Highly Emissive Dye-Sensitized Upconversion ... - ACS Publications

Mar 20, 2017 - bined with mesoporous silica, which has Ce6 (red-light-excited photo- sensitizer) and ... excitation lights, 980 nm light is the most w...
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Highly Emissive Dye-Sensitized Upconversion Nanostructure for Dual-Photosensitizer Photodynamic Therapy and Bioimaging Jiating Xu,† Piaoping Yang,*,† Mingdi Sun,† Huiting Bi,† Bin Liu,† Dan Yang,† Shili Gai,† Fei He,† 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, P. R. China ‡ State Key Laboratory of Rare Earth Resource Utilization, Changhcun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China S Supporting Information *

ABSTRACT: Rare-earth-based upconversion nanotechnology has recently shown great promise for photodynamic therapy (PDT). However, the NIRinduced PDT is greatly restricted by overheating issues on normal bodies and low yields of reactive oxygen species (ROS, 1O2). Here, IR-808-sensitized upconversion nanoparticles (NaGdF4:Yb,Er@NaGdF4:Nd,Yb) were combined with mesoporous silica, which has Ce6 (red-light-excited photosensitizer) and MC540 (green-light-excited photosensitizer) loaded inside through covalent bond and electrostatic interaction, respectively. When irradiated by tissue-penetrable 808 nm light, the IR-808 greatly absorb 808 nm photons and then emit a broadband peak which overlaps perfectly with the absorption of Nd3+ and Yb3+. Thereafter, the Nd3+/Yb3+ incorporated shell synergistically captures the emitted NIR photons to illuminate NaGdF4:Yb,Er zone and then radiate ultrabright green and red emissions. The visible emissions simultaneously activate the dual-photosensitizer to produce a large amount of ROS and, importantly, low heating effects. The in vitro and in vivo experiments indicate that the dual-photosensitizer nanostructure has trimodal (UCL/CT/MRI) imaging functions and high anticancer effectiveness, suggesting its potential clinical application as an imaging-guided PDT technique. KEYWORDS: IR-808, upconversion, dual-photosensitizer, photodynamic therapy, bioimaging relatively narrow.34 Besides, the continuous-wave 980 nm laser usually causes significant water absorption and thus leads to overheating issues of biobodies, which causes undesired injury to normal cells and tissues and further imperfect penetration depth.35−38 An effective method to solve the above issues is to dope Nd3+ ions in Yb3+-sensitized UCNPs, which can successfully modulate the excitation wavelength at 980 nm to a more biocompatible wavelength at 808 nm where the tissue transparency is maximized and thus the heating effect is minimized.39−43 In addition, the absorption cross section of Nd3+ is 1 order of magnitude higher than that of Yb3+,44 which can markedly boost the capture ability of excitation photons.45−47 Meanwhile, the energy transfer efficacy from Nd3+ to Yb3+ can be up to 70% according to previous reports.48−50 Thus, doping Nd3+ into the conventional Yb3+-

P

DT is an emerging therapy modality for cancer treatment.1−8 However, the conventional photosensitizers applied in PDT are usually triggered by visible light, which cannot penetrate thick biotissues.9−15 The utilization of these visible-light-excited photosensitizers is confined to treating tumors on the lining of internal organs or cavities or just under the skin and is less efficacious when treating deep-seated and large tumors.16−18 The application of NIR laser to PDT can achieve deeper penetration than that of visible light because most biomolecules absorb minimally in the NIR range (700−1100 nm);19−23 thus, a vehicle that can transduce NIR laser to visible light is extremely desired for this technology. Fortunately, tremendous progress in rare-earth-based upconversion nanoparticles (UCNPs) provides an alternative way to realize NIR-to-visible conversion.24−30 Among various NIR excitation lights, 980 nm light is the most widely used to produce upconversion luminescence (UCL) in Yb3+-sensitized UCNPs.31−33 However, due to the parity-forbidden character of intra-4f transitions, the absorption band of the Yb3+ ions is © 2017 American Chemical Society

Received: February 10, 2017 Accepted: March 20, 2017 Published: March 20, 2017 4133

DOI: 10.1021/acsnano.7b00944 ACS Nano 2017, 11, 4133−4144

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Figure 1. TEM images of OA-NaGdF4:Yb,Er (a), core−shell OA-UCNPs (b), NOBF4-UCNPs (c), UCS (d), and UCSM (e) (insets are the corresponding high-resolution images and particle size distribution); XPS spectra of UCNPs (f) and USCM (g), XRD patterns (the standard pattern of β-NaGdF4 is given for comparison) (h), and FT-IR spectra (i) of as-prepared OA-UCNPs, NOBF4−UCNPs, UCS, and UCSM nanoparticles.

make the nanoparticles represent ultrabright UCL under low 808 nm excitation. To make full utilization of the enhanced multicolor (red and green) upconversion emissions for PDT, the IR-808-sensitized UCNPs were further encapsulated with a mesoporous silica layer, which has Ce6 and MC540 loaded inside through covalent bond and electrostatic interactions, respectively. The design of UCNPs with a layer of silica can not only allow a high loading of photosensitizers but also avoid direct contact with the cells in the biobodies and protect them from being degraded by the harsh in vivo microenvironment.63−65 In addition, according to Shao’s report,46 these dyesensitized nanoparticles may endow the nanosystem with photothermal conversion capability, which is beneficial for PDT because a suitable hyperthermia can increase intratumoral oxygen content and consequently accelerate tumor oxygenation. Such design of dual-photosensitizer nanomedicine affords a potential strategy to induce excellent cancer cell apoptosis and improve antitumor efficiency. Furthermore, due to the unique atomic property of Gd3+/Yb3+ elements and their codoping in UCNPs, the innovated nanoplatform also poses UCL/CT/ MRI trimodal imaging functions, thereby realizing imagingguided PDT.

sensitized UCNPs holds great promise to shift the excitation wavelength into 808 nm and keep the ideal UCL efficacy. Accordingly, in these upconversion materials, the Nd3+ ions absorb 808 nm photons, whereas the Yb3+ ions serve as an energy bridge, receiving the energy from the Nd3+ ions and transferring it to illuminate the activators. Thus, the 808 nm light excited UCNPs can efficiently enhance the penetration depth in biotissues and reduce unwanted overheating effects. However, the biggest challenge of Nd3+-sensitized upconversion nanomaterials is the severe quenching effect caused by energy back-transfer from activators to 4IJ energy levels of Nd3+ (Figure S1).51−54 Thus, the design of core−shell structure with Nd3+ and emitters separated in distinct layers is extremely essential to eliminate the detrimental quenching effects. To make the UCNPs readily applicable in PDT and imaging fields, scientists are still on the way to develop nanomaterials with high upconversion emission under low excitation power density. Following this trend, some recent papers reported that utilizing NIR dye to sensitize UCNPs can significantly improve upconversion performance.55−59 In these literature examples, we have learned that the NIR dyes have much higher absorption coefficients due to their wider absorption band and higher extinction coefficient than lanthanide sensitizers. Furthermore, the dominating NIR-emissive peaks of IR-808 overlap perfectly with the absorption of Yb3+ and Nd3+ ions,60−62 so we envisage that a suitable combination of IR808 and UCNPs with Nd3+/Yb3+ doped shell could afford superior UCL upon 808 nm laser excitation. In this paper, the IR-808, which has a broad absorption band with a maximum at 808 nm, was synthesized and applied to sensitize NaGdF4:Yb,Er@NaGdF4:Nd,Yb nanoparticles. The core−shell structure and IR-808 sensitization synergistically

RESULTS AND DISCUSSION The core NaGdF4:Yb,Er nanoparticles were prepared by a modified thermal decomposition method according to a previous report. 66 In the TEM image of OA-capped NaGdF4:Yb,Er nanoparticles (Figure 1a), the sample consists of uniform and monodisperse nanoparticles with a mean diameter of 24.6 nm. To enable the efficient upconversion of the IR-808-sensitized nanoparticles under 808 nm light excitation, a sensitizing layer (NaGdF4:Nd,Yb) was coated on 4134

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Scheme 1. Schematic Illustration of the Proposed Energy Level Diagram (a) and the Energy-Transfer Mechanisms in DyeSensitized UCNPs upon 808 nm Excitation (b), Functionalization of Dye-Sensitized UCNPs with Mesoporous Silica and the Dual-Photosensitizer of Ce6 and MC540 for Imaging-Guided PDTa (c)

a

Insets are the synthetic process of the APTES-modified Ce6 and the molecule structure of MC540.

the core component via an epitaxial growth method.67 The TEM image (Figure 1b) indicates that the uniformity and dispersity of the UCNPs have been kept well, and the average size is about 28.2 nm. Afterward, a very short ionic NOBF4 ligand was first used to replace the original oleic ligand and then further exchanged with the carboxylic-functionalized IR-808 dye.68 The IR-808 dye was prepared using the commercial dye IR-783 (Scheme S1), and its HNMR and FT-IR spectra are provided in Figures S2 and S3, respectively. Notably, the uniformity and dispersity are retained very well after NOBF4 treatment, and the size distribution data is similar to that of OA-UCNPs (Figure 1c). The IR-808 can harvest the photons around 808 nm, transfer the energy to the Nd3+- and Yb3+doped shell, and finally migrate to the activator Er3+ through Yb3+ ions (Scheme 1a,b). The photosensitizer Ce6 was premodified with APTES and then covalently conjugated inside the mesoporous silica along with the silica-coating process (top inset of Scheme 1c). The UCNPs with silica shell and Ce6 were named UCS. After the silica layer was coated, the negatively charged MC540 molecules (down inset of Scheme 1c) were inserted into the mesopores through electrostatic interaction, and thus, the dual-photosensitizer nanomedicine (abbreviated as UCSM) was obtained for trimodal (MRI, CT and UCL) imaging-guided PDT (Scheme 1c). The TEM images in Figure 1d,e indicate that UCS and UCSM consist of well-dispersed nanoparticles with an average size at about 46.5 nm. In addition, close observation reveals the wormlike channels on the mesoporous silica layer. X-ray photoelectron spectroscopy (XPS) analysis (Figure 1f,g) and the EDS spectrum (Figure S4) verify the elemental composition of

UCSM, and the Er3+ cannot be detected due to its low doping concentration. The XRD patterns of the OA-UCNPs, NOBF4UCNPs, UCS, and UCSM samples and the standard pattern of hexagonal NaGdF4 are presented in Figure 1h. Obviously, all of the diffraction peaks of these four samples can readily correspond to pure hexagonal NaGdF4. As accepted, the luminescent material having a hexagonal crystal phase is beneficial for photoinduced biomedical application due to its high upconversion efficacy. Fourier transform infrared (FT-IR) spectroscopy is used to identify the functional groups on the surface and give additional evidence for successful modification, and the FT-IR spectra of OA-UCNPs, NOBF4−UCNPs, UCS, and UCSM are presented in Figure 1i. As shown, the OA-UCNPs exhibit bands at 1463 and 1564 cm−1, which associates with the vibrations of the carboxylic groups. The strong transmission bands at 2854 and 2924 cm−1 are assigned to the asymmetric and symmetric stretching vibrations of methylene (−CH2), respectively, and the broad band at around 3450 cm−1 originates from the O−H stretching vibration. After NOBF4 treatment, the C−H stretching vibration of OA molecules has a noticeable intensity decrease at 2800−3000 cm−1. In addition, a great reduction in the vibrational peak of the carboxylic groups (1500−1200 cm−1) can be seen, along with the appearance of a vibrational peak of NOBF4 (1084 cm−1). These results confirm the successful ligand exchange of NOBF4 with the OA ligand on the surface of UCNPs. The stretching vibrations of CO in the DMF molecules result in a new peak at around 1650 cm−1. When further treated with the IR-808 dye, new peaks appear at 1000 to 1500 cm−1, indicating that IR-808 dyes are anchored 4135

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Figure 2. Absorption spectra of IR-783 and IR-808 (a), emission spectrum of IR-808 and the absorption spectrum of UCNPs (b), UC emission spectra of OA-Er@NaGdF4:0.1Yb,xNd nanoparticles (c), and NOBF4 treated UCNPs with increased amounts of IR-808 (d).

functionalized derivative (Scheme S1). The IR-808 absorbs photons mainly locate between 650 and 850 nm, with a maximum at 808 nm (Figure 2a, red line). Notably, ignorable absorption of IR-808 below 650 nm is significant for this study because the antenna molecules should be transparent to the upconverted photons. From Figure 2b, we can find that the absorption peaks of Nd3+ and Yb3+ ions doped in the UCNPs overlap strongly with the NIR-emissive peaks of IR-808. These overlaps allow for nonradiative energy transfer from IR-808 to the Nd3+ and Yb3+ ions in the active-shell and finally to the Yb3+/Er3+ coupled upconverting zone through Yb3+ ions, which act as energy bridges for migrating photons from antenna to Er3+ ions. When excited by 808 nm light, the IR-808 has very significant NIR absorptivity, and thereafter a broadband NIR peak emitted. The emitted NIR photons have wavelengths varying from 750 to 1050 nm, of which 808 and 980 nm photons can be synergistically captured by the Nd3+ and Yb3+ ions, combined with the core−shell structure which avoids the detrimental energy back transfer from activators to Nd3+ ions to achieve the ideal UCL. To achieve the brightest upconversion in IR-808-sensitized core−shell nanoparticles, the shell composition should be first determined. In consideration of the bigger NIR absorption section of Nd3+ and the essential role of Yb3+ in transferring energy among nanocrystal lattice, the doping content of Yb3+ in active shell was selected at 10%. According to the result in Figure 2c, it is obvious that NaGdF4:0.3Nd,0.1Yb has the highest emission intensity, which is most suitable for further sensitization. The three characteristic emission peaks of Er3+ correspond to 2H11/2 → 4I15/2 (510−530 nm), 4S3/2 → 4I15/2 (530−570 nm), and 4F9/2 → 4I15/2 (630−680 nm) transitions. Note that the NOBF4 treatment brings an improvement to emission intensity (Figure S9). In this study, OA-capped UCNPs (OA-UCNPs) usually possess a slight cloudy appearance in cyclohexane (upper left insets in Figure S9),

on the UCNP surface through both the sulfate and carboxylic groups (Figure S3). After silica coating, there are typical Si− O−Si deformation (797 and 463 cm−1) and asymmetric stretching (1088 cm−1) vibrations. When hydrophilic MC540 molecules are loaded in the mesopores, a new peak at 1630 cm−1 represents the benzene ring on MC540.69 The N2 adsorption/desorption isotherms and the pore-size distributions of UCS and UCSM are shown in Figure S5. These two samples represent typical type IV isotherms, implying the mesopore structure of silica channels.65 The BET surface area of the UCS is 680.7 m2 g−1, and the average size of the pore is identified to be 3.88 nm using the Barrett−Joiner−Halenda (BJH) method (Figure S5a,b). The suitable pore size and the large surface area will be ideal to save cargos. To load MC540 molecules easily, we modified the UCS with an amino group and therefore endow it with positive surface property, and then the MC540 was inserted into the mesopores of silica layer through electrostatic interaction effect. As shown in Figure S6, the UCS initially has a zeta potential of −9.8 mV and increased to 21.8 mV after amino modification. After MC540 was trapped into the silica mesopores, the obtained UCSM sample has a zeta potential of 12.3 mV, which demonstrates the successful MC540 loading. Consistent with this, the UCSM has the BET surface area of 536.4 m2 g−1 and a mean pore size of 3.48 nm, which are slightly lower than that of UCS sample (Figure S5c,d). Notably, the loading of MC540 through an electrostatic interaction effect is very stable (Figure S7). In addition, the loading amounts of IR-808, Ce6, and MC540 in the UCSM nanostructure are, respectively, calculated to be 0.22, 36, and 27 μg mg−1 (Figure S8). The IR-783 with the maximum absorption peak at 783 nm (Figure 2a, black line) was used as the initial material to fabricate antenna molecules IR-808 to be attached onto the surface of the UCNPs. Nucleophilic substitution of the chlorine atom in IR-783 was executed to prepare a carboxylic acid 4136

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Figure 3. Upconversion emission spectra of IR-808-sensitized UCNPs, UCNPs@mSiO2, UCS, and UCSM and UV−vis absorption spectra of Ce6, MC540, and UCSM (a); the variation trend of the absorbance of DPBF mixed with the UCS (b), USM (c), and UCSM (d) nanoparticles under 808 nm laser irradiation for different times. Confocal laser scanning microscopy (CLSM) images of HeLa cells with oxidized DCF fluorescence under different irradiation times. All laser pump powers are 0.72 W cm−2 (e).

0.9:1000. The upconversion luminescence of UCNPs-IR-808 has been enhanced approximate 12 times. According to theory, the luminescent donor would have an emission intensity decrease with the combination of the energy acceptor of the photosensitizers. Figure 3a represents the upconversion emission spectra of IR-808-sensitized UCNPs, UCNPs@mSiO2, UCS, and UCSM nanoparticles and UV−vis absorption spectra of Ce6, MC540 and USCM nanoparticles. As shown, there are obvious overlaps in the red- and greenemissive peaks of UCNPs@mSiO2 and the absorbance peaks of Ce6 and MC540 molecules. In comparison with UCNPs@ mSiO2, the red emission peak decreases dramatically for the Ce6-conjugated UCS, and then the green peak decreases obviously after loading MC540. These results in combination with the lifetime measurements in Figure S10 verify the efficient energy transfer between the dye-sensitized UCNPs and dual-photosensitizer. Thereafter, a question that should be considered is whether the absorbed energy activates the Ce6 and MC540. Parts b−d of Figure 3 show the absorbance variation trend of DPBF solution mixed with UCS, UCNPs@mSiO2@MC540 (USM), and UCSM under 808 nm laser irradiation for various times. The absorption peaks of the DPBF decrease gradually at the wavelength of 350−470 nm, which indicates the efficient production of ROS. In other words, the photosensitizers in the UCS, USM, and UCSM samples can be respectively and

which originates from the severe light scattering due to the low nanoparticle solubility. However, when the nanoparticles are transferred into DMF solvent after NOBF4 treatment, the solution becomes transparent because of the improved solubility (upper right insets in Figure S9). The NOBF4modified UCNPs are highly dispersive in DMF due to the dualstabilization effect from DMF and NOBF4, and therefore, the UCNPs’ solubility is even higher than the initial solubility in cyclohexane.67 Thus, we speculate that the improved UCNP solubility reduces the laser light scattering, which inevitably enhances the excitation efficacy and therefore improves the emission intensity. In Figure 2d, the optimized IR-808 coverage rate was determined so as to realize the brightest upconversion of UCNPs. As shown, the upconversion emission first increases with addition of IR-808 to a DMF solution containing UCNPs, which is consistent with the increasing absorptivity of the 808 nm photons by an enhancing number of antenna molecules on the surface. However, further increase in the IR-808/UCNPs ratio leads to a reduced luminescence beyond a certain concentration. Two factors may account for the observed decrease: an increasing concentration of excess antenna molecules and increased mutual interactions between IR-808 molecules on the surface of nanoparticles, which absorb the excitation photons but do not transfer it to UCNPs.45,47,58 The optimal UCNPs-IR-808 weight ratio is determined to be 4137

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The in vivo CT imaging is displayed in Figure 4c,d. As shown, after injection with the UCSM sample, the tumor site has a CT value of 554.9 HU, which is obviously higher than that without injection (27.6 HU). Figure 5 represents the upconversion

simultaneously activated by 808 nm light. Notably, the dualphotosensitizer loaded nanoparticles have higher ROS generation efficacy because the reduction rate of DPBF absorbance in Figure 3d is faster than those in Figure 3b,c. In addition, we investigated the intracellular ROS generation of UCSM by DCFH-DA. The DCFH-DA can be taken up by cells but cannot radiate fluorescence. However, the DCFH molecules could be oxidized to DCF with luminous green fluorescence under 488 nm light radiation after hydrolysis to DCFH by intracellular esterase.70 Figure 3e exhibits the CLSM images of HeLa cells with oxidized DCF fluorescence. The stronger and stronger green fluorescence also indicates that the significant ROS generation of the UCSM upon 808 nm laser irradiation. Before practical utilization, it is necessary to assess the biocompatibility of the as-fabricated sample. Figure S11 shows the L929 cell viabilities after incubating with the UCSM sample having various concentrations tested by MTT assays. The UCSM sample shows a high viability of 98.0−99.4% in the entire concentration range even at 500 μg mL−1, indicating the excellent biocompatibility of UCSM. Moreover, the UCSM can act as an MRI contrast agent because the Gd3+ ions have a positive enhancement ability toward the T1 signal. In Figure S12, the signals are positively enhanced with sample concentrations changing from 0 to 5 mg mL−1. The longitudinal relaxivity (R1) value is 0.3489 mg−1 s−1. As accepted, the CT-imaging technique is reliable because of its high resolution in the three-dimensional structure and deep tissue penetration. In addition, the Yb element contained material has potency to be utilized as CT imaging contrast agents.71 In vitro CT imaging performance of UCSM is presented in Figure 4a. As presented, with the Gd/Yb concentrations enhanced, the CT signal increases obviously, and the CT values show positive enhancement versus the sample concentrations with a large slope of 14.23 (Figure 4b).

Figure 5. UCLM images of HeLa cells incubated with UCSM nanoparticles at 37 °C for 0.5, 1, and 3 h. Scale bars for all images are 50 μm.

luminescent microscopy (UCLM) images of HeLa cells incubated with UCSM for 0.5, 1, and 3 h at 37 °C. The DAPI, which has blue emission, is used to label the cell nuclei. It is obvious that the UCSM nanoparticles in the cells radiate green luminescence upon 808 nm laser irradiation. With the incubation time prolonged, the intensity of the green emission increases. Meanwhile, there are no signals found outside of the cells, whereas the green luminescence located at the intracellular region implies that the as-prepared sample has been swallowed into the cells rather than merely adhered on the cell surface. In addition, most of the UCL fluorescence signal is located in the cytoplasm, which verifies that the nanoparticles are engulfed by endocytosis through lysosomes and endosomes into the cells instead of passive adsorption. These results indicate that the UCSM sample is an effective UCL imaging contrast agent with an ignorable background. Figure 6 exhibits the CLSM images of HeLa cells treated with FITC-modified USCM for 0.5, 1, and 3 h at 37 °C, which are used to reveal the cell-uptake process. The FITC, which has green fluorescence, is used to track the USCM nanoparticles, and the overlay images of DAPI and FITC were also exhibited. As shown in the first 0.5 h, there is only weak luminescence of FITC, which indicates that few UCSM-FITC nanoparticles were swallowed by cancer cells. With the incubation time prolonged, the stronger green fluorescence is observed in the cell nucleus and cytoplasm, revealing that more FITC-modified nanoparticles are located in the cells. These results further validate that the UCSM-FITC nanoparticles can be effectively internalized by cancer cells. To compare the anticancer efficacy of the prepared samples, four groups of HeLa cells were treated under different conditions for 24 h, and then cell viability was quantitatively tested using the MTT method (Figure 7). The viability of cells treated with the laser irradiation only indicates that the 808 nm light shows no obvious negative impact on the cells. After USM incubation and then 808 nm light irradiation, a number of

Figure 4. In vitro CT images with different Gd/Yb concentrations (a), CT values of UCSM aqueous solutions versus the Gd/Yb concentrations (b), and CT images (c, d) of tumor-bearing Balb/c mouse preinjection (up) and after injection (bottom). 4138

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Figure 6. CLSM image of HeLa cancer cells incubated with USCM-FITC for 0.5 h, 1, and 3 h at 37 °C. Scale bars for all images are 50 μm.

were treated with UCS sample and then NIR light irradiation, the viabilities were lower than those in the USM and NIR lasertreated group; this may be due to the closer energy-transfer distance between the Ce6 and the dye-sensitized UCNPs. Obviously, the UCSM- and NIR-treated group has the highest cell killing efficacy, which demonstrates that the dual-photosensitizer nanostructure makes the best utilization of the greenand red-emissive photons for producing ROS. In addition, PI, which could dye dead cells with a red color, and calcein AM, which could dye living cells with a green color, were applied to differentiate cancerous cells under various conditions to verify the cell-killing efficiency. In the left image of Figure 7b, almost only green cells can be seen for the cells only irradiated by 808 nm light, which indicates that the pure 808 nm laser irradiation has no obvious damage to Hela cells. However, in the right picture of Figure 7b, for cells incubated with a UCSM sample and irradiated by an 808 nm laser, almost only red cells can be observed, implying that the cancer cells were effectively killed due to the PDT effects derived from the UCSM nanoparticles. Here, a comparison experiment was conducted to reveal the difference between 980 nm light and 808 nm light when applied to PDT and UCL imaging (Figure S13). Obviously, the 980 nm laser-irradiated group has lower cell-killing efficacy than that of 808 nm laser-irradiated group after penetrating a biotissue. Consistent with this finding, the in vivo UCL images achieved in pork muscle tissues (Figure S13c) indicate that the upconverted light from 808 nm laser excited sample can be more clearly visualized at about 8 mm. The tumor inhibition efficacy of the as-synthesized samples was investigated through the in vivo experiments. Here, one group of U14 tumor-bearing mice was kept as control

Figure 7. In vitro HeLa cell viabilities after incubation with culture, UCS, USM, or UCSM at different concentrations under NIR irradiation (a) and the CLSM image of HeLa cancer cells after incubation with culture alone and UCSM with NIR irradiation, dyed with calcium AM and PI (b). Scale bars for all images are 50 μm.

HeLa cells are killed with obviously lower viability than those treated with laser irradiation only, which may be caused by the PDT effect derived from MC540. However, when the cells 4139

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Figure 8. Changes in the body weights (a) and the relative tumor volume (b) achieved from mice with varying treatments; photographs of mice and excised tumors from representative mice (c); H&E stained images of tumor tissues obtained after 14 days of treatment (d) (***p < 0.001).

Figure 9. H&E stained images of heart, liver, spleen, lung, and kidney collected from different groups after treatment for 2 weeks. The scale bars in all images correspond to 50 μm.

experiment, another one injected with saline only, and an additional three injected with USM, UCS, and UCSM solution, respectively. In addition, the 808 nm laser irradiation of tumor sites should be executed for the latter four groups after the injection. The body weight and the tumor size were recorded every 2 days after the initial treatments. Parts a and b of Figure 8 exhibit the average body weights and the average tumor volume of each group during the 2 week treatments. As presented, there is no body weight decrease in these five groups, indicating that the samples have no adverse drug reaction to the mice. Compared with the control group, the

growth of tumors on NIR light irradiated mice is slightly inhibited over the course of 2 weeks of treatment, which may be caused by the heat effect originating from the hemoglobin absorbance. Meanwhile, the mice injected with the USM nanoparticles and exposed to the NIR light show a much smaller tumor size than the NIR-irradiated group, which may originate from the PDT effect of MC540. The tumor growth on UCS- and NIR-treated mice is inhibited to a certain degree after 2 weeks, which may be ascribed to the PDT effect of Ce6. The UCS sample has higher anticancer efficacy than USM, perhaps due to the closer distance between UCNPs and Ce6. It 4140

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(CF3COONa) and N,N-dimethylformamide (DMF) (from Tianjin Kermel Chemical Co., Ltd.); and trifluoroacetic acid (CF3COOH) (from Beijing Chemical Regent Co., Ltd.) were of analytical grade without purification. Synthesis of OA-Stabilized NaGdF4:Yb,Er Core Nanoparticles. Core and core−shell nanoparticles were synthesized by a general decomposition strategy using lanthanide oleates as precursors. Typically, 1 mmol of lanthanide oleates (Er/Yb/Gd = 2:20:78) was added to a reaction vessel having 15 mL of OA and 15 mL of ODE. The mixture was degassed at 110 °C for 0.5 h with magnetic stirring. Thereafter, the mixture was heated to 300 °C and kept for 1.5 h under N2 protection. After the temperature was cooled to about 40 °C, ethanol and cyclohexane were used to centrifuge the product. The obtained nanoparticles were dissolved in cyclohexane for further use. Synthesis of OA-Stabilized NaGdF4:Yb,Er@NaGdF4:Nd,Yb Nanoparticles. Briefly, the above cyclohexane containing core nanoparticles was added to a reaction vessel containing 15 mL of OA and 15 mL of ODE. Then 1 mmol of CF3COONa, 0.50 mmol of Gd(CF3COO)3, Nd(CF3COO)3, and Yb(CF3COO)3 with various mole ratios were added. The mixture was degassed at 120 °C for 40 min with magnetic stirring. After being flushed with N2, the reaction system was heated to 310 °C and maintained for 1 h. The obtained nanoparticles were dissolved in cyclohexane (10 mg mL−1) for further use. Synthesis of IR-808 Dye. The reaction was performed in a threeneck vessel under a dry N2 atmosphere using a modified procedure. 4Mercaptobenzoic acid (80 mg, 0.24 mmol) and IR-783 (100 mg, 0.12 mmol) were mixed with 5 mL of DMF at 25 °C and stirred for 24 h. Then the mixture was vacuum dried at 40 °C to evaporate DMF. Following that, 50 mL of diethyl ether was added to wash the product three times. After the liquid was filtered through a PTFE syringe (0.45 μm), the product was centrifuged and finally vacuum-dried to yield IR808 dye. Preparation of NOBF4-Modified UCNPs. The OA-stabilized UCNPs were first replaced with sub-nanometer ligands of NOBF4 using a modified procedure. During this process, 5 mL of a DMF solution of NOBF4 (0.01 M) was mixed with 5 mL of OA-UCNPs in cyclohexane (10 mg mL−1) at room temperature. The mixture was shaken gently for 10 min, allowing an extraction of nanoparticles from the upper cyclohexane layer into the bottom DMF layer. After the cyclohexane layer was disposed, the nanoparticles in DMF were purified by adding a large amount of toluene and cyclohexane (v/v = 1:1) and centrifuged for 5 min at 4500 rpm. Subsequently, the NOBF4-modified UCNPs (NOBF4-UCNPs) were weighted and redissolved in DMF for dye-sensitization experiments. Preparation of Dye-Sensitized UCNPs. A 1 mL portion of NOBF4-UCNPs in DMF (10 mg mL−1) was mixed with 1 mL of DMF having various amounts of IR-808 dye. Synthesis of UCNPs@Ce6@mSiO2. First, 0.5 mL of DMSO, which contains 2.0 mg of Ce6, 4 mg of NHS, 6 mg of EDC HCl, and 12 μL of APTES, was stirred at room temperature for 2 h. After that, a vessel containing deionized water (20 mL) and CTAB (0.1 g) was put in an ultrasonic cleaner to obtain a transparent solution, and then 2 mL of DMF solution containing NOBF4-UCNPs (∼10 mg mL−1) was added. The mixture was then stirred vigorously for 2 h, leading to a homogeneous solution. Thereafter, 300 μL of NaOH (2 M), 6 mL of ethanol, and 40 mL of deionized water were added to the above solution. The mixture was heated to 70 °C in water bath under vigorous stirring, and then 0.5 mL of DMSO having 200 μL of TEOS and 2.0 mg of APTES-premodified Ce6 was added slowly into the solution and maintained for 10 min. Then the mixture was centrifuged and washed three times with ethanol. The obtained nanoparticles were transferred to 50 mL of ethanol having NH4NO3 (0.3 g) and stirred at 60 °C for 2 h to extract CTAB templates. Finally, the UCNPs@Ce6@ mSiO2 (abbreviated as UCS) nanoparticles were dispersed in deionized water. For UCNPs@mSiO2 preparation, a similar procedure was conducted except APTES-modified Ce6 was used. Synthesis of UCNPs@Ce6@mSiO2-MC540. The MC540 molecules were loaded into the mesoporous layer through electrostatic interaction. First, 0.5 mL of APTES and the obtained UCS was added

is noted that the mice treated with UCSM injection and NIR irradiation display tumor volume even smaller than the initial size. Moreover, during the investigation period, the body weight of five groups was not obviously affected, which demonstrates an ignorable side effect of the as-prepared sample to the mice. In Figure 8c, the digital photographs of tumors collected from representative mice also confirm that the tumor volume upon UCSM and NIR treatment is the smallest, revealing its highest tumor inhibition efficiency. As a further proof, hematoxylin and eosin (H&E) stained tumor sections displayed in Figure 8d show the highest level of tissue damage for the group injected with a UCSM sample and irradiated with an NIR laser, illustrating good consistence with the data of tumor growth. The pathomorphological analysis of kidney, lung, liver, heart, and spleen is given in Figure 9. It can be seen that there is no evident organ damage to all five of the groups, indicating that the product has excellent biocompatibility in vivo. Additionally, the biochemical results of UCSM nanoparticles including total protein (TP), alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (CRE), which is closely related to the function of kidney and liver, are listed in Table S1. Compared with the control group, there is no obvious damage to kidney or liver. Moreover, the complete blood tests indicate that the injected sample has no obvious interference with the physiological regulation of immune or heme response. All in all, the developed UCSM holds great promise as a biocompatible nanomedicine for PDT clinical applications.

CONCLUSIONS In summary, we utilized IR-808-sensitized UCNPs carrying dual-photosensitizer as PDT agent. The nanoparticles with optimal IR-808 coverage and the core−shell structure can efficiently transduce the 808 nm photons to green and red light. The APTES-modified Ce6 was covalently conjugated along with the growing process of mesoporous silica shell, and then MC540 was electrostatically loaded inside the silica channels. The adopted drug-loading process can ensure high loading content, avoiding the undesired leakage and decreasing the distance between the energy donor and the acceptors. The spectral overlaps between the maximum absorption of the dualphotosensitizer and the upconverted visible emissions take full advantage of the highly emissive upconversion to activate PDT agents to generate cytotoxic ROS for antitumor therapy. The experimental results validate that the dual-photosensitizer UCSM nanostructure exhibits higher PDT efficacy than those of the single-load UCS or USM nanosystem. Moreover, the UCSM showed the UCL, CT, and MRI imaging properties under a single 808 nm light excitation, revealing its potency in imaging-guided PDT fields. EXPERIMENTAL SECTION Materials. Sodium fluoride (NaF), Nd2O3 (99.99%), Yb2O3 (99.99%), Gd2O3 (99.99%), Er2O3 (99.99%), hydrochloric acid (HCl), Chlorin e6 (Ce6), and nitrosonium tetrafluoroborate (NOBF4) (from Sinopharm Chemical Reagent Co., Ltd.); oleic acid (OA), 1-octadecene (ODE), IR-783, 4-mercaptobenzoic acid, Nhydroxysuccinimide (NHS), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), merocyanine 540 (MC540), aminopropyltrimethoxysilane (APTES), 4′,6-diamidino-2-phenylindole (DAPI), folic acid (FA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI) and calcein AM (from Sigma-Aldrich. Co. LLC); sodium trifluoroacetate 4141

DOI: 10.1021/acsnano.7b00944 ACS Nano 2017, 11, 4133−4144

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ACS Nano

intraperitoneal injection. Then 100 μL of USCM solution was injected intratumorally into the mice for scanning. In Vitro Cellular Uptake andUCLM Observation. The cellular uptake process was investigated on HeLa cells using a CLSM. Typically, HeLa cells were put into a six-well plate and incubated at 37 °C with 5% CO2. After 12 h incubation, 1 mL of USCM-FITC sample (1 mg mL−1) was added to each well and incubated for 0.5, 1, and 3 h, respectively. After the cells were washed with PBS three times, 1 mL of glutaraldehyde (2.5%) was used to treat the cells for 10 min. Then the cells were further washed with PBS three times, and 1 mL of DAPI solution (20 μg mL−1) was added and kept for 10 min. After washing three times, the cells were observed using a Leica TCS SP8 instrument. For the UCLM observation, the slides were prepared using a similar process except that the cells were detected using an inverted fluorescence microscope and a 808 nm continuous wave laser was used to radiate the sample. In Vitro Cytotoxicity. HeLa cells (6000−7000 well−1) were put into of a 96-well plate and incubated at 37 °C with 5% CO2. After the cells were incubated for 24 h, the UCS, USM, and UCSM samples were dissolved in culture and diluted to various concentrations (15.62, 31.25, 62.5, 125, 250, and 500 μg mL−1), and then the cells were treated with following treatments: NIR irradiation only, UCS and NIR irradiation, USM and NIR irradiation, UCSM and NIR irradiation, respectively. Before the NIR irradiation was carried out, the samples were added and the cells were incubated for another 6 h to finish the cell uptake. Subsequently, 20 μL of MTT solution (5 mg mL−1) was added to each well followed by incubation for 4 h. Finally, 150 μL of DMSO was added to the wells, and the absorbance at 490 nm was recorded using a microplate reader for calculation. In Vivo Toxicity. The U14 cells were injected subcutaneously in the left axilla of each mouse to obtain tumors. When the tumors grew to 6−8 mm, the mice were randomly divided into five groups (5 group−1) and injected in vein with the saline, UCS, USM, and UCSM under NIR irradiation, respectively. Another group without treatment was control group. The tumor focus was irradiated with an 808 nm laser for 10 min every time (pump power is 0.72 W cm−2). The body weights and the tumor sizes were measured and recorded. Histological Examination. The histological analysis was carried out after 2 weeks of treatment. Less than 1 cm × 1 cm of tissues of each representative organs of the heart, liver, spleen, lung, kidney, and tumor tissues of the mice in five groups were excised. Then the excised tissues were successively dehydrated using buffered formalin, ethanol of various concentrations, and xylene. Thereafter, the dehydrated tissues were embedded in liquid paraffin and sliced to 3 × 5 mm for hematoxylin and eosin (H&E) staining. The final stained slices were observed using an optical microscope.

to 20 mL of water and stirred for 12 h. Afterward, the mixture was centrifuged and washed three times with water. Finally, the sample was mixed with 2 mg of MC540 and stirred in 20 mL of water for 12 h to obtain the UCNPs@Ce6@mSiO2-MC540 (abbreviated as UCSM). To decorate FITC on UCSM for cellular uptake experiment, 20 mg of UCSM and 0.5 mg of FITC were stirred in 20 mL of water for 12 h. Then the mixture was centrifuged and washed three times to remove free FITC molecules. For comparison, we synthesized UCNPs@ mSiO2-MC540 (abbreviated as USM) using similar procedure. Before the in vivo experiment, the as-prepared nanoparticles (including UCM, USM, and UCSM) were further decorated by folic acid (FA). Typically, 20 mg of UCSM was dispersed in 20 mL of deionized water. One mL of FA (10 mg mL−1), 1 mL of NHS (2 mg mL−1), and 1 mL of EDC HCl (6 mg mL−1) were mixed together and stirred for 2 h. Then the mixture was added to the UCSM solution and stirred for 12 h in dark. Finally, the product was centrifuged and cleaned with water three times to remove the free molecules. Characterization. Upconversion emission spectra were measured on an Edinburgh FLS 980 apparatus using 808 nm laser diode module (K98D08M-30W, China) as the excitation source. UV−vis absorption spectra were acquired by a TU-1901 dual beam UV−vis spectrometer. Fourier-transform infrared (FT-IR) spectra were tested on a Vertex PerkinElmer 580BIR spectrophotometer (Bruker), using the KBr pellet technique. X-ray diffraction patterns of the particles were collected on a D8 Focus diffractometer (Bruker) using CuKα radiation (λ = 0.15405 nm). The samples for XRD analysis were prepared by depositing the nanoparticle solution on the glass slides and drying at 80 °C in a vacuum. Transmission electron microscopy (TEM) micrographs were obtained from a FEI Tecnai G2 S-twin transmission electron microscope with a field emission gun operating at 200 kV. ROS Detection of Samples. Typically, 1 mL of sample solution (1 mg mL−1) was mixed with 1 mL of ethanol having 1 mg of DPBF and put into a 24-well plate. The solution was kept in the dark and irradiated by a 808 nm light (0.72 W cm−2) for various times (0, 2, 5, 7, and 10 min). Then the supernatant was collected for UV−vis measurements. The intracellular ROS generation ability of the UCSM was studied using DCFH-DA. HeLa cells were incubated in the sixwell plate with coverslips for 24 h, and 1 mL of UCSM (1 mg mL−1) was added. After incubation for 3 h, DCFH-DA was added and incubated for 10 min and then washed with PBS three times. After irradiation with an 808 nm laser for 1, 3, and 5 min, the fluorescence images were achieved at a wavelength of 488 nm. In Vitro Viability of UCSM. L929 fibroblast cells (6000−7000 well−1) were put into a 96-well plate and incubated at 37 °C with 5% CO2 for 24 h to obtain monolayer cells. Among them, 8 wells were left with culture only for the control group. The samples were diluted into various concentrations (7.81, 15.62, 31.25, 62.5, 125, 250, and 500 μg mL−1) and then added into the wells and incubated for another 24 h. Thereafter, 20 μL of MTT solution (5 mg mL−1) was added into each well. After incubation for 4 h, 150 μL of DMSO was added into each well. The absorbance at 490 nm was detected using a microplate reader for calculation. In Vitro T1-Weighted MR Imaging. The in vitro MR imaging experiments were conducted in a 0.5 T MRI magnet. The UCSM nanoparticles were diluted into various concentrations. T1 was acquired using an inversion recovery sequence. T1 measurements were conducted using a nonlinear fit to changes in the mean signal intensity within each well as a function of repetition time (TR) using a Huantong 1.5 T MR scanner. Finally, the r1 relaxivity values were determined through the curve fitting of 1/T1 relaxation time (s−1) versus the sample concentration (mg mL−1). In Vitro and in Vivo X-ray CT Imaging. The in vitro CT imaging was performed on a Philips 64-slice CT scanner at a voltage of 120 kV. The sample was diluted into various concentrations and then placed in a line for CT imaging experiment. All of the mouse experiments were conducted in compliance with the criteria of the Guide for the Care and Use of Laboratory Animals (China). Female Balb/c mice were purchased from Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Harbin, China). The mice were first anesthetized with 10% chloral hydrate (0.03 mL g−1 of mouse) by

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00944. Figures S1−S13, Scheme S1, and Table S1 (PDF)

AUTHOR INFORMATION Corresponding Authors

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

Piaoping Yang: 0000-0002-9555-1803 Jun Lin: 0000-0001-9572-2134 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC 21401032, 51472058, 4142

DOI: 10.1021/acsnano.7b00944 ACS Nano 2017, 11, 4133−4144

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51332008, 51422209, 51602072, and 51572258), China Postdoctoral Science Foundation (2014M560248, 2016T90269, 2015M581430, and 2015T80321), the Natural Science Foundation of Heilongjiang Province (B201403 and B2015020), Outstanding Youth Foundation of Heilongjiang Province (JC2015003), Heilongjiang Postdoctoral Fund (LBHZ14052, LBH-Z14070, LBH-TZ1606 and LBHTZ0607), and the Fundamental Research funds for the Central Universities is greatly acknowledged.

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DOI: 10.1021/acsnano.7b00944 ACS Nano 2017, 11, 4133−4144