800 nm-driven intense red-emitting upconversion nanophosphors with

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Biological and Medical Applications of Materials and Interfaces

800 nm-driven intense red-emitting upconversion nanophosphors with core/double-shell structure for dual-modal upconversion luminescence and magnetic resonance in vivo imaging applications A-Ra Hong, Youngsun Kim, Tae Sup Lee, Sehoon Kim, Kwangyeol Lee, Gayoung Kim, and Ho Seong Jang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18078 • Publication Date (Web): 16 Mar 2018 Downloaded from http://pubs.acs.org on March 19, 2018

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ACS Applied Materials & Interfaces

800 nm-driven intense red-emitting upconversion nanophosphors with core/double-shell structure for dual-modal upconversion luminescence and magnetic resonance in vivo imaging applications A-Ra Hong,†,‡ Youngsun Kim,§ Tae Sup Lee,ǁ Sehoon Kim,§ Kwangyeol Lee,‡ Gayoung Kim,§ and Ho Seong Jang†,┴,* †

Materials Architecturing Research Center, Korea Institute of Science and Technology, 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea

‡

Department of Chemistry, Korea University, 145 Anam-ro, Seoul 02841, Republic of Korea

§

Center for Theragnosis, Korea Institute of Science and Technology, 5, Hwarang-ro 14-gil,

Seongbuk-gu, Seoul 02792, Republic of Korea ǁ

Division of RI Convergence Research, Korea Institute of Radiological & Medical Sciences

(KIRAMS), 75, Nowon-ro, Nowon-gu, Seoul 01812, Republic of Korea ┴Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea

ACS Paragon Plus Environment

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*To whom correspondence should be addressed

E-mail: [email protected]

KEYWORDS: core/shell/shell, upconversion, nanophosphors, in vivo fluorescence imaging, in vivo magnetic resonance imaging


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ABSTRACT In this study, intense single-band red-emitting upconversion nanophosphors (UCNPs) excited with 800 nm near infrared (NIR) light are reported. When a NaYF4:Nd,Yb active-shell is formed on the 12.7 nm-sized NaGdF4:Yb,Ho,Ce UCNP core, the core/shell (C/S) UCNPs show tunable emission from green to red depending on the Ce3+ concentration under excitation with 800 nm NIR light. Thirty mol% Ce3+-doped C/S UCNPs exhibit single-band red emission peaking at 644 nm via a 5F5 → 5I8 transition of Ho3+. A high Nd3+ concentration in the shell results in strong absorption at around 800 nm NIR light even though the shell thickness is not large, and smallsized C/S UCNPs (16.3 nm) emit bright red light under 800 nm excitation. The formation of a thin NaGdF4 shell on the C/S UCNPs further enhances the upconversion (UC) luminescence, and sub-20 nm-sized core/double-shell (C/D-S) UCNPs exhibit 2.8 times stronger UC luminescence compared with C/S UCNPs. Owing to strong UC luminescence intensity and Gd3+ ions on the nanocrystals’ surface, they can be applied as a UC luminescence imaging agent and a T1 contrast agent for magnetic resonance (MR) imaging. In vivo UC luminescence and high contrast MR images are successfully obtained by utilizing the red-emitting C/D-S UCNPs.


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1. INTRODUCTION Lanthanide ion-doped upconversion nanophosphors (UCNPs) are regarded as one of promising fluorescence imaging agents because of their unique optical properties, such as anti-Stokes shift emission and sharp emission peaks, compared with conventional fluorophores.1,2 The optical features of the UCNPs, such as anti-Stokes shift luminescence, long life time, and non-blinking and non-bleaching luminescence, make it a great benefit to apply UCNPs to bio-imaging applications.3-9 In addition, the use of near infrared (NIR) light as an excitation source for upconversion (UC) luminescence is another advantage in fluorescence imaging utilizing UCNPs since biomolecules do not exhibit autofluorescence under the NIR light illumination, giving rise to a high signal-to-noise ratio.9 Owing to these advantages of UCNPs in bio-related applications, there have been many reports on the synthesis and application of UCNPs over the previous decade.10-22 The UCNPs used in most previous studies absorb 980 nm NIR light and emit visible light.12,16,17 Unfortunately, the utilization of 980 nm excitation results in the heating of cells and tissues irradiated with the NIR light23,24 because water molecules, which constitute a significant part of the biological system, exhibit a large absorption cross-section in the 980 nm spectral region.25 In addition, because water molecules strongly absorb incident 980 nm light, the penetration depth of the incident light into the tissue becomes short, making deep tissue imaging difficult. These problems can be solved by using Nd3+- or dye-sensitized UCNPs that exhibit UC luminescence under 800 nm excitation, located in the biologically transparent window.24,26,27 In particular, Nd3+-sensitized UCNPs are more favorable than dye-sensitized UCNPs, owing to the low photostability of organic dyes.28 Yan’s group reported green-emitting NaGdF4:Yb,Er/NaGdF4:Nd,Yb,24 and Liu’s group reported a NaYF4:Yb,A,Nd(1%)/NaYF4:Nd(20%) (A = Tm, Er, and Ho) core/shell (C/S) structure23 in


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which the activator ions are geometrically separated from the Nd3+ ions to prohibit luminescence quenching of activator ions by energy transfer from the activators to the Nd3+ ions. Since these studies, there have been several reports on the 800 nm NIR light-driven blue/green UC luminescence.29-33 Huang and Lin reported bright green- and blue-emitting UCNPs with activecore/active-shell structure under 808 nm excitation.29 Wang et al. applied Nd3+-sensitized NaYF4:Yb,Ho-based UCNPs for fluorescence imaging and photodynamic therapy utilizing 808 nm NIR light.34 Zhong et al. enhanced UC green and blue luminescence from Nd3+-sensitized Er/Tm/Ho-activated UCNPs by using quenching shield sandwich structure.28 Despite these investigations of ~800 nm NIR light-driven UCNPs, reports on Nd3+-sensitized red-emitting UCNPs are relatively lacking. Recently, Chen et al. reported Nd3+-sensitized single-band redemitting UCNPs with C/S structure35 and Shen et al. reported strong red-emitting UCNPs with an intercalated nanostructure.36 The realization of single-band red light under 800 nm NIR light is believed to be highly advantageous for in vivo bio-imaging because both NIR light from an excitation source and red light emitted from the UCNPs lie in the optically transparent window in biological system.25 However, the particle size for the C/S structure reported previously by Chen et al. was approximately 50 nm, which is too large for in vivo application because small UCNPs are desirable for renal clearance.37 Additionally, the intercalate nanostructure makes the synthetic procedure too complicated and gives rise to larger particle size.36 In a study by Wang et al., in vitro cell imaging was conducted by utilizing a red emission peak from NaYF4:Yb,Hobased UCNPs, which show UC luminescence peaks at green and red spectral regions; the particle size was also large (45.2 nm), and the green peak showed higher intensity than the red peak.34 Thus, although 800 nm NIR light-driven red emission is advantageous for in vivo imaging, in


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vivo imaging utilizing single band red-emitting UCNPs under ~800 nm excitation has not been reported yet. In this study, we report on NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 core/double-shell (C/D-S) structured UCNPs with a very small size (< 20 nm) in which the inorganic NaGdF4 shell was grown on the C/S UCNPs to strengthen UC red light under 800 nm NIR light illumination. The NaGdF4 was selected as the outermost shell since the Gd3+ ions on the UCNP’s surface can act as an agent for magnetic resonance (MR) imaging.38 We optimized red luminescence under 800 nm light irradiation and then applied C/D-S UCNPs to in vivo UC luminescence imaging. Additionally, we investigated the feasibility of the application of the intense red-emitting C/D-S UCNPs to in vivo MR imaging. These results indicate that the sub-20 nm red-emitting C/D-S UCNPs can be a promising dual-modal UC luminescence and MR imaging agent.

2. EXPERIMENTAL SECTION 2.1. Materials. For C/D-S UCNPs’ synthesis, GdCl3·6H2O (99%), HoCl3·6H2O (99.9%), YbCl3·6H2O (99.9%), CeCl3·7H2O (99.999%), YCl3·6H2O (99.99%), NdCl3·6H2O (99.9%), NaOH (99.99%), and NH4F (≥ 99.99%) were purchased from Sigma-Aldrich and were used as raw materials. Furthermore, 1-octadecence (ODE, 90% technical grade) and oleic acid (OA, 90% technical grade) were also obtained from Sigma-Aldrich. 2.2. Synthesis of NaGdF4:Yb,Ho,Ce UCNPs. Red-emitting NaGdF4:Yb,Ho,Ce UCNPs were synthesized with slight modification of the previously reported method by Zhang’s group.39 First, 1 mmol of GdCl3·6H2O, YbCl3·6H2O, HoCl3·6H2O, and CeCl3·7H2O was mixed with OA (6 mL)


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and ODE (15 mL) and then heated to 150 °C for 40 min. After the solution was cooled down to 50 °C, NaOH (2.5 mmol) and NH4F (4 mmol)-dissolved methanol (MeOH) solution (10 mL) was added to the reaction solution followed by stirring for 40 min. After the removal of MeOH, temperature of the reaction medium was raised to 300 °C and maintained for 90 min. The core UCNPs were precipitated by adding ethanol (EtOH) and washed with EtOH several times. Finally, they were dispersed in CHCl3 (10 mL). 2.3. Synthesis of NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs. One mmol of LnCl3·6H2O (Ln = Y (95-x)%, Nd x%, and Yb 5%) was dissolved in mixed solvents of OA (6 mL) and ODE (15 mL) through heat-treatment (150 °C and 40 min) and then, temperature of the reaction solution was decreased to 80 °C. NaGdF4:Yb,Ho,Ce CHCl3 solution (10 mL) was added to the reaction solution followed by the addition of 10 mL of MeOH solution containing 2.5 mmol of NaOH and 4 mmol of NH4F. The reaction mixture was stirred at 50 °C for 40 min. After removal of MeOH, the temperature of the reaction medium was raised to 300 °C and maintained for 110 min. After finishing heat-treatment, the synthesized C/S UCNPs were precipitated by adding EtOH and washed with EtOH several times. The resultant nanocrystals were dispersed in CHCl3 (10 mL) for further use. 2.4. Synthesis of NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs. First, GdCl3·6H2O (1mmol) was dissolved in mixed solvents of OA (6 mL) and ODE (15 mL) by heattreatment at 150 °C for 40 min. After the solution was cooled down to 80 °C, C/S UCNP solution (10 mL) was added to the reaction flask, followed by the addition of 10 mL of MeOH solution containing NaOH (2.5 mmol) and NH4F (4 mmol) to the reaction solution. The mixed solution was stirred at 50 °C for 40 min. After removal of MeOH, the temperature of the reaction medium was raised to 300 °C and maintained for 110 min. After finishing heat-treatment, the


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synthesized C/D-S UCNPs were precipitated by adding EtOH and washed with EtOH several times. Finally, the C/D-S UCNPs were dispersed in CHCl3 (10 mL). 2.5. Synthesis of ligand free-NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs. The ligand free C/D-S UCNPs (LF-C/D-S UCNPs) were prepared by adopting the method reported by Bogdan et al. with slight modification. Typically, 3 mL of oleate ligand (OL)-capped C/D-S UCNP CHCl3 solution was mixed with 1.5 mL of 2 M HCl solution followed by ultrasonication for 5 min. And then the solution was centrifuged at 16,500 rpm for 20 min to collect the LF-C/DS UCNPs. After washing with EtOH several times, the LF-C/D-S UCNPs were dispersed into 6 mL of deionized (DI) water. 2.6. In Vivo UC luminescence imaging. Animal studies were permitted by the animal care and use committee of the Korea Institute of Science and Technology (KIST). The mice were treated under the institution-established guidelines. The 60 μL of LF-C/D-S UCNPs (4 mg mL-1) were administrated into the back and left femoral regions, respectively, of BALB/c nude mice. The UC luminescence images were obtained by using an INV-16M Davinch Invivo Imaging System (Davinch K, Republic of Korea)8 with an external laser source (808 nm) for excitation. To exclude noise from the laser and mouse, a 650/45 nm bandpass filter on the detection part and an 800/40 nm bandpass filter on the NIR laser were used. 2.7. In Vivo MR imaging. Whole-body MR images were obtained by using a 3 T MR (Magnetom Trio Tim, Siemens) with an animal coil. Three mice per group were used in all animal experiment. Isoflurane in oxygen (2%) was used for anesthesia of the mice and MR images were obtained after subcutaneous or intramuscular injection of 100 µL of red-emitting C/D-S UCNP (6 µg mL-1) in normal BALB/c athymic mice. The T1-weighed gradient echo


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imaging was performed under the following conditions: repetition time (TR) 10.2 ms, echo time 3.6 ms, matrix 216 × 256, field of view (FOV) 5 × 6 cm2, slice thickness 0.8 mm, gap 0 mm, scan time 3 min. MR transverse cross-section images were aquired. 2.8. Characterizations. Absorption and photoluminescence (PL) spectra of all UCNP samples were collected using a Perkin-Elmer Lambda25 UV/VIS spectrometer (scan speed = 480 nm min-1) and a Hitachi F-7000 spectrophotometer (scan speed = 240 nm min-1) coupled with 980 and 800 nm NIR light-emitting continuous wave diode lasers, respectively. PL excitation (PLE) spectra were collected by using a wavelength tunable (690 ~ 1040 nm) Mai Tai HP Ti:sapphire laser (Spectra-Physics, USA). The tunable NIR light was focused onto the Hellma 111-QS cell containing UCNP solution and emitted light was measured at right angle of incident NIR light. Laser power was calibrated in terms of the wavelength of the laser. A Tecnai F20 G2 transmission electron microscope (FEI co., USA) with an operation voltage of 200 kV was used to acuire transmission electron microscopy (TEM) images, and a Talos 200X scanning transmission electron microscope (S/TEM) (FEI co., USA) with an operation voltage of 200 kV was used to obtain an energy dispersive X-ray spectroscopy (EDS) line scan profile. High resolution scanning transmission electron microscopy (HRSTEM) images and electron energy loss spectroscopy (EELS) spectra were acquired using an aberration-corrected Titan 80/300 S/TEM (FEI co., USA) with an operation voltage of 300 kV. The X-ray diffraction (XRD) characterzation was conducted with a Bruker D8 ADVANCE diffractometer using Cu Kα radiation.8 T1 measurements were carried out using an inversion recovery method with a variable inversion time (TI) at 3 T MR (Magnetom Trio Tim, Siemens). The MR images were obtained at 35 different TI values ranging from 50 to 1,750 ms. T1 relaxation times were obtained from the non-linear least square fit of the signal intensity measured at each TI and echo time (TE) and


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plotted as 1/T1 vs Gd3+ molar concentration. The slopes of the graph provided the molar relaxivity r1.

3. RESULTS AND DISCUSSION As shown in Figure 1a, water shows strong absorption at the wavelength range of 900 – 1000 nm. Thus, as mentioned above, 980 nm NIR light is largely absorbed by the water molecules. The strong absorption of the NIR light in this wavelength range by water causes the temperature of the water to rise. As shown in Figure S1, the temperature of water in the cuvette under irradiation with a 980 nm laser was significantly increased compared with the irradiation with an 800 nm laser. In addition, HbO2 and Hb show strong absorption bands at wavelength regions shorter than 600 nm.25 Liu’s group calculated the absorption of human skin, and the simulation result showed that the absorption of whole skin is low in the range of 630 – 900 nm.23 Thus, it is desirable for excitation and emission band of UCNPs to reside in the spectral region of 630 – 900 nm for efficient in vivo imaging, and the 800 nm NIR light-driven red-emitting UCNPs can satisfy this condition. As shown in Figure 1b, 800 nm NIR light-driven strong red-emitting UCNPs can be obtained by following the synthetic procedure shown in the schematic illustration. First, redemitting core UCNPs, which are excited by NIR light of 980 nm, are synthesized. Then, Nd3+doped active-shell is grown on the core UCNP. The Nd3+ ions are absorb NIR light at around 800 nm and transfer the absorbed energy to the core. Finally to enhance red light, optically inert shell is grown on the core/active-shell UCNPs, resulting in the formation of core/active-shell/inert shell, i.e., C/D-S UCNPs.


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To obtain red-emitting UCNPs, NaGdF4:Yb,Ho,Ce UCNPs were synthesized as ultrasmall core materials because the red emission could be realized from the UCNPs via cross-relaxation between Ho and Ce.35,40 When Ce3+ was co-doped into the NaGdF4:Yb,Ho UCNPs, green emission of Ho3+ due to the 5S2, 5F4 → 5I8 electronic transition is quenched by cross-relaxation between Ce3+ and Ho3+ [Ho3+:5I6 + Ce3+:2F5/2 → Ho3+:5I7 + Ce3+:2F7/2 and Ho3+:5S2, 5F4 + Ce3+:2F5/2 → Ho3+:5F5 + Ce3+:2F7/2].35,40 In our experimental conditions, when Ce3+ concentration was higher than 30 mol%, polydispersed nanoparticles were synthesized. As shown in Figure S2, both small and large particles were observed. In addition, when the Ce3+ concentration varied from 0 to 30 mol%, the synthesized core UCNPs showed single hexagonal phase as shown in Figure S3. Thus, we synthesized NaGdF4:Yb(18%),Ho(2%),Ce(x)/NaYF4:Nd(50%),Yb(5%) C/S UCNPs where x was varied from 0 to 30%. The synthesized C/S UCNPs exhibit characteristic PL peaks under excitation with both 800 and 980 nm NIR light (See Figures 2 and S4). In our study, Yb3+ was co-doped together with Nd3+ into the shell because Nd3+ and Yb3+ co-doping was effective for efficient energy transfer from Nd3+ to Ho3+ as supported by the PL spectra shown in Figure S5. Under 800 nm excitation, the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs showed even stronger UC luminescence intensity than the NaGdF4:Yb,Ho,Ce/NaYF4:Nd C/S UCNPs. When 800 nm NIR light illuminates the C/S UCNPs, it is absorbed by Nd3+ ions in the shell, followed by the energy transfer to Yb3+ ions in the shell. Via an energy migration process, the excited energy is transferred from Yb3+ in the shell to Yb3+ in the core,24 and some of the excited energy may be transferred directly from the Nd3+ ions, which are located in the vicinity of the core, to Yb3+ in the core.23 Then, ground state electrons in the 5I8 state of Ho3+ are excited to the 5S2, 5F4 states through the 5I6 intermediate energy level by successive energy transfer from


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Yb3+ to Ho3+.35 In addition, ground state electrons of Ho3+ ions can be excited to the 5F3 state via excited state absorption through a three-step UC process (Figure S6). The C/S UCNPs showed three emission peaks at 484, 539, 644 nm, respectively, via 5F3 → 5I8, 5S2,5F4 → 5I8, and 5F5 → 5

I8 electronic transitions in Ho3+ ions. Interestingly, weak emission band peaking at 582 nm was

also observed in the PL spectra shown in Figure 2a. This emission band was only observed under 800 nm NIR light excitation. In Figure S4, only Ho3+-related peaks were observed under 980 nm NIR light excitation. According to Li et al., the emission peak centered at ~585 nm can be observed from the Nd3+ ions under 800 nm NIR light excitation.41 Thus, the weak emission band between the two Ho3+ emission peaks at 539 and 644 nm is attributed to the 4G7/2 → 4I11/2 electronic transition in Nd3+ ions.41 As shown in Figure 2a, the ratio of intensity of red to green peaks increased with increasing Ce3+ concentration in the host nanocrystals, owing to the cross-relaxation process discussed above (also see Figure S6). As a result, the Commission Internationale de l’Éclairage (CIE) color coordinates of the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs moved from the green to the red region on the CIE 1931 chromaticity diagram under 800 nm laser irradiation (Figure 2b). When 30 mol% of Ce3+ was doped into the NaGdF4:Yb,Ho, the NaGdF4:Yb(18%),Ho(2%),Ce(30%)/NaYF4:Nd(50%),Yb(5%) C/S UCNPs displayed highly pure red light under 800 nm laser irradiation (see Figure 2b). Owing to the suppression of the green emission peak of Ho3+ by Ce3+ co-doping, the NaGdF4:Yb(18%),Ho(2%),Ce(30%)/NaYF4:Nd(50%),Yb(5%) C/S UCNPs exhibited highly pure red light and the color purity was found to be 97.9% by using following equation,42

Pe =

(x − x w )2 + (y − y w )2 (xb − x w )2 + (yb − y w )2

(1).


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In equation 1, x and y are the color coordinates of the C/S UCNPs, and xw and yw are the color coordinates of the standard white light. Also, xb and yb are the color coordinates of the point where the line connecting the color coordinates of the white light and the color coordinates of the C/S UCNPs meets the spectrum locus. For strong UC luminescence under 800 nm excitation, larger absorption at 800 nm NIR light is desirable and requires a higher concentration of Nd3+ ions. Nonetheless, there is a limitation for Nd3+ doping concentration. According to Zhong et al., the Nd3+ may quench UC luminescence from the activators (in this case, Ho3+ ions) even though Nd3+ is doped into the shell.28 The NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs exhibited lower PL intensity than NaGdF4:Yb,Ho,Ce/NaYF4 core/inert shell UCNPs, as shown in Figure S7. Thus, a trade-off between the enhancement and quenching of the UC luminescence will exist at a certain Nd3+ concentration. To achieve strong red UC luminescence under 800 nm laser irradiation, the Nd3+ concentration in the shell was optimized. PL properties of the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb UCNPs with varying Nd3+ concentrations were investigated and the PL spectra were shown in Figure 3. NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb UCNPs did not show significant changes in morphology and size under various Nd3+ concentrations, as shown in Figure S8. Thus, it can be noted that the PL intensity of the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb UCNPs is affected by the Nd3+ concentration. In our experimental conditions, the 50 mol% of Nd3+-doped C/S UCNPs (i.e., NaGdF4:Yb(18%),Ho(2%),Ce(30%)/ NaYF4:Nd(50%),Yb(5%), referred to as NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb for simplicity) exhibited the strongest red emission intensity under excitation with 800 nm NIR light (Figure 3a). High concentration of the Nd3+ in the activeshell resulted in strong absorption of NIR light at 800 nm (Figure S9). This significant increase in the absorption of NIR light at 800 nm led to a strong UC red luminescence under 800 nm


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excitation even though the active-shell thickness was not large. Figure 3b depicts the 3dimensional (3D) PL spectrum of the C/S UCNPs with variation in both emission and excitation wavelengths. PL excitation (PLE) spectrum for the red emission of the C/S UCNPs can be obtained by using the 3D PL spectrum (see Figure S10). As expected from the absorption spectrum of Figure S9, the C/S UCNPs showed a stronger PL intensity under 730 – 820 nm NIR light excitation than under ~980 nm NIR light excitation. Indeed, strong PLE bands peaking at 742 and 796 nm were observed for the C/S UCNPs. As shown in Figure S10, the PLE intensity of the C/S UCNPs at 800 nm was higher than that at 980 nm, indicating that the red luminescence of the C/S UCNPs under 800 nm light irradiation was stronger than that under 980 nm light irradiation. To further enhance UC red luminescence, an NaGdF4 inert shell was grown on the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs. The formation of the outermost inert shell on the C/S UCNPs can be confirmed by TEM analysis, as shown in Figure 4. When the NaYF4:Nd,Yb active-shell and NaGdF4 outermost shell were grown on the core UCNP, particle size was gradually increased, as shown in Figure 4a. The NaGdF4:Yb,Ho,Ce core, NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S, and NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs exhibited hexagonal phase, as verified from the XRD patterns of the UCNPs (Figure S11). Figure 4a insets show high angle annular dark field (HAADF) HRSTEM images of the core, C/S, and C/D-S UCNPs, confirming that the synthesized UCNPs exhibit a highly crystalline hexagonal structure, as can be seen from the highly clear lattice fringes. In addition, the HRSTEM images indicate that active and inert shells were epitaxially grown on the core and C/S UCNPs, respectively. After the formation of NaYF4:Nd,Yb shell on the UCNP core, the size of the UCNPs increased from 12.7 nm for core UCNPs to 16.3 nm for C/S UCNPs. In addition,


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formation of the NaYF4:Nd,Yb shell can be directly confirmed by comparing EELS spectrum of the core with that of the C/S UCNPs (see Figure S12). Nd M4,5 core-loss edge peaks were observed in the EELS spectrum of the C/S UCNPs, while no Nd-related core-loss edge peak was not observed in that of the core UCNPs. The growth of NaGdF4 outermost shell further increased the particle size to 19.5 nm. Since all elements constituting the outermost shell overlapped with elements constituting core materials, EELS or EDS spectra of the C/D-S UCNPs cannot verify the formation of the C/D-S structure. Therefore, the EDS line scan profile was obtained to confirm the growth of the NaGdF4 outermost shell. Figure 4d shows the EDS line scan profiles across NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs. In the Ce Lα line profile, there is one peak in the core region and the Nd Lα profile showed two peaks in the shell region. In the Gd Lα line profile, three peaks were observed in the core and the outermost shell regions. This result confirms the formation of the C/D-S structure. The formation of the NaGdF4 outermost shell significantly enhanced the PL intensity of the red emission attributed to the 5F5 → 5I8 transition in Ho3+, as shown in Figure 4e. The intensity of the 5F5 → 5I8 peak of C/D-S UCNPs was 2.8–fold that of C/S UCNPs. The decreased number of surface quenching sites owing to the formation of the outermost inorganic shell on the Nd3+-doped active-shell layer highly suppresses surface quenching. This result means that protection of the sensitizer in the activeshell from the surface quenching as well as that of the activator in the UCNP core from the surface quenching is very important for achieving strong UC luminescence of UCNPs with an Nd3+-sensitized active-shell layer under 800 nm NIR light irradiation. Consequently, the growth of the thin active-shell containing high Nd3+ concentration on the core and thin outermost shell on the active-shell allowed the synthesis of bright red-emitting C/D-S UCNPs with sub-20 nm size.


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This 800 nm NIR light-driven red-emitting C/D-S UCNP with small size (< 20 nm) is desirable for bio-imaging applications. However, as-synthesized C/D-S UCNPs have hydrophobic surfaces, and therefore, these UCNPs are not dispersed in water. Hence, to enable bio-imaging applications, water-soluble C/D-S UCNPs were obtained by removing the surface ligand, i.e., oleate ligand on the surface of the C/D-S UCNPs using the method previously reported by Capobianco’s group.43 No agglomeration was observed after the removal of the oleate ligand (Figure 5a). As shown in the inset of Figure 5a, the LF-C/D-S UCNP-dispersed DI water solution is highly clear and background letters are clearly seen. There was little change in the clearness of the UCNP DI water solution to the naked eye for two weeks, as shown in Figure S13, indicating that the LF-C/D-S UCNPs were stable in DI water. When we investigated zeta potential of LF-C/D-S UCNPs in DI water, the zeta potential values exhibited the average value of 23.77 mV with small fluctuation of standard deviation of 1.38 mV for 8 days. This result supports the colloidal stability of LF-C/D-S UCNPs (Figure S14). The Fourier transform-infrared (FT-IR) spectroscopy spectra support the conclusion that the surface oleate ligand was removed from the C/D-S UCNPs (Figure S15). The OL-capped C/D-S UCNPs showed two bands at 1462 and 1562 cm-1 due to symmetric and asymmetric stretching of the COO- group, respectively, while the LF-C/D-S UCNPs did not exhibit peaks at 1462 and 1562 cm-1. Although PL intensity was lowered after ligand removal, bright red UC luminescence was still observed in the LF-C/DS UCNPs under 800 nm laser irradiation (Figure 5b inset). To investigate the toxicity of the LF-C/D-S UCNPs, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay was performed by using NIH-3T3 and HeLa cells (Figure S16). According to assay, the LF-C/D-S UCNPs have low cytotoxicity. To demonstrate the feasibility of the use of the C/D-S UCNPs for in vivo UC luminescence imaging, 60 µL of the


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LF-C/D-S UCNPs (4 mg mL-1) were administrated subcutaneously and intramuscularly into a nude mouse, respectively. To investigate the effect of 808 nm laser exposure on the mouse before the injection of the C/D-S UCNPs into the mouse, the mouse was illuminated with the 808 nm laser with the relatively high power density of 1.7 W cm-2 for 5 min. As shown in Figure 6a, the mouse was not damaged under this laser irradiation conditions. After the C/D-S UCNPs were subcutaneously injected into the mouse, intense UC luminescence signal was observed from the injected area (back of the nude mouse) under the irradiation with the 808 nm NIR laser (power density ~0.66 W cm-2) as shown in Figure 6b. In addition, UC luminescence signal was still observed for the intramuscular injection into femoral region of the nude mouse under the same irradiation conditions (Figure 6c). In our study, the cooling temperature of the chargecoupled device camera was -25 °C, resulting in less sensitivity of the equipment for the in vivo imaging. If a more sensitive imaging system such as an IVIS imaging system is used, a stronger UC luminescence signal could be acquired.44 To evaluate the magnetic properties of the red-emitting C/D-S UCNPs, relaxation times were measured for the LF-C/D-S UCNP aqueous solutions with various concentrations. Figure 7a exhibits the MR images which show that T1 contrast effect of the C/D-S UCNPs depends on the Gd3+ concentration ([Gd3+]). Plotting T1-1 as a function of [Gd3+] gives rise to linear relationship between [Gd3+] and contrast effect. The relaxivity (r1) for the red-emitting C/D-S UCNPs was determined to be 2.6 mM-1s-1. Considering this relaxivity value, we investigated the capability of the red-emitting C/D-S UCNPs for MR imaging. As shown in Figure 7b, a 3 T human clinical scanner was used to estimate the T1-weighted MR images. Analysis of the MR images revealed an MR signal enhancement with an increase of concentration of C/D-S UCNPs in a Gd3+ concentration dependent manner. With the increase in the concentration of the C/D-S UCNP, T1-


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weighted MR imaging intensity was considerably enhanced, indicating that the red-emitting C/D-S UCNPs could act as a contrast agent for T1-weighted MR imaging. We further investigated if they can be utilized for MR imaging using a normal mouse model. The C/D-S UCNPs were subcutaneously, intramuscularly, and intravenously injected into a female athymic mouse. As illustrated in Figures 7c and 7d, T1-weighted images exhibit the evident enhancement effect in the injection sites. The contrast-to-noise ratio (CNR) of C/D-S UCNPs was 19 ± 4.8 in subcutaneous injection sites and 18.5 ± 1.9 in intramuscular injection sites (Figure S17). In Figure S18, T1-weighted MR images showed that intravenously injected C/D-S UCNPs were accumulated in the liver at 4 h post injection, due to nonspecific binding to serum proteins and localizing in the reticuloendothelial system (RES). The intravenously injected C/D-S UCNPs were also excreted through kidney, because the CNR values of liver and kidney were peaked at 4 h post injection and those were gradually decreased at 24 h post-injection. These results suggest that the red-emitting C/D-S UCNPs can be utilized as a contrast agent for T1-weighted MR imaging. In addition, the in vivo toxicity was assessed by monitoring body weights of mice and histological conditions of major organs after intravenous administration of the LF-C/D-S UCNPs. During the monitoring of mice for a week, body weight declined for initial several days (Figure S19). In the histological examination, mild and multifocal inflammation was observed in liver while no abnormality was detected in heart, lung, spleen and kidney, compared to the control as shown in Figure S20. Taken altogether, the UCNPs exhibit some mild in vivo toxicity, especially for hepatic regions where the UCNPs mainly accumulate. Although in vivo imaging and animal toxicity results using LF-C/D-S UCNPs were shown in this study, further functionalization of the C/D-S UCNPs with biomolecules can be possible for


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targeting application because the surface the C/D-S UCNPs can be capped with carboxyl group (Figure S21). After functionalization of the C/D-S UCNPs with biomolecules, the biomolecule functionalized C/D-S UCNPs can be utilized in disease-targeted in vivo applications via intravenous injection of the C/D-S UCNPs. Further studies about surface fabrication and conjugation of various targeting moieties to disease lesions can be developed.

4. CONCLUSION In conclusion, sub-20 nm single band red-emitting NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs were synthesized. With varying Ce3+ concentrations, the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs exhibited green-to-red multicolor emission under 800 nm excitation. A high concentration of Nd3+ in the active-shell caused strong absorption of 800 nm NIR light and the C/S UCNPs emitted strong UC luminescence under 800 nm excitation. The UC red light was further enhanced by the formation of the NaGdF4 thin layer on the C/S UCNPs which can act as an MR contrast agent. The C/S and C/D-S structure was confirmed by EELS spectra and EDS line profile analyses. The water-dispersed ligand free C/D-S UCNPs exhibited low toxicity, and in vivo UC luminescence imaging was successfully performed by utilizing the C/D-S UCNPs and an 808 nm NIR laser. In addition, the C/D-S UCNPs showed an evident MR contrast effect, and the T1-weighted enhancement effect was observed in the in vivo MR images. This study suggests that the 800 nm NIR light-driven red-emitting C/D-S UCNPs could serve as a contrast agent for both UC luminescence and T1-weighted MR imaging. For practical utilization of the C/D-S UCNPs developed in this study, further studies on the renal


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clearance and in vivo toxicity are required after surface functionalization of the C/D-S UCNPs with biomolecules for targeting.

ASSOCIATED CONTENT Supporting Information. TEM images of NaGdF4:Yb,Ho,Ce UCNPs and NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs, XRD patterns of core, C/S, C/D-S UCNPs, PL spectra of C/S UCNPs, PLE and EELS spectra of core and C/S UCNPs, MTT assay and in vivo toxicity results, and additional MR images, FT-IR spectra of OL-capped, ligand free, and PAAs-capped C/D-S UCNPs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Telephone: +82-2-958-5263, Fax: +82-2-958-5599. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2016R1A2B2013629), the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (NRF-2013M3C1A3065040), and a grant of the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by Ministry of Science and ICT (MIST) (50536-2018;50461-2018), Republic of Korea. We thank Dr. Ji-Hoon Kyhm for technical help for PLE measurement.

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Figure captions Figure 1. (a) Absorption spectrum of DI water which was measured in a path length of 1 cm. (b) Schematic illustration showing procedure to produce 800 nm NIR light-driven strong red lightemitting UCNPs with C/D-S structure whose excitation and emission wavelengths lie in the optically transparent window. Figure

2.

(a)

Normalized

PL

spectra

and

(b)

CIE

color

coordinates

of

NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs with varying Ce3+ concentrations under 800 nm excitation. Inset shows photograph showing UC luminescence from the C/S UCNPs with various Ce3+ concentrations of 0, 3, 5, 10, 20, and 30% from left to right under irradiation with an 800 nm NIR laser. Figure 3. (a) PL spectra of the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs with varying Nd3+ concentrations of 10, 20, 30, 40, 50, and 60% under 800 nm excitation. Inset shows integrated PL intensity of the C/S UCNPs with various Nd3+ concentrations. (b) 3D PL spectrum of the NaGdF4:Yb(18%),Ho(2%),Ce(30%)/NaYF4:Nd(50%),Yb(5%)

C/S

UCNPs

obtained

with

varying excitation laser wavelength from 720 to 1040 nm. Figure 4. TEM images of (a) NaGdF4:Yb,Ho,Ce core, (b) NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S, and (c) NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs and insets show corresponding HRSTEM images. (d) EDS line profiles of the C/D-S UCNPs and inset shows HAADF STEM image of the C/D-S UCNPs indicated with line profile direction. (e) PL spectra of the core, C/S, and C/D-S UCNPs under excitation with 800 nm NIR light. Inset shows photograph showing UC red luminescence from the core, C/S, and C/D-S UCNP solutions (from left to right) under irradiation with a continuous wave 800 NIR laser.


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Figure 5. (a) TEM image of ligand free C/D-S UCNPs. Inset shows photograph of the DI waterdispersed LF-C/D-S UCNP solution. (b) PL spectra of OL-capped C/D-S UCNPs and LF-C/D-S UCNPs under excitation with 800 nm NIR light. Inset shows photograph showing the UC red luminescence from the OL-capped and ligand free C/D-S UCNP solutions. Figure 6. (a) Bright field image of a nude mouse after irradiation with an 808 nm NIR laser for 5 min (power density ~1.7 W cm-2). In vivo UC luminescence images under irradiation with an 808 nm NIR laser (power density ~0.66 W cm-2) after (b) subcutaneous and (c) intramuscular injections of the LF-C/D-S UCNPs (4 mg mL-1, 60 µL). The signal intensities were measured to be 2.10 ± 0.44 × 1014 photons/s/cm2/sr (average value ± standard deviation) for ROI 1 and 0.52 ± 0.15 × 1014 photons/s/cm2/sr for ROI 2 for 3 mice. Figure 7. (a) T1 relaxation rates (T1-1) versus Gd3+ concentration, (b) T1-weighted in vitro MR image with various Gd3+ concentrations. T1-weighted in vivo MR images for (c) subcutaneous and (d) intramuscular injection sites.


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Figure 1. (a) Absorption spectrum of DI water which was measured in a path length of 1 cm. (b) Schematic illustration showing procedure to produce 800 nm NIR light-driven strong red lightemitting UCNPs with C/D-S structure whose excitation and emission wavelengths lie in the optically transparent window.


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Figure

2.

(a)

Normalized

PL

spectra

and

(b)

CIE

color

coordinates

of

NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs with varying Ce3+ concentrations under 800 nm excitation. Inset shows photograph showing UC luminescence from the C/S UCNPs with various Ce3+ concentrations of 0, 3, 5, 10, 20, and 30% from left to right under irradiation with an 800 nm NIR laser.


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Figure 3. (a) PL spectra of the NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S UCNPs with varying Nd3+ concentrations of 10, 20, 30, 40, 50, and 60% under 800 nm excitation. Inset shows integrated PL intensity of the C/S UCNPs with various Nd3+ concentrations. (b) 3D PL spectrum of the NaGdF4:Yb(18%),Ho(2%),Ce(30%)/NaYF4:Nd(50%),Yb(5%)

C/S

UCNPs

obtained

with

varying excitation laser wavelength from 720 to 1040 nm.


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Figure 4. TEM images of (a) NaGdF4:Yb,Ho,Ce core, (b) NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb C/S, and (c) NaGdF4:Yb,Ho,Ce/NaYF4:Nd,Yb/NaGdF4 C/D-S UCNPs and insets show corresponding HRSTEM images. (d) EDS line profiles of the C/D-S UCNPs and inset shows HAADF STEM image of the C/D-S UCNPs indicated with line profile direction. (e) PL spectra of the core, C/S, and C/D-S UCNPs under excitation with 800 nm NIR light. Inset shows photograph showing UC red luminescence from the core, C/S, and C/D-S UCNP solutions (from left to right) under irradiation with a continuous wave 800 NIR laser.


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Figure 5. (a) TEM image of ligand free C/D-S UCNPs. Inset shows photograph of the DI waterdispersed LF-C/D-S UCNP solution. (b) PL spectra of OL-capped C/D-S UCNPs and LF-C/D-S UCNPs under excitation with 800 nm NIR light. Inset shows photograph showing the UC red luminescence from the OL-capped and ligand free C/D-S UCNP solutions.


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Figure 6. (a) Bright field image of a nude mouse after irradiation with an 808 nm NIR laser for 5 min (power density ~1.7 W cm-2). In vivo UC luminescence images under irradiation with an 808 nm NIR laser (power density ~0.66 W cm-2) after (b) subcutaneous and (c) intramuscular injections of the LF-C/D-S UCNPs (4 mg mL-1, 60 µL). The signal intensities were measured to be 2.10 ± 0.44 × 1014 photons/s/cm2/sr (average value ± standard deviation) for ROI 1 and 0.52 ± 0.15 × 1014 photons/s/cm2/sr for ROI 2 for 3 mice.


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Figure 7. (a) T1 relaxation rates (T1-1) versus Gd3+ concentration, (b) T1-weighted in vitro MR image with various Gd3+ concentrations. T1-weighted in vivo MR images for (c) subcutaneous and (d) intramuscular injection sites.


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TOC figure


37

800 nm-driven intense red-emitting upconversion nanophosphors with

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