Energy Transfer Highway in Nd3+

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Energy transfer highway in Nd -sensitized nanoparticles for efficient near-infrared bioimaging Cong Cao, Meng Xue, Xingjun Zhu, Pengyuan Yang, Wei Feng, and Fuyou Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 13, 2017

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ACS Applied Materials & Interfaces

Energy transfer highway in Nd3+-sensitized nanoparticles for efficient near-infrared bioimaging Cong Cao, Meng Xue, Xingjun Zhu, Pengyuan Yang, Wei Feng*, Fuyou Li* Department of Chemistry & Institute of Biomedicine Sciences & State Key Laboratory of Molecular Engineering of Polymers & Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan road, Shanghai, P.R. China.

ABSTRACT: Despite the large absorption cross-section of Nd3+ dopant as a sensitizer in lanthanide doped luminescence system, the strong cross-relaxation effect of it impedes the promotion of doping concentration and thus reduces the utilization of excitation light. In this work, we introduce a highly efficient acceptor, Yb3+ ion, which can quickly receive energy from Nd3+ ions, to construct an energy transfer highway for the enhancement of near infrared emission. By using the energy transfer highway, the doping amount of Nd3+ ions in our NaYF4:Yb,Nd@CaF2 core/shell nanoparticles (CSNPs) can be markedly elevated to 60%. The quantum yield of CSNPs was determined to be 20.7%, which provides strong near infrared luminescence for further bioimaging application. Remarkably, deep tissue penetration depth (~10 mm) in in vitro imaging and high spatial resolution of blood vessel (~0.19 mm) in in vivo imaging were detected clearly with weak autofluorescence, demonstrating that probes can be used as excellent NIR biosensors. KEYWORDS: Nd3+-sensitized, NIR probe, Energy transfer, Optimization luminescence, High spatial resolution, Bioimaging

■ INTRODUCTION Fluorescence-based biological imaging attracts more and more attention for diverse applications in clinical diagnosis and therapy because of its high spatial resolution and fast sensitivity.1-4 The concept of tissue transparent window in near-infrared region (NIR, 700-1700 nm) has been proposed recently, which exhibits so many merits: deep tissue penetration, low scattering and absorption of light, weak autofluorescence from biological organisms, etc.5-9 Differ from other luminescence materials (e.g., carbon nanomaterials, quantum dots, fluorescent dyes),7-11 lanthanide doped luminescence nanocrystals are promising probe for their outstanding photochemical properties, including narrow emission bands, relatively low toxicity and superior photostability.12-15 Previous research have focused on upconversion nanoparticles (anti-Stokes shift emission) for biomedical imaging,14-19 while Nd3+ or Er3+ ions doped nanocrystals with large Stokes shift emission are also ideal NIR probes and need more investigation.13, 20-24 However, the doping concentration of rare earth activator ions is largely restricted by drastic cross-relaxation. In traditional Nd3+ doped luminescence probe, the optimal doping amount is only around 5%.22-28 The concentration quenching impedes the system effectively harvesting more excitation light and thus obtains limited emission light, which would be unfavorable for luminescence probes.21, 25-28

High efficiency energy transfer (up to 70%) in Nd3+ sensitized Yb3+ system has been reported in many kinds of host materials.22, 29-37 The sensitizing process makes it possible for elevating Nd3+ doping amount.22 However, Nd3+ and Yb3+ ions were always co-doped with other activators (Er3+, Tm3+, Ho3+, Tb3+) to optimize upconversion luminescence rather than NIR emission. Moreover, the doping concentration of Nd3+ was still restricted to utilize the excitation energy. Therefore, we just introduce Yb3+ ions into Nd3+ doped system without other activators, to construct an energy transfer highway for promoting the energy utilization efficiency. On this transfer highway, Yb3+ ions acted as the only activators for NIR emission, which can quickly receive energy from Nd3+ ions. Thus it can reduce the amount of excited-state-Nd3+ in the luminescence process, and store much excitation energy for the following luminescence process.22, 30-32, 34, 36 The energy transfer highway will give rise to increasing the doping concentration of Nd3+ and simultaneously enhancing the NIR emission. Based on this concept, we designed an Nd3+sensitized-Yb3+ system to maximize the energy utilization efficiency. In addition, surface-related NIR quenching in the aforementioned nanosystem may be significantly suppressed by the core/shell structure just like upconversion luminescent nanoparticles.13-14, 17, 38-39

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After luminescence optimization, cubic phase of NaYF4:7%Yb,60%Nd@CaF2 nanocrystals, with small size (~12 nm) and high quantum yield (~20.7%) were finally synthesized. The high efficiency of energy transfer in this Nd3+-sensitized-Yb3+ system, exciting at 808 nm, providing NIR emission at 980 nm, which would be a promising candidate for biological imaging.30, 34-35 Ethylenediamine tetramethylenephosphonic acid (EDTMP) (Figure S1) was further modified on the nanoparticles to improve the watersolubility and extend blood circulation time.40-42 Two Vshaped capillaries can be distinguished through 10 mm pork tissue. Finally, an in vivo vascular imaging in the mouse hindlimb with high spatial resolution down to 0.19 mm at a depth of 2-3 mm was detected clearly. ■ RESULTS AND DISCUSSION To optimize energy transfer between Nd3+ and Yb3+ ions, we chose cubic phase NaREF4 and CaF2 outer layer to construct classical and distinguishing core/shell structure (Figure 1a). We synthesized NaYF4:x%Yb,y%Nd (x=0, 1, 3, 5, 7, 10, y=0, 30, 45, 60, 75, 95) NPs by a thermolysis method. The transmission electron microscopy (TEM) images (Figure 2a, Figure S2 and S3) showed that all prepared NaYF4:x%Yb,y%Nd NPs have the spherical shape ~6 nm in diameter. The dopant of Nd3+ ions did not change the shape and phase of the nanoparticles (Figure S2). It was verifed by the X-ray powder diffraction (XRD) measurement that the core was cubic phase of NaNdF4 (Figure S4). Meanwhile, the high-resolution transmission electron microscopy (HRTEM) image (Figure 2b) was in agreement with XRD pattern. A small shift of the diffraction peaks compared with standard pattern of cubic phase NaNdF4 (JCPDS: 27-0757) was due to the larger ionic radius of Nd3+ than Y3+ ions.43 TEM images of the oleic acid capped NaYF4:7%Yb,60%Nd@CaF2 core/shell nanoparticles (OA-CSNPs) showed that the diameter of OA-CSNPs was 12 nm and the thickness of the CaF2 layer was 3 mm (Figure 2d). In addition, the little diffraction peak centered at 32 ° of NaYF4:Yb,Nd core nanoparticle can be attributed to the cubic phase of NaNdF4, indicating the difference between standard cubic phase of NaNdF4 and standard cubic phase of CaF2 (JCPDS: 35-0816) (Figure S4 and Figure 2c). After coating the thick CaF2 shell, the little peak centered at 32 ° of NaYF4:Yb,Nd@CaF2 (content ratio=1:4) core/shell NPs was not obvious (Figure 2c).39 Meanwhile, the HRTEM image (Figure 2e) was in agreement with XRD pattern. The core/shell structure was quiet discernible under high-angle annular dark-field scanning TEM (HAADF-STEM) (Figure 2f). The energy dispersive X-ray analysis (EDXA) also demonstrated the successful synthesis of the OA-CSNPs, in which Ca was coexist with other elements (Figure S5). As a contrast, the Nd3+-based NPs was first synthesized, in which Nd3+ acted simultaneously as the sensitizer and emitter. Conventional small and unusual

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large amounts of Nd3+ ions were separately doped into cubic phase NaYF4

Figure 1. (a) Illustration of NaYF4:7%Yb,60%Nd@CaF2 core/shell structures under 808 nm excitation. (b) Absorption spectra of different NPs: NaYF4:7%Yb,60%Nd, NaYF4:5%Nd, NaYF4:60%Nd and NaYF4:7%Yb (all dispersed in cyclohexane). (c) NIR luminescence spectra of above mentioned NPs, under an 808 nm laser (~200 mW/cm2) at room temperature. NPs. There are three NIR absorption peaks, which located at 740 nm, 800 nm, and 860 nm, corresponding to transition from 4I9/2 state to 4F7/2, 4F5/2, and 4F3/2 states (Figure 1b). NIR emissions peak of Nd3+ ions were separately at 860 nm (4F3/2 → 4I9/2), 890 nm (4F3/2→4I11/2), 1060 nm (4F3/2→4I13/2) and 1330 nm (4F3/2→4I15/2) (Figure 1c).24, 30 Notably, the integrated absorption at 800 nm of NaYF4:60%Nd was about 17 times higher than the optimized NaYF4:5%Nd (Figure 1b), while the luminescence was lower than the latter (Figure S6a). It can be seen that the optimal doping amount


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of Nd3+-based system was surely about 5%, coincide with traditional reports.25, 28 Higher doping concentration truly

Figure 2. TEM and HRTEM images of the NaYF4:7%Yb,60%Nd nanoparticles (a) and (b), and the NaYF4:7%Yb,60%Nd@CaF2 NPs (d) and (e) (all dispersed in cyclohexane). (c) XRD pattern of NaYF4:7%Yb,60%Nd NPs, NaYF4:7%Yb,60%Nd@CaF2 NPs and standard pattern of cubic phase of CaF2. (f) The HAADF-STEM image of the NaYF4:7%Yb,60%Nd@CaF2 NPs. caused deleterious concentration quenching, weak luminescence and low energy utilization efficiency. Considering concentration quenching in Nd3+-based NPs, large absorption cross-section of Nd3+ ions and high Nd3+→Yb3+ energy transfer efficiency make Nd3+-sensitizedYb3+ system more feasible in enhancing the utilization of excitation energy and overcoming concentration quenching. We introduced Yb3+ ions as activators to construct Nd3+-sensitized photoluminescence system. The designed structure of NPs and proposed energy transfer pathway for this sensitized system were shown in Figure 1a and 3a. A decrease of emission peak from Nd3+ and simultaneous appearance of peak at 980 nm from Yb3+ were observed in NaYF4:Yb,Nd nanoparticles (Figure 3b, 3e and S6). This observation also illustrated that Nd3+ effectively transferred energy to the Yb3+ ions. The doping concentration adjustment was investigated to achieve the maximum NIR emission intensity. Activators were usually at low concentration, therefore we fixed the Yb3+ ion dopant concentration at 7% (Figure 3b). In comparison with activators, Nd3+ ions, served as light absorber and sensitizers, doping concentration was

regulated in a large range (from 30% to 95%). We observed that the luminescence intensity was also increased along with Nd3+ doping amount increasing. It means that much more energy is absorbed from the excitation light by Nd3+ ion dopant elevating. Nevertheless, more sensitizer doping would lead to deleterious cross-relaxation thus the emission was weakened with Nd3+ doping concentration increasing right along (Figure 3e and S6c). On this occasion, Nd3+ ions doping concentration was finally determined at 60%, which issued brightest NIR emission. The luminescence mechanism was proposed as a single photon process in the energy transfer highway. To determine the mechanism, NIR luminescence of nanoparticles under gradually increased excitation power density was detected. The 808 nm laser excited Nd3+ ions to its 4F5/2 state, followed by a non-radiation transition to its 4F3/2 state, and then the energy transferred to the state 2F5/2 of Yb3+, then quickly emitting at 2F7/2 (Figure 3a). In theory, the luminescence emission is proportional to the increased exciting pump power density. The results revealed its truly a single photon process (Figure S7).



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It should be noted that both the absorption and near infrared emission of Nd3+-sensitized-Yb3+ system are enhanced by contrast with only Nd3+ ions doped system. The integrated absorption peak centered at 800 nm for Nd3+ was about 25 times larger than the absorption peak at 980 nm for Yb3+ ions in the OA-CNPs (Figure 1b). The integrated emission peak at 980 nm for OA-CNPs was ~8

Figure 3. (a) In NaYF4:Yb,Nd@CaF2 nanoparticles, illustration of energy transformation mechanism. The emission peaks were centered at 860 nm (4F3/2 to 4I9/2), 890 nm (4F3/2 to 4I11/2), 980nm (2F5/2 to2F7/2) and 1060m (4F3/2 to 4I13/2) as well as 1330m (4F3/2 to 4 I15/2), respectively. (b) NIR luminescence spectrum of NaYF4:x%Yb,60%Nd (x=3, 5, 7, 10) NPs dispersed in cyclohexane. (c) The relationship between emission peak centered at 980nm and the doping amount of Yb3+. (d) The summarize of NIR luminescence optimization at different dopant concentration and the brightest is the NaYF4:7%Yb,60%Nd NPs. (e) NIR luminescence spectrum of NaYF4:7%Yb,y%Nd (y=30, 40, 50, 60, 70, 80, 93) NPs dispersed in cyclohexane at room-temperature. (f) The relationship between emission peak centered at 980nm and the doping amount of Nd3+. times higher than that at 1060 nm for NaYF4:60%Nd3+ even though they have almost same absorption intensity at 800 nm (Figure 1c). In contrast, there was no obvious NIR emission from NaYF4:7%Yb NPs under 808 nm excitation. Above results implied the feasibility of using Nd3+-sensitized NPs which rendered NIR luminescence surpassing the conventional Nd3+-based NPs. This conclusion is supported by the excitation spectra from the state 4F7/2 of Yb3+ ions in OA-CNPs (Figure S8). The observed excitation peaks matched well with three NIR absorption peak of Nd3+ ions at 740, 800, 860 nm and one peak of Yb3+ ions at 980 nm. CaF2 optical innert layer was coated to protect inner sensitizer and activator from surface quenching. The growth of CaF2 shell on OA-CNPs suppressed nonradiative transition at the nanoparticle surface and reduced surface defects. The integrated NIR luminescence intensity from

the OA-CSNPs was 2.5 times higher than that of OA-CNPs (Figure 4a) under excitation of an 808 nm laser. Furthermore, CaF2 shell (rare earth ions free) could depress RE ionic leakage to reduce the potential toxicity in biomedical applications.29, 39 To further certify the effect of core/shell structure, we measured the lifetime of Nd3+ and Yb3+ ions, respectively. When the CaF2 shell was applied to NaYF4:7%Yb,60%Nd NPs, the lifetime of Yb3+ (2F5/2 → 2F7/2) was recorded from 52 s increased to 251 s (Figure 4d). While the lifetime of Nd3+ ions (4F3/2→4F9/2) has a slight change after coating with CaF2 (from 31 s to 22 s, Figure 4c). It suggested that the CaF2 shell could effectively prevent surface quenching mainly from Yb3+ ions and little surface quenching from Nd3+ ions. Both enhanced emission intensity and lengthened


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lifetime of Yb3+ in core/shell nanoparticles verified the protective effect of CaF2 layer. The absolute quantum yield (QY) was defined as the ratio of the quantity of emitted photons to the quantity of absorbed photons, determined to be 20.7% for OA-CSNPs, under a high energy power xenon lamp (150 W) at the excitation of 808 nm. As we all known, absorbance and quantum efficiency are the determinants of fluorescence emission. This new luminescent probe has significantly enhanced absorption band and high quantum yield, and work in the near infrared window, indicating the high energy utilization efficiency and potential application as a sensitive biomedical imaging probe. In order to validate the in vitro and in vivo biological imaging performance, the OA-CSNPs need to be transferred

OA-CSNPs (Figure 4a), which is another great demonstration of the protective effect of CaF2 shell. The result of ligand exchange was further verified by Fourier-transform infrared (FTIR) spectroscopy, as the asymmetric and symmetric stretching vibration bands (2925 and 2854 cm-1) of -CH2- of OA disappeared, meanwhile the asymmetric vibration peaks (1126 and 1007 cm-1) referred to as P-O bonds of EDTMP appeared (Figure S9). The photograph of OA-CSNPs dispersed in cyclohexane and EDTMP-CSNPs dispersed in water (Figure 4b) showed good monodispersity and no aggregation after few days. The dynamic light scattering (DLS) analysis (Figure S10) suggested that the EDTMP-CSNPs in aqueous solution were about 18 nm in diameter. In addition, the zeta-potential of EDTMP-CSNPs was measured to be -26.6±1.4 mV, which

Figure 4. (a) Room temperature NIR emission spectra of OA-CSNPs, OA-CSNPs, EDTMP-CNPs and EDTMP-CSNPs. (b) Photographs of OA-CSNPs (organic phase) and EDTMP-CSNPs (aqueous phase), and corresponding luminescence photos taken by NIR camera under the 808 nm laser. (c) Nd3+ decay luminescence (ex=808 nm, em=1060 nm) and (d) Yb3+ decay luminescence (ex=808 nm, em=980 nm) observed of the OA-CNPs and the OA-CSNPs (both dispersed in cyclohexane).

Figure 5. (a) Simplified diagram depicting the experimental setup for the tissue penetration experiment. (b), (c) and (d) The cross sectional fluorescence intensity profiles were corresponding to the capillaries filled with EDTMP-CSNPs under no pork tissue, 5 mm and 10 mm pork tissue (marked with white dash lines). Red dashed curves were Gaussian functional fitting lines. The below figure showed the full width at half maximum (FWHM), which was used here for representing the calculated width of the capillaries.

to the hydrophilic phase. EDTMP was coated to replace the surface oleic acid ligand of the NPs, forming EDTMP-modified NaYF4:7%Yb,60%Nd nanoparticles (called EDTMP-CNPs) and EDTMP-modified NaYF4:7%Yb,60%Nd@CaF2 nanoparticles (EDTMP-CSNPs) by using a ligand exchange method.41-42 The luminescence of EDTMP-CNPs was only 20% of the OA-CSPs because of quenching in water. Here, CaF2 inert layer isolated inner sensitizer and activator from surface quencher in solution, so that EDTMP-CSNPs remained 70% luminescence of the

can be attributed to the negatively charged EDTMP ligands. One of the most important advantages of rare earth NPs compared to fluorescent dyes is the superior photostability. The photostability analysis of the EDTMP-CSNPs and ICG dyes showed that the nonoparticles exhibited remarkably high stability compared with ICG dyes (Figure S11). Next, we investigated cytotoxicity of EDTMP-CSNPs through a methyl thiazolyl tetrazolium (MTT) assay experiment. The biocompatibility of the EDTMP-CSNPs was carried on Hela cells after incubation with different concentrations of EDTMP-CSNPs (0, 50, 100, 200, 400, 800



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g/mL) for 24 hours. The MTT results (Figure S12) suggested that Hela cells remained good viability under different concentrations of NPs. Regarding to the potential toxicity, we further investigated the histology test. No obvious lesion was detected, revealing that no noticeable toxicity to mice (Figure S13). The frightful challenges of in vivo imaging are the limited tissue penetration depth and poor resolution. They are closely related to the complicated biological environment and affected by the photon absorption and scattering of tissue. Here, we designed an in vitro simulation experiment to evaluate tissue penetration and spatial resolution systematic (Figure 5). Pork tissue with different thickness (0, 5 mm and 10 mm) was separately covered onto two V-shaped capillaries (0.50 mm inner diameter) filled by

Figure 6. (a) In vivo NIR imaging was collected by intravenous injecting EDTMP-CSNPs (15 mg/kg per nude mouse). (b) In vivo imaging of the bright field was displayed. (c) In vivo NIR imaging was collected after mouse liver covered. (d)(f) In situ NIR imaging of the mouse was collected. (e) In situ imaging of the bright field was collected. The cross sectional fluorescence intensity profiles in (g), (h) and (i) were corresponding to the white dash lines marked in (d), (c) and (f). Red dashed curves represented Gaussian functional fitting lines and the corresponding FWHM was displayed.


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EDTMP-CSNPs solution. Then the capillaries were illuminated with an 808 nm laser, while the luminescence signals were collected by the camera (Silicon based). It is noteworthy that the detection QY of the camera is about 20% at 980 nm (Figure S14). As shown in Figure 5c, the V-shape of the capillaries was detectable unambiguously with sharp peaks even under 5 mm pork slices, meanwhile the calculated diameter (0.54 mm) also matched well with the actual diameter, demonstrating high resolution even under 5 mm tissue. The V-shaped capillaries were still visible through 10 mm pork tissue, but the broad peaks showed weak resolution, reflecting absorbance and scattering of photons by the tissue (Figure 5e). An in vivo imaging experiment was conducted by intravenously injecting EDTMP-CSNPs. The liver of the nude mouse can be observed clearly after injection within 1 minute (Figure 6a). The luminescence intensity profiles of blood vessels were shown in Figure 6, in which peaks could be identified and fit to Gaussian functions (marked with red dash curves).28 Here, the FWHM was used for estimating the width of the vessels. Femoral arteries in in vivo imaging were distinguished unambiguously with a diameter of 0.19 mm (Figure 6h). The penetration depth of femoral arteries was measured to be about 2-3 mm by subsequent dissection experiment. The calculated values of other blood vessels width extracted from luminescence image were 0.26 mm, 0.42 mm and 0.54 mm, agreed well with the in situ bright field picture (Figure 6g and 6i). This NIR imaging was performed on our EMCCD camera with a pixel size of 0.13 mm in the view without additional high magnification objective lens. These results proved that the EDTMP-CSNPs can realize visualization of small blood vessel, which will be promising to vascular related disease imaging and angiogenesis diagnosis in tumor. ■ CONCLUSIONS In conclusion, we synthesized Nd3+-sensitized-Yb3+ core/shell nanoparticles, which have an average diameter of 12 nm, strong NIR emission at 980 nm and high quantum yield up to 20.7%. Nd3+ ions acted as activators, with increased amount (60%) make it absorb more excitation energy at 808 nm, while Yb3+ ions manifesting energy transfer highway effect. After coating with CaF2 inert shell and ligand exchange, the water-soluble EDTMP-CSNPs showed bright NIR luminescence, good biocompatibility and low biotoxicity. Two V-shaped capillaries can be distinguished clearly even through 10 mm pork tissue, showing deep penetration. Finally, an in vivo vascular imaging spatial resolution down to 0.19 mm in the mouse hindlimb was obtained, demonstrating that NaYF4:7%Yb,60%Nd@CaF2 core/shell nanocrystal can be used as an efficient NIR probe.

■ EXPERIMENTAL SECTION

Materials. Lanthanide oxides, Y2O3 (>99.999%), Nd2O3 (>99.999%) and Yb2O3 (>99.999%) were all brought from Shanghai Yuelong Rare Earth New Materials Co., Ltd. Trifluoroacetic acid sodium salt (CF3COONa, 99%), oleylamine (OM; >90%), oleic acid (OA; >90%) and 1octadecene (ODE; >90%) were always bought from SigmaAldrich Co.. Ethylenediamine tetra (methylene phosphonic acid) (EDTMP) were purchased from TCI (Shanghai) Development Co., Ltd. CaCO3, cyclohexane solvent, ethanol solvent and trifluoroacetic acid (TFA) were always brought from Sinopharm Chemical Reagent Co., China. Ln(CF3COO)3 solid powders were all obtained through corresponding oxide reacting with excessive TFA. Ca(CF3COO)2 was obtained by CaCO3 reacting with excessive TFA. The chemical reagents in experiments were all used of analytical grade with no further purification. In all experiments, deionized water was used all along. Characterization. TEM images, HRTEM images, HAADFSTEM image and EDXA spectra of the as-prepared nanocrystals were determined by a Tecnai G2 F20 S-Twin (FEI, America) operates at 200kV. XRD patterns were carried out on D8 advance diffractometer (Bruker, Germany) with a scanning rate of 0.5 ° per minute by using Cu Kα radiation (λ= 0.15406 nm). Absorption spectrum was measured with Lambda 750 (Perkin-Elmer, America). The NIR photoluminescence spectrum were measured on QM 40 (PTI, America) with an external 808 nm continuous wavelength (CW) laser. The photostability of EDTMPCSNPs and ICG dyes are measured by continuously excited by an 808 nm CW laser (~200 mW/cm2) for 30 minutes. The quantum yield of OA-CSNPs powder was measured on Quantaurus-QY Plus from Hamamatsu Co. with a high energy xenon lamp (150 W) as excitation source. We characterized the dynamic light scattering (DLS) and zetapotential of the NPs on ZS90 (Malvern, England). An InGaAs CCD camera from Hamamatsu Co. collected the NIR luminescence photographs under an 808nm CW laser (~200 mW/cm2). Synthesis of NaYF4:x%Yb,y%Nd NPs Capped with OA. In the case of NaYF4:x%Yb,y%Nd NPs: 1 mmol CF3COONa, , x% mmol of Yb(CF3COO)3, y% mmol of Nd(CF3COO)3 and (100-x-y)% mmol of Y(CF3COO)3 were mixed into a 100 mL three-neck flask containing with OM( 10 mmol), OA (10 mmol) and ODE (20 mmol). The mixed solution was heated to 130 °C under vacuum for several minutes with continuous stirring until the mixture turned to transparent. Then the transparent solution was heated to 310 °C and kept for 45 minutes under nitrogen protecting. The nanoparticles were precipitated by adding much ethanol and centrifugation, and finally dispersed in 10 mL of cyclohexane for further application. Synthesis of NaYF4:7%Yb,60%Nd@CaF2 Core/shell NPs Capped with OA (OA-CSNPs). The 0.5 mmol as-prepared optimized NaYF4:7%Yb,60%Nd core nanoparticles and 2 mmol Ca(CF3COO)2 powder were added into a 100 mL



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three-neck flask containing with ODE (20 mmol) and OA (20 mmol). The mixed solution was heated to 110 °C to remove cyclohexane with constant stirring under vacuum until formed a transparent mixture. Then the solution was heated to 310 °C and kept for 30 min under nitrogen protecting. The OA-CSNPs were precipitated by adding much ethanol and centrifugation. Finally, the products were dispersed in 5 mL of cyclohexane. Synthesis of NaYF4:7%Yb,60%Nd@CaF2 NPs Modified with EDTMP. The 0.2 mmol OA-CSNPs was added with excessive dichloromethane solution of NOBF4 at room temperature. The precipitated nanoparticles was dispersed in water after removing the supernatant by centrifugation. Ethanol and deionized water were added to further purify the dispersion. Then ligand-free nanoparticles were dispersed in EDTMP solution (50 mg/mL) for 30 minutes stirring, washed with water, and collected by centrifugation. Cytotoxicity Assay. The in vitro cytotoxicity was performed by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay. Human epithelial adenocarcinoma Hela cells were incubated with different concentrations of EDTMP-CSNPs (0, 50, 100, 200, 400, 800 g/mL) in a 96 well cell culture plate for 24 hours with a constant temperature at 37 °C under 5% CO2 atmosphere. Subsequently, 10 L MTT (5 mg/mL) solution was separately added to every well and then incubated for four hours. After removing the medium and adding dimethyl sulfoxide solution (DMSO, 100 L) to each well, a Tecan Infinite M200 monochromator was used to collected the optical density OD570 value (Abs.) of per well with background subtraction at 690 nm. The formula for calculating viability of cell growth is shown as follows: Cell viability (%) = (mean of Abs. of treatment groups/mean of Abs. of control groups) *100%. Histological Assessment. All animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee, Department of Pharmacy, Fudan University. In the test group, three four-week-old BALB/c female mice were all intravenously injected with EDTMPCSNPs (2.0 mg/mL * 0.15 mL). Moreover, three BALB/c female mice with no injection were set as the blank group. Tissue samples were harvested from test group and control group after 7 days. Both two groups of mice were all sacrificed, and the liver, spleen, lung, heart, and kidney of mice were collected and immersion in paraformaldehyde, then embedded into paraffin, finally sectioned, and stained by hematoxylin and eosin for further observation. In Vitro and in Vivo NIR Luminescence Bioimaging. All mouse luminescence imaging experiments were carried on the in vivo luminescence imaging system reported by our group. To determine penetration depth and resolution of EDTMP-CSNPs in NIR luminescence bioimaging, we tested the luminescence signals of the two capillaries of V-shaped arrangement filled with the EDTMP-CSNPs (2.0 mg/mL in

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water) under different thickness of pork (0 mm, 5mm, 10 mm). We used an 808 nm CW laser as excitation resource (~200 mW/cm2); meanwhile a 980 nm long-pass filter from Semrock Co. was used to collected signals in front of an Andor DU897 EMCCD camera (512x512-pixel silicon charge-coupled device) for NIR imaging. Then EDTMPCSNPs (2.0 mg/mL, 0.20 mL) were injected intravenously into a nude female mouse for imaging. The in vivo bioimaging experiment was done with the same conditions with the capillaries imaging experiment. Signals obtained from luminescent images were all analyzed with AndorSolis software, Bruker Molecular Imaging Software (Bruker MI SE) and Origin 8.0® software. ■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. (1) Additional characterizations of nanoparticles; (2) Experimental details for EDXA and the size distribution; (3) NIR luminescence of nanoparticles; (4) MTT and H&E stains results; (5) QY curve of EMCCD camera. ■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. (W. Feng) *E-mail: [email protected]. (F. Y. Li) Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENT The authors are very grateful for the NIR photoluminescent lifetime measurements from Professor Xueyuan Chen and Doctor Datao Tu, in Fujian Institute of Research on the Structure of Matter (Chinese Academy of Sciences, CAS). The authors acknowledge the financial support from National Basic Research Program of China (2015CB931801, 2013CB733703), National Natural Science Foundation of China (21527801, 21671042, and 21231004), Shanghai Sci. Tech. Comm. (15QA1400700) and the CAS/SAFEA International Partnership Program for Creative Research Teams. ■ REFERENCES 1. Hilderbrand, S. A.; Weissleder, R., Near-infrared Fluorescence: Application to In Vivo Molecular Imaging. Curr. Opin. Chem. Biol. 2010, 14, 71-79. 2. Dang, X. N.; Gu, L.; Qi, J. F.; Correa, S.; Zhang, G. R.; Belcher, A. M.; Hammond, P. T., Layer-by-layer Assembled Fluorescent Probes in the Second Near-Infrared Window for Systemic Delivery and Detection of Ovarian Cancer. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 5179-5184. 3. Frangioni, J. V., In Vivo Near-Infrared Fluorescence Imaging. Curr. Opin. Chem. Biol. 2003, 7, 626-634. 4. Chen, G. Y.; Roy, I.; Yang, C. H.; Prasad, P. N., Nanochemistry and Nanomedicine for Nanoparticle-based


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Energy Transfer Highway in Nd3+

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