead status testing is carried out in 6-well plates. Typically, the MCF-7 cells were incubated to obtain a monolayer of cells, and then UCNP@mSiO2−ZnPc was added. After incubation with UCNP@mSiO2−ZnPc under 5% CO2 atmosphere at 37 °C for 12 and 36 h, the coverslips were washed with PBS several times. Finally, the microphotographs were recorded on a Leica DMI 4000 B instrument. In Vitro Photodynamic Therapy Effect of UCNP@mSiO2− ZnPc. MCF-7 cancer cell lines were cultured in a 96-well plate at 37 °C and 5% CO2 for 16 h. The cells were incubated with different concentrations of UCNP@mSiO2−ZnPc for another 3 h, and then each well was washed with PBS several times to remove the medium. Then, they were irradiated with an 808 nm NIR laser (1 W cm−2) for different times (1, 2, 3, 5, 7, and 10 min). The cancer cells treated with laser irradiation only and MCF-7 cells without treatment were

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RESULTS AND DISCUSSION Synthesis and Characterization of NaYF4:x%Er@ NaLuF4. The synthetic procedure is illustrated in Scheme 1a, and the nanoparticles consist of a NaErF4 core and a NaLuF4 inert shell. The photo of the colloidal dispersion of the

Figure 1. Representative TEM images of (a) NaErF4 core-only nanoparticles and (b) NaErF4@NaLuF4 core@shell nanoparticles. (c) Scanning TEM image of the NaErF4@NaLuF4 core@shell nanoparticles. (d) Representative high-magnification TEM image of the NaErF4@NaLuF4 nanoparticles. C

DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 2. Upconversion luminescence spectra of (a) core-only and (b) core@shell (NaYF4:x%Er@NaLuF4) nanoparticles with variable doped Er3+ concentrations in the core (NaYF4:x% Er3+, x = 5, 25, 50, and 100) under 980 nm laser excitation. (c) Upconversion luminescence spectra of core@shell nanoparticles (NaYF4:x%Er@NaLuF4) under 808 nm laser excitation spectra. (d) Schematic diagram of the energy level of upconversion and downshifting luminescence at various excitation fluxes.

Table 1. Luminescence Decay of Different Nanoparticles with Varied Er3+ Doping Concentrationa and Their Corresponding Lifetime Values 543 nm core only 5% 25% 50% 100%

530 403 380 330

μs μs μs μs

Er@Lu 820 630 450 420

μs μs μs μs

650 nm Er@Y 599 516 381 361

μs μs μs μs

core only 450 366 336 326

μs μs μs μs

Er@Lu

Er@Y

1.16 1.06 0.43 0.41

1.2 0.8 0.6 0.2

ms ms ms ms

ms ms ms ms

NaYF4:x%Er@NaLuF4 (x = 5, 25, 50, and 100) nanoparticles under 808 nm laser excitation shown in Scheme 1b, indicating the emission color could be controlled by the Er3+ concentration in the core. As is shown in Figure S2a and Scheme 1c, the TEM image indicates that NaErF4@NaLuF4@mSiO2 consists of well-dispersed nanoparticles with an average diameter of 57 nm. EDS results are as shown in Figure S2b, there are Na, Er, F, Lu, Si, and Zn in the sample. The outmost mesoporous shell has the pores with an average diameter of 2−3 nm. The average sizes of obtained NaErF4 (Figure 1a), NaErF4@NaLuF4 (Figure 1b), and NaErF4@NaYF4 (Figure 1c) nanoparticles are 21, 31.5, and 30.6 nm, respectively. High-resolution TEM in Figure 1d confirm that the synthesized core@shell nanoparticles are highly uniform with good crystallinity and hexagonal phase. As shown in Figure 2a, the UCL intensity of the core only under 980 nm laser excitation reduces with increasing Er3+

Figure 3. Luminescence decay of the red emission with variable doped erbium concentration in the (a) NaYF4:x%Er core-only nanoparticles and (b) NaYF 4 :x%Er@NaLuF 4 nanoparticles. (c) Schematic diagram of the energy level of Er3+ under 808 nm excitation and downshifting emission (1550 nm) level. (d) NIR-II images of NaYF4:x%Er@NaLuF4 (x = 5, 25, 50, and 100) nanoparticles at 808 nm excitation wavelengths. D

DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. (a) Optical microscopyimages of MCF-7 cells incubated with UCNP@mSiO2−ZnPc powders with the concentration of 125 and 500 μg mL−1 for (a1 and a3) 12 h and (a2 and a4) 36 h, respectively. MTT assay of cell viability of MCF-7 cells incubated with UCNP@mSiO2− ZnPc with different concentrations for (b) 48 h and (c) 24 h.

for the core only and core@shell nanoparticles were further detected. For the core NaYF4:x%Er, the red UCL emission lifetime reduced from 450 to 326 μs with Er3+ concentration increased from 5 to 100% (Figure 3a), while the green UCL emission lifetime was reduced from 530 to 330 μs (Figure S6). The distance between adjacent doped ions decreases when the Er3+ concentration increases and increases the possibility of energy transfer/migration to the surface. The red UCL emission lifetime increased from 450 to 1162 μs for the core doped with 5% Er3+ after epitaxial shell growth (Figure 3b). Similarly, the lifetime of green emission is increased from 530 to 820 μs (Figure S7). As prospected, the lifetime values of green emission level (4S3/2) are shorter than red emission level (4F9/2). The lifetime of NaYF4:x%Er@NaYF4 nanoparticles with different Er3+ doping concentrations under 808 nm laser excitation are presented in Figure S8. The luminescence decay of the Er@Lu and Er@Y nanoparticles with variable Er3+ doping concentrations and their relevant lifetime values have been listed in Table 1, demonstrating that no concentration quenching is detected in the lifetime of core@shell nanoparticles. The data of emission intensity and lifetime values in the core@shell nanoparticles suggest that energy migration is the leading mechanism in concentration quenching. Thus, when the energy transfer/ migration to the surface is inhibited by an epitaxial shell, the concentration quenching is restrained by the original core. Furthermore, the inhibition of both surface quenching and concentration quenching in the core@shell nanoparticles would increase the upconversion emission and downshifting emission in high doped nanoparticles. The downshifting emission wavelength centered at 1550 nm of Er3+ doped nanoparticles was generated through the excited photons transfer from energy level of 4I13/2 to 4I15/2 (Er3+ ions level) (Figure 3c). When the Er3+ concentration increases, the emission intensity at the wavelength 1550 nm shows a strong enhancement (Figure 3d), and the highly doped (100% Er3+) core@shell nanoparticles are the brightest. Similar to the enhancement of downshifting emission with increase Er3+ concentration is also suitable for an excitation

concentration in the core, which is due to the concentration quenching. The core@shell nanoparticles with different shell sizes were synthesized. TEM images of the nanoparticles and the upconversion emission spectra under 980 nm were are shown in Figure S3. The emission intensity increased with the shell thickness enhanced from 2 to 5 nm, and there was almost no further enhancement between the thickness of 5 and 5.5 nm. Thus, we chose the 5 nm as the optimized shell thickness. After epitaxial shell was coated on the heavily doped Er3+ core, we see that the UCL emission enhances with increasing Er3+ percentage and that the red emission bands (4F9/2 → 4I15/2) are higher than the green emission bands (2H11/2/4S3/2 → 4I15/2; Figure 2b). The spectra of other series of nanoparticles coated with yttrium epitaxial shells are shown in Figure S4. Then, we explored the upconversion emission properties of NaYF4:x% Er@NaLuF4 (x = 5, 25, 50, and 100) core@shell nanoparticles under another excitation wavelength of 808 nm (Figure 2c). The direct transition of Er3+ from the ground state level (4I15/2) to the excited state level (4I9/2) was excited at 808 nm. Similar to the excitation of 980 nm, the spectra of UCL shows enhancement of the red band with increasing Er3+ doped concentration (Figure S5a). The spectra of other series of nanoparticles coated with Yttrium epitaxial shells are shown in Figure S5b. Interestingly, the UCL spectra of the core@shell nanoparticles show the UCL intensity increases with increasing Er3+ doped concentration in the core. Under excitations of 808 and 980 nm laser, NaYF4:Er3+@NaLuF4 shows two property luminescence emission bands centered at ∼543 and ∼650 nm originating from green (2H11/2 → 4I15/2 and 4S3/2 → 4I15/2) and red (4F9/2 → 4I15/2) transitions of Er3+ ions. Downshifting and upconversion luminescence under various wavelength excitations for the Er3+ doped as shown in the energy level diagram (Figure 2d) were methodically probed for these core or core@shell nanostructures. It is known that the concentration quenching or surface quenching would reduce the lifetime of emission.53−57 The lifetime of red and green emissions under 808 nm excitation E

DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 5. (a) Viability of MCF-7 cells incubated with UCNP@mSiO2−ZnPc under 808 nm laser (1 W cm−2). Microscope images of MCF-7 cancer cells incubated (b) with culture only under 808 nm laser, (c) with UCNP@mSiO2−ZnPc in the dark as the control, and (d−i) with UCNP@mSiO2−ZnPc (100 μg mL−1) under 808 nm irradiation(1 W cm−2) for different times (1, 2, 3, 5, 7, and 10 min).

the liver samples were normal, and no pulmonary fibrosis was detected in all the lung samples. There was no necrosis in any of the organs. These data indicate the good biocompatibility of the as-prepared UCNP@mSiO2−ZnPc nanoparticles. There are several advantages of the 808 nm excitation compared with 980 nm: (a) The water has less absorption of 808 nm, resulting evident lower heating effect and hurt to the normal cells. (b) The 808 nm excitation has deeper penetration depth than that of 980 nm, and more excitation energy could arrive in the which is more suitable for in vivo or in vitro photoactive bioapplication. Moreover, NaErF4@NaY/LuF4 could emission higher upconversion luminescence under 808 nm than that of 980 nm. As shown in Figure S12, BALB/c mice was injected with the NaErF4@NaLuF4@mSiO2−ZnPc nanoparticles (100 μL, 200 μg mL−1) at the right hind limb for luminescence imaging. Under 808 nm laser, yellow upconversion luminescence (no optical filter used) and strong downshifting NIR-II luminescence emissions were detected. Also, the BALB/c mice was injected. Also, the lutecium element in the nanoparticles may have a CT imaging effect. As shown in Figure S13, there was obvious CT imaging signals after injection. The light-triggered PDT efficacies of UCNP@mSiO2−ZnPc using the MTT assay were evaluated. Six groups of MCF-7 cells were treated under different concentrations (15.6−500 μg mL−1) for 16 h, and then the MTT assay was carried out for quantitative

at 654 nm, we find that highly doped Er3+ concentration in the core@shell nanoparticles is the brightest (Figure S9). This downshifting emission in the NIR-II biological window is explored for bioimaging, indicating this UCNP could be applied as the dual-modal imaging agent. In Vitro/in Vivo Imaging and PDT Effect. The biocompatibility of UCNP@mSiO2−ZnPc powder was first assessed. The as-synthesized UCNP@mSiO2−ZnPc were dissolved into different physiological solutions of water, PBS, serum, and medium. As shown in Figure S10, it could be well-dispersed into the different physiological solution and kept stable after 6 h. Furthermore, the in vitro studies were performed on MCF7 cells lines. Optical microscope images of MCF-7 cells incubated with UCNP@mSiO2−ZnPc powders with the concentration of 125 μg mL−1and 500 μg mL−1 for 12 and 36 h, respectively (Figure 4a). With increasing culture time from 12 to 36 h, metabolic waste of cells increased with the morphology slightly changed. MTT assay was further carried out for different time points of 24 and 48 h, respectively (Figure 4b,c). When the cells incubated with the nanoparticles for 48 h, the viability was 65.78−88.91%, while the viability is 81.65−93.45% when the cells incubated with the nanoparticles for 24 h. Finally, in vivo toxicity of the UCNP@mSiO2−ZnPc nanoparticles were detected. When the mice injected with nanoparticles for 14 days, H&E stained images were shown in Figure S11. Hepatocytes in F

DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 6. (a) Photographs of mice and excised tumors. Changes in the (b) body weight and (c) tumor size. (d) H&E stained images of different organs in the PDT treatment group.

group was much smaller than that in other groups. H&E stained tumor sections presented in Figure 6d shows there were no necrosis in any of the organs in the PDT treatment group.

test of the cell viability (Figure 5a). There were almost no dead cancer cells under laser irradiation alone or only incubated with UCNP@mSiO2−ZnPc (250 μg mL−1) in the dark, while 74.12% cells were killed after irradiation for 5 min at the concentration of 250 μg mL−1. Even under a lower pump power of 0.7 W cm−2, about 55.0% of cells were killed with the concentrations of 125−500 μg mL−1 (Figure S14). The PDT effects of UCNP@mSiO2−ZnPc on MCF-7 cells were further verified using Calcein AM (green) and PI (red) costaining. Upon increased 808 nm laser irradiation (1 W cm−2) time, green fluorescence are decreased, indicating the cell viability gradually decreased with increasing time of laser irradiation. After 10 min, all the cells were killed, as indicated by the intense homogeneous red fluorescence (Figure 5b−i). Further experiments were carried out to investigate the PDT efficacy of UCNP @mSiO2−ZnPc in vivo. Herein, four groups of H22 cancer-bearing mice were treated as follows: with saline injection, with PBS, with UCNP@mSiO2−ZnPc in the dark, with NIR irradiation, and with UCNP@mSiO2−ZnPc under NIR irradiation. The mice body weights and tumor sizes were measured every 2 days after the treatments, and the final tumors and mice at day 14 were shown in Figure 6a. As shown, there was no significant decrease of mice weight in the four groups (Figure 6b), indicating UCNP@mSiO2−ZnPc does not show obvious toxicity to normal growth of mice. The tumor size curves versus the treatment days were shown in Figure 6c, revealing that the tumor growth in the best PDT treatment

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CONCLUSION In summary, after NaXF4 (X = Y and Lu) growth on those core nanoparticles, we see that the UCL and downshifting emission enhance with increasing percentage of Er3+ doping. The luminescence lifetime curves of the highly doped UCNP demonstrate that the leading restrainer pathway at concentration quenching is energy migration to the surface rather than crossrelaxation between adjacent doped ions. The designed UCNP combining Er3+ assisted energy migration and an active core@ shell structure exhibited red UCL of Er3+ upon NIR excitation. After coating a mesoporous silica layer and storing the therapy agents of ZnPc, the emission light transmit energy to ZnPc, which provides 1O2. In vitro results indicate that the sample exhibits an efficacious tumor inhibition effect and dual-modal imaging (UCL and NIR-II).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b02257. G

DOI: 10.1021/acs.inorgchem.8b02257 Inorg. Chem. XXXX, XXX, XXX−XXX

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TEM image and EDS of the NaErF4@NaLuF4@mSiO2 nanoparticles; TEM images and upconversion spectra under 980 nm laser of core@shell nanoparticles with different shell sizes; UCL spectra of NaYF4:x%Er@ NaYF4 nanoparticles with variable Er3+ doping concentrations under 980 nm excitation. UCL spectra of NaYF4:x%Er@NaLuF4 and NaYF4:x%Er@NaYF4 nanoparticles with variable Er3+ doping concentrations under 808 nm laser excitation; luminescence decay of the green emission of the NaYF4:x%Er and NaYF4:x%Er@NaLuF4 with variable Er3+ doped concentration under 808 nm laser excitation; luminescence decay of red emission and green emission of the NaYF4:x%Er@NaYF4 with variable Er3+ doped concentration under 808 nm CW laser diode excitation; downshifting luminescence spectra of the NaYF4:x%Er@NaLuF4 with variable Er3+ doped concentration. UCNP@mSiO2−ZnPc were dissolved into different physiological solutions for different time; H&E stained images of different organs in the control and nanoparticles-injected group; upconversion luminescence and downshifting luminescence photographs of mice after injected nanoparticles under 808 nm laser excitation; CT images of the mouse before and after injection; viability of MCF-7 cells incubated with UCNP@mSiO2−ZnPc under 808 nm laser (0.7 W cm−2) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (R.L.). *E-mail: [email protected] (P.Y.). *E-mail: [email protected] (J.T.). ORCID

Ruichan Lv: 0000-0002-6360-6478 Bo Hu: 0000-0001-5193-3511 Piaoping Yang: 0000-0002-9555-1803 Jie Tian: 0000-0002-9523-2271 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (NSFC 81227901, 81772011, and 81801744), the National Key R&D Program of China Grant (Nos. 2016YFC0102000, 2017YFA0205202, and 2017YFA0205200), and the Fundamental Research Funds for the Central Universities (XJS17011 and JBX181202).

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