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the axillary lymph node, no PL signal was detected even when irradiated with a 980 nm laser (Figure S13). These findings show that persistent luminesc...
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Hybrid Nanoclusters for Near-Infrared to Near-Infrared Upconverted Persistent Luminescence Bioimaging Xiaochen Qiu, Xingjun Zhu, Ming Xu, Wei Yuan, Wei Feng,* and Fuyou Li* Department of Chemistry & Institute of Biomedicine Science & State Key Laboratory of Molecular Engineering of Polymers & Collaborative Innovation Center of Chemistry for Energy Materials, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China S Supporting Information *

ABSTRACT: Persistent luminescence (PL) bioimaging provides an optimal method of eliminating autofluorescence for a higher resolution and sensitivity because of the absence of excitation light. However, ultraviolet light is still necessary in common energy charging processes, which limits its reactivation in vivo because of its low penetration depth. In the present study, we introduce a type of hybrid nanocluster (UCPL-NC) composed of upconversion nanoparticles, β-NaYbF4:Tm@NaYF4, and persistent nanoparticles, Zn1.1Ga1.8Ge0.1O4:0.5%Cr, which can be activated by a 980 nm laser and exhibits an afterglow at 700 nm to realize near-infrared (NIR) to NIR UCPL bioimaging. The PL of the UCPL-NCs can be reactivated even when covered with a 10 mm pork. We demonstrate that these polyethylene glycol-modified phospholipid-functionalized UCPLNCs can be reactivated in vivo and applied in the PL lymphatic imaging on small animals. KEYWORDS: hybrid nanoclusters, imaging agents, nanoparticles, self-assembly, UCPL

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INTRODUCTION Optical bioimaging has become a powerful tool in current biology and medicine as it allows researchers to investigate the morphological details of biological systems at the molecular and cellular level.1−4 Optical bioimaging offers a number of distinct merits such as high sensitivity, portability, noninvasiveness, and ease to operate.5−9 However, autofluorescence is still a limitation in further increasing sensitivity and resolution.9−15 To improve the signal-to-noise ratio (SNR), persistent luminescence (PL), which can last for several minutes and even hours after removing the excitation source,16 has recently been applied in bioimaging.17−24 The separation of excitation and signal acquisition is an optimal way of eliminating autofluorescence in optical bioimaging. In the past decade, developments in the field of PL nanomaterials have been witnessed because of their potential applications in medical diagnostics. Despite these developments, PL materials still face some challenges. To date, it is still difficult to synthesize nanostructured PL materials using wet chemistry methods.25−28 Recently, a direct hydrothermal method to synthesize sub-10 nm ZnGa2O4:Cr nanocrystals was developed and used in bioimaging.29 However, the wet chemistry synthesis of PL materials with other matrix hosts has not yet been reported. In addition, short-wavelength excitation sources, such as ultraviolet (UV) light, are still needed in the excitation process of PL, which has limited tissue penetration depth.30,31 To solve this problem, a near-infrared (NIR) photostimulated PL was obtained using the Cr3+-doped LiGa5O8 material.32 However, in this case, the material needs to be preirradiated with a UV light © 2017 American Chemical Society

(250−360 nm), and the photostimulated emission will accelerate the decay of afterglow until the precharged energy is exhausted. Recently, ZnGa2O4:Cr was found to be activated by red light,33 but the emission intensity and penetration depth were still limited because of the poor recharging efficiency and relatively short reactivated wavelength. Upconversion luminescence (UCL) can convert low-energy light, usually in the NIR region, into higher-energy visible light even in the UV region.34,35 Therefore, upconversion nanoparticles (UCNPs) can be used as an ideal wavelength converter to efficiently charge PL nanoparticles (PLNPs) to obtain an NIR-excited PL. In bulk materials, UCPL was achieved by excitation with a 980 nm laser.36,37 However, it is still difficult to construct UCPL in a nanoplatform. In the present study, we introduce a UCPL hybrid nanocluster (UCPL-NC) composed of UCNPs and PLNPs. The UCPLNCs were effectively activated using a 980 nm wavelength light source in vivo and gave a persistent afterglow at 700 nm (Figure 1). This means that the energy can be effectively recharged in vivo and that the luminescence decay lifetime of the phosphor is no longer a limitation in NIR PL imaging. Notably, the signal at 700 nm emission could still be collected even when the UCPL-NC aqueous solution was covered by a 10 mm layer of pork after being reactivated at 980 nm. It should be noted that the intensity remained the same after Received: July 20, 2017 Accepted: August 31, 2017 Published: August 31, 2017 32583

DOI: 10.1021/acsami.7b10618 ACS Appl. Mater. Interfaces 2017, 9, 32583−32590

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Research Article

RESULTS AND DISCUSSION

Synthesis and Characterization of UCPL-NCs. To obtain intensive UV emission, pure hexagonal-phase oleic acid (OA)-stabilized core−shell β-NaYbF4:0.5%Tm@NaYF4 nanoparticles (Tm-UCNPs) with an average diameter of 21 nm (Figure 2b) were synthesized using a well-established solvothermal method.38 The structure of the Tm-UCNPs was confirmed by the X-ray powder diffraction pattern and highresolution transmission electron microscopy (TEM) (Figure S2). Zn1.1Ga1.8Ge0.1O4:0.5%Cr nanoparticles (ZGGO-PLNPs) were synthesized via a modified hydrothermal method.39 PLNPs are sub-10 nm (Figure 2c) and hydrophilic, which can be dispersed in water stably without aggregating (Figure S3). It should be noted that the size of ZGGO-PLNPs can be changed from sub-10 to almost 100 nm by increasing the doping concentration of germanium (Figure S4). The codoping of germanium was confirmed by energy-dispersive X-ray spectroscopy (EDS) (Figure S5). The as-prepared ZGGOPLNPs were then functionalized with OA to improve oil dispersity and facilitate further assembly of the UCPL-NCs in a two-phase system (Figure S6). Multicomponent self-assembly is a powerful method for producing various advanced materials with novel integrated functions and new properties because of the interactions between neighboring nanoparticles.40−42 In the present study, the synthesis of UCPL-NCs was via an evaporation-induced self-assembly method. In brief, spherical OA-coated ZGGO-PLNPs and Tm-UCNPs were simultaneously encapsulated within micelles by dodecyltrimethylammonium bromide (DTAB). Also, the optimal mass ratio of ZGGO-

Figure 1. Schematic diagram of NIR-light-charged upconversion persistent luminescence (UCPL) and traditional UV-light-charged PL.

several cycles of reactivation. The UCPL-NCs were further used as optical bioimaging agents in lymphatic imaging.

Figure 2. TEM image of (a) as-prepared β-NaYbF4:0.5%Tm nanoparticles, (b) β-NaYbF4:0.5%Tm@NaYF4 nanoparticles, (c) Zn1.1Ga1.8Ge0.1O4 nanoparticles, and (d) UCPL-NCs. (e) High-resolution TEM image of UCPL-NCs. (f and g) TEM image and the corresponding EDS elemental mapping image of UCPL-NCs. 32584

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radiative reabsorption, we compared the PL spectra of UCPLNCs and the mixture of UCNP and PLNP solutions. The PL intensity of UCPL-NCs was about 10 times higher than that of the mixture (Figure 3a, inset), owing to the reduction of the energy-transfer distance between UCNPs and PLNPs in the hybrid structure of UCPL-NCs. The result revealed that the UCPL was obtained through a nonradiative energy-transfer process. To verify the PL property of UCPL-NCs, the afterglow spectrum was investigated under CW preactivation at 980 nm. The afterglow spectrum (Figure 3b) shows that UCPL-NCs can be charged by a 980 nm laser to give an NIR emission band at 700 nm, which was attributed to the 2E to 4A2 transition of luminescence center Cr3+ ions in gallogermanate.43 The activation stability of the UCPL-NC aqueous solution was studied by repeated activation for 10 s with a 980 nm laser. The results (Figure 3b, inset) show that UCPL-NCs can be repeatedly activated to restore the PL signal lasting for more than 5 min, which is long enough for imaging. PL Imaging of UCPL-NCs. Compared with conventional fluorescence imaging strategies, PL imaging without excitation improves the SNR by avoiding the excitation light and autofluorescence of tissues that arises from in situ continuous excitation. We compared the imaging contrast between the PL imaging without excitation light and the UCL imaging obtained by the conventional filter-based approach with continuous excitation (Figure 4). A band-pass filter (800 ± 6 nm) was used to reduce the excitation at 980 nm and other optical backgrounds, to ensure that the NIR emission near 800 nm was mainly collected. However, in reality, the background intensity up to 2524 counts was observed in UCL imaging and the SNR was only 6.6 because of the much higher photon flux of ultraintensive excitation than emission. Although the conventional filter-based approach is always applied to reduce the background, because of the elimination of the most excitation light at 980 nm, residual excitation and autofluorescence are still the main limitations in reducing the background noise in UCL bioimaging. By contrast, PL imaging without excitation light reduced the background intensity to 412 counts, which almost approached the background detected from the environment with no sample, and the SNR increased to 327.4. For UCPL-NCs, excitation with 980 nm light, which lies in the biological transparency window (600−1100 nm), has a deep biological tissue penetration. As shown in Figure 5, UCPL makes it possible to achieve PL bioimaging activation in vivo and the decay time is not a limitation to PL bioimaging anymore because of the increased penetration depth of NIR excitation. In a proof-of-concept experiment (Figure S11), 200 μL of the 3 mg/mL UCPL-NC aqueous solution was added to a 96-well plate, and a meat-covering method was used to simulate the environment in vivo. The penetration depths of different activated light sources were then compared under the same conditions. As shown in Figure 6a, UCPL-NCs can be activated at 254 nm, 365 nm, or white LED light without pork but are hardly detected when covered with 5 mm of pork. Importantly, when the UCPL-NCs were activated with a 980 nm laser, the PL signals were still detected even when covered with 10 mm of pork. We next carried out in vivo imaging experiments during the lymph node dissection (Figures 6b, S12, and S13). UCPL-NCs (50 μL, 3 mg/mL) were injected intradermally into the right paw of the mouse. We irradiated the area around the sentinel lymph node with a 254 nm, 365 nm, white LED light or 980 nm laser for 10 s. An intense PL

PLNPs to Tm-UCNPs was 6:4 (Figure S7). The resultant clear micelle aqueous solution was then injected into a poly(vinylpyrrolidone) (PVP) and polyethylene glycol (PEG) solution under vigorous stirring. After that, the UCPL-NCs were isolated by centrifugation and redispersed into deionized water. The average size of the UCPL-NCs was approximately 100 nm, which was shown by the TEM image (Figure 2d) and dynamic light scattering (DLS) measurements (Figure S8). The TEM image also showed that each UCPL-NC was composed of a PLNP “core” and a wavelength convertor UCNP “shell”. EDS elemental mapping (Figure 2g) further confirmed the core− shell structure. We speculate that the formation of this “core− shell” structure was most probably caused by the different sizes and solvophobic interactions of the Tm-UCNPs and ZGGOPLNPs.38 UCPL Properties. UCL spectra of the Tm-UCNPs and UCPL-NCs were investigated under continuous-wave (CW) excitation at 980 nm. The prominent upconversion emission bands centered at 345, 360, 452, 475, and 800 nm corresponded to the 1I6 → 3F4, 1D2 → 3H6, 1D2 → 3F4, 1G4 → 3H6, and 3H4 → 3H6 transitions of Tm3+ ions, respectively (Figure 3a). For UCPL-NCs, the upconversion emissions in the

Figure 3. (a) Comparison of the UCL spectra of Tm-UCNPs and UCPL-NCs; the inset shows the PL spectra of UCPL-NCs and the simple mixture of UCNP and PLNP solutions. (b) PL spectrum of UCPL-NCs and the inset shows the recycle afterglow decay curves of the UCPL-NC nanoparticle aqueous solution (1 mL, 3 mg/mL) after irradiation for 10 s with a 980 nm laser. PL intensity was monitored at 700 nm as a function of time.

UV and blue regions decreased sharply. However, by contrast, the NIR emissions remained the same. This may be due to the efficient energy transfer from Tm-UCNPs to ZGGO-PLNPs in the UV and blue regions. In addition, the excitation spectrum (Figure S10) shows that the ZGGO-PLNPs can be excited in the UV and blue regions. To confirm whether PLNPs receive energy from UCNPs through nonradiative energy transfer or 32585

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Figure 5. Schematic diagram of (a) traditional UV-light-charged PL bioimaging and (b) NIR-light-charged UCPL bioimaging.

indicated that UCPL-NCs exhibited low toxicity in living systems. In methyl thiazolyl tetrazolium (MTT) assays, HeLa cells were incubated with UCPL-NCs at different concentrations varying from 100 to 500 μg/mL for 24 h. The cell viability was over 90%, even with a concentration up to 500 μg/ mL, which indicates the low cytotoxicity of UCPL-NCs (Figure 8a). To further compare the damages to cells with different light sources, the killing effects for 980 nm lasers and UV light as the excitation source were further studied on HeLa cells. It should be noted that the cell vitality was only 80% following irradiation with 254 nm light for 10 s with a power density of 100 mW/cm2, whereas almost all of the cells survived following irradiation with a 980 nm laser even at a power density of 300 mW/cm2 (Figure 8b). Histological analysis of the tissues obtained from the harvested organs (heart, lung, liver, spleen, kidney, and lymphatic node) was also employed to evaluate the potential toxicity of UCPL-NCs. Hematoxylin and eosin (H&E) stains of these organs showed no apparent histological changes caused by the adverse effects of the nanoparticles (Figure S14). All these above experimental results indicated that UCPL-NCs exhibited low toxicity on living systems.

Figure 4. Comparison of the in vitro imaging contrast between the UCL imaging and the PL imaging of UCPL-NCs. The intensity profile along the line across the 16-bit grayscale (a) UCL and (b) PL imaging of the 1 mL UCPL-NC aqueous solution (3 mg/mL). The UCL imaging was collected through a band-pass filter (800 ± 6 nm) under the excitation of CW 980 nm. The PL imaging was obtained after removing the band-pass filter and excitation source. The noise curve (red curve) of the dark current was then obtained after removing the sample. SNR = [(mean luminescence intensity of the UCL or PL signal) − (mean luminescence intensity of the laser-off background)]/ [(mean luminescence intensity of the UCL or PL background) − (mean luminescence intensity of the laser-off background)].

signal was collected at 700 nm only from the sentinel lymph node after removing the light source. The SNR of 980 nm light activated PL imaging was 124.5, which was approximately fivefold higher than that when activated directly by 254 nm light with the same power intensity of 100 mW/cm2. Moreover, the PL signal was still detected 5 min after ceasing excitation by increasing the exposure time. A higher intensity can be obtained with a stronger activation light; however, the heat effect of 980 nm light will also be more obvious.44 The position of the lymph node was clearly shown by PL imaging after the removal of the skin (Figure S12). Following the removal of the axillary lymph node, no PL signal was detected even when irradiated with a 980 nm laser (Figure S13). These findings show that PL imaging could potentially assist in the location of lymph nodes and facilitate the lymph node dissection. In control experiments, UCNPs (30 μL, 1 mg/mL) and PLNPs (30 μL, 4 mg/mL) were injected intradermally into the paw of the mouse, respectively, and no PL signal was detected when activated with a 980 nm laser (Figure 7). Toxicity of UCPL-NCs. For potential bioapplication, the toxicity of UCPL-NCs was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (Figure 8) and histological analysis (Figure S14). The experimental results

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CONCLUSIONS In summary, UCPL-NCs were obtained via a simple and effective strategy to assemble NaYbF4:0.5%Tm@NaYF4 with ZGGO-PLNPs in a two-phase system. A persistent emission of 700 nm was obtained following activation with a 980 nm laser. Importantly, PL bioimaging was achieved following activation in vivo because of the increased penetration depth of NIR excitation. We further applied the UCPL-NCs as optical bioimaging agents in lymphatic imaging, which could potentially assist in the location of lymph nodes and facilitate the lymph node dissection. Moreover, the cytotoxicity measurements of UCPL-NCs suggested that the nanoparticles had few side effects on living systems. In conclusion, we believe 32586

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wt %), and sodium hydroxide (NaOH) were bought from Sinopharm Chemical Reagent Co., China. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was obtained from Shanghai Ponsure Biotechnology Co., Ltd. ReCl3 (YbCl3, TmCl3, or YCl3) was prepared by dissolving the corresponding oxides in concentrated hydrochloric acid (35 wt %) at an elevated temperature and then by evaporating the water completely. Ga(NO3)3 solution was prepared by dissolving Ga2O3 in 1:1 concentrated nitric acid at an elevated temperature and then by removing the water and excess HNO3 through evaporation at 105 °C. The solutions of 2 mol/L Ga(NO3)2, 2 mol/L Zn(NO3)2, and 0.008 mol/L Cr(NO3)3 were stored as the precursor solutions. The size and morphologies of the nanoparticles were characterized through a transmission electron microscope (JEOL, JEM-2010F) at a working voltage of 200 kV. EDS elemental mapping was also performed by TEM (JEOL, JEM-2010F). Samples were prepared by placing a drop of the diluted dispersion in cyclohexane or ethanol on the surface of a copper grid. Powder X-ray diffraction (XRD) measurements were measured with a Bruker D4 X-ray diffractometer (Cu Kα radiation, λ = 0.15406 nm). DLS and zeta potential were carried out on a Malvern Zetasizer Nano ZS system. Excitation and emission spectra of UCNPs, PLNPs and UCPL-NCs were measured with an Edinburgh LFS-920 fluorescence spectrometer, and UCL spetra were measured by using an external 0−800 mW adjustable CW laser at 980 nm (Connet Fiber Optics, China), as the excitation source, instead of the xenon source in the spectrophotometer. Fouriertransform infrared spectroscopy was performed using an IR Prestige21 spectrometer (Shimadzu) from samples in KBr pellets. Synthesis of β-NaYbF4:0.5%Tm Nanoparticles. β-NaYbF4:0.5% Tm nanoparticles were synthesized via a well-established solvothermal method as per the literature reported before. In a typical procedure of β-NaYbF4:0.5%Tm, YbCl3 (0.995 mM) and TmCl3 (0.005 mM) were added to a mixture of OA (7.4 g, 26 mmol) and ODE (11.4 g, 45 mmol) in a three-necked flask at room temperature. The mixture was heated to 140 °C with vigorous magnetic stirring under vacuum until a transparent solution was formed. Sodium hydroxide (100 mg) was added to the round-bottomed flask and then dissolved at 90 °C (1 h). After that, ammonium fluoride (148 mg) was added at room temperature. Air and moisture were then removed from the reaction mixture by attaching a vacuum and then injecting N2 gas into the round-bottomed flask three times. The reaction solutions were then heated to 300 °C (1 h). After cooling to room temperature, the mixture was centrifuged and washed with cyclohexane (10 mL) and absolute ethanol (10 mL) three times. The as-prepared nanoparticles were dispersed in cyclohexane (10 mL) for further coating. Synthesis of β-NaYbF4:0.5%Tm@NaYF4 (Core−Shell) Nanoparticles. YCl3 (1 mM) and the prepared β-NaYbF4:0.5%Tm were added to a mixture of OA (7.4 g, 26 mmol) and ODE (11.4 g, 45 mmol) in a three-necked flask at room temperature. The mixture was heated to 140 °C with vigorous magnetic stirring under vacuum for 30 min to remove moisture and oxygen, thus forming a transparent solution. The solution was then heated to 300 °C and maintained for 1 h under N2 protection. After cooling to room temperature, the mixture was centrifuged and washed with cyclohexane (10 mL) and absolute ethanol (10 mL) three times. The as-prepared nanoparticles were dispersed in cyclohexane (10 mL).

Figure 6. (a) Mean intensity of PL imaging activated at different wavelengths of light in vitro, 200 μL of the 3 mg/mL UCPL-NC aqueous solution in a 96-well plate activated at 254 nm light, 365 nm light, white light-emitting diode (LED) light, or 980 nm laser covered without pork, with 5 mm of pork, or 10 mm of pork. The power intensity of 254 nm light, 365 nm light, and 980 nm laser was 100 mW/cm2, and the power of the white LED light was 10 W. (b−e) In vivo lymphatic images were detected at the lymph nodes of the nude mice after the injection of UCPL-NCs (50 μL, 3 mg/mL) activated at 254 nm light (b), 365 nm light (c), LED light (d), or 980 nm laser (e).

that UCPL is ideal for long-term monitoring of the biological processes in real time.

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METHODS

Materials and Characterization. Rare earth oxides, Yb2O3 (99.999%), Tm2O3 (99.999%), and Y2O3 (99.999%), were purchased from Shanghai Yuelong New Materials Co. Ltd. Gallium oxide (Ga2O3; 99.99%) was obtained from Alfa Aesar. OA (90%), 1octadecene (ODE; >90%), and germanium oxide (GeO2; 99.99%) were purchased from Sigma-Aldrich. Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), chromic nitrate nonahydrate (Cr(NO3)3·9H2O), absolute ethanol, aqueous ammonia (28 wt %), cyclohexane, methyl alcohol, chloroform, hydrochloric solution (35 wt %), nitric acid (65

Figure 7. In vivo lymphatic images were detected at the lymph nodes of the nude mice after the injection of UCNPs (30 μL, 3 mg/mL) and PLNPs (30 μL, 3 mg/mL), respectively. (a) 980 nm laser-excited UCL imaging; (b) 980 nm excited PL imaging; and (c) 365 nm light-excited PL. 32587

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Figure 8. Cell viability values (%) estimated by the MTT proliferation test vs (a) concentrations of UCPL-NCs (100−500 μg/mL) after 24 h incubation at 37 °C and (b) different power intensities of the 254 nm light or 980 nm laser incubated with 300 μg/mL UCPL-NCs. μg/mL; diluted in RPMI 1640), and another group was only irradiated by different light sources. After 24 h of further incubation, MTT (20 μL, 5 mg/mL) was added to each well, and the cells were subsequently incubated for an additional 4 h at 37 °C under 5% CO2. The produced purple formazan product was dissolved in dimethyl sulfoxide and quantified by the absorbance at 570 nm, with background subtraction at 690 nm, which was measured by means of a Tecan Infinite M200 monochromator-based multifunction microplate reader. The following formula was used to calculate the inhibition of cell growth: cell viability (%) = (mean of absorbance value of treatment group/mean of absorbance value of control) × 100%. Histological Analysis. In the test group, nude mice (n = 3) were intravenously injected with UCPL-NCs at a total dose of 3 mg/mL (200 μL). Also, the nude mice (n = 3) with no injection were selected as the control group. The tissues were harvested from the test and control groups after 24 h. The heart, liver, spleen, lung, and kidney were removed, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Investigation of the Penetration Depth. The tissue penetration ability of UCPL-NCs with different excitation light sources was investigated through the meat-covering method. The aqueous solutions of UCPL-NCs were covered with different thicknesses of pork. Then, the PL signal was collected after 10 s irradiation with 254 nm, 365 nm, white LED, or 980 nm light source. The power density of the 254 nm and 365 nm light and the 980 nm laser was 100 mW/cm2, and the power of the white LED light was 10 W. PL imaging was performed with a modified luminescence in vivo imaging system designed by our group. In this system, an Andor DU897 electron multiplying charge-coupled device was used as the signal collector. Lymphatic PL Imaging In Vivo. In vivo, in situ, and ex vivo PL imagings were taken 30 s after irradiation for 10 s with different light sources (254 nm light, 365 nm light, white LED light, or 980 nm laser). In vivo lymphatic PL imaging was performed 30 min after the intradermal injection of UCPL-NCs (50 μL, 3 mg/mL) into the paw of the nude mouse. The lymphatic drainage in situ PL imaging after the removal of skin and fatty tissues and the ex vivo PL imaging after the removal of the lymph node from the body were also measured. The power density on the surface of the nude mouse was 100 mW/ cm2. The images of luminescent signals were analyzed with Kodak Molecular Imaging Software.

Synthesis of Zn 1+x Ga 2−2x Ge x O 4 :0.5%Cr Nanoparticles (PLNPs). Zn1+xGa2−2xGexO4:0.5%Cr nanoparticles were synthesized via a hydrothermal process. In a typical synthesis, GeO2 (10.5 mg) was dispersed in 10 mL of deionized water with vigorous stirring first, then several drops of aqueous ammonia (28 wt %) were added, and the solution turned transparent immediately. A mixture of Ga(NO3)3 (1.8 mmol), Zn(NO3)2 (1.1 mmol) and Cr(NO3)3 (0.005 mmol) was added to (NH4)2GeO3 solution dropwise under vigorous stirring. Then, the resulting solution was adjusted to pH 9.0 with concentrated aqueous ammonia (28 wt %). The mixture was transmitted into a Teflon-lined autoclave (25 mL) and sealed. The autoclave was put in an oven at 220 °C for 10 h and was then naturally cooled until reaching room temperature. The resulting precipitate was washed sequentially with ultrapure water and ethanol and dried at 70 °C for 2 h. Finally, hydroxylated PL nanoparticles (PLNPs-OH) were obtained. Surface Modification of PLNPs. Hydrophobic PLNPs were synthesized through the modification of OA. The prepared PLNPsOH were added to OA (20 mL) at 120 °C for 30 min under the protection of N2. After cooling to room temperature, the mixture was centrifuged and washed with cyclohexane (10 mL) three times. The as-prepared nanoparticles were dispersed in CHCl3 (10 mL). Synthesis of UCPL-NCs. UCPL-NCs were synthesized through the method as previously reported with modified.3 Typically, 1 mL of the chloroform solution containing 6 mg of OA-coated PLNPs and 4 mg of UCNPs was added to 4 mL of the DTAB solution (4 mg/mL). The solution was mixed by ultrasonication for 10 s. Then, the mixture was stirred vigorously overnight at room temperature to remove the chloroform solvent. This clear PLNP-UCNP-micelle aqueous solution was added into a 5 mL EG solution containing 20 mg of DSPE-PEG2000 and 550 mg of PVP (molecular weight: 55 000) under vigorous stirring for 30 min at room temperature. The resulting UCPL-NCs were isolated by centrifugation and redispersed in ethanol and chloroform (2 mg/mL), and 100 μL of OA-coated UCNPs in cyclohexane (2 mg/mL) was mixed with 200 μL of DSPE-PEG-2000 in chloroform (10 mg/mL). After keeping the solution overnight under vigorous stirring at room temperature to evaporate the organic solvent completely, 1 mL of deionized water was added into the synthesis vessel at an elevated temperature, and a transparent solution was obtained after being placed in an ultrasonic bath for 5 min at the temperature of 70 °C. Cell Culture and Cytotoxicity of UCPL-NCs. A human cervical carcinoma cell line (HeLa cells) was obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). The HeLa cells were grown in modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. In vitro cytotoxicity was measured by performing an MTT assay on HeLa cells. Briefly, the cells were plated in 96-well flat-bottomed plates with a concentration of 5 × 104 cells per well and allowed to grow at 37 °C and 5% CO2 for 24 h. Then, one group was incubated with UCPL-NCs of different concentrations (0, 100, 200, 300, 400, and 500

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b10618. XRD; high-resolution TEM and DLS images of UCPLNCs; TEM and high-resolution TEM of Zn1+xGa2−2xGexO4:0.5%Cr (x = 0−0.5); and in situ and ex vivo lymphatic UCPL images (PDF) 32588

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (W.F.). *E-mail: [email protected] (F.L.). ORCID

Wei Feng: 0000-0002-8096-2212 Fuyou Li: 0000-0001-8729-1979 Author Contributions

X.Q. and X.Z. carried out the experiments. X.Q., W.F., and F.L. wrote the paper. X.Q., X.Z., W.F., and F.L. designed the experiments. W.F. and F.L. analyzed the data. All authors read and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support by the National Key R&D Program of China (2017YFA0205100), MOST of China (2015CB931800), NSFC (21231004, 21527801, 21671042), and Shanghai Sci. Tech. Comm. (15QA1400700) is gratefully acknowledged. Professor Tao Yi from the Department of Chemistry at the Fudan University is thanked for helpful advice to revise the manuscript. Dr. Hua Zhu from the Department of Chemistry at the Brown University is thanked for useful discussion.

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