High Contrast Upconversion Luminescence Targeted Imaging in

In the process of in vivo imaging, however, a high autofluorescence background could mask the signals from the fluorescent probes. Herein, a high cont...
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Anal. Chem. 2009, 81, 8687–8694

Articles High Contrast Upconversion Luminescence Targeted Imaging in Vivo Using Peptide-Labeled Nanophosphors Liqin Xiong,† Zhigang Chen,† Qiwei Tian,† Tianye Cao,† Congjian Xu,‡ and Fuyou Li*,† Department of Chemistry and The Obstetrics and Gynecology Hospital, Fudan University, Shanghai, 200433, P. R. China Fluorescence targeted imaging in vivo has proven useful in tumor recognition and drug delivery. In the process of in vivo imaging, however, a high autofluorescence background could mask the signals from the fluorescent probes. Herein, a high contrast upconversion luminescence (UCL) imaging protocol was developed for targeted imaging of tumors based on RGD-labeled upconversion nanophosphors (UCNPs) as luminescent labels. Confocal Z-scan imaging of tissue slices revealed that UCL imaging showed no autofluorescence signal even at high penetration depth (∼600 µm). More importantly, region of interest (ROI) analysis of the UCL signal in vivo showed that UCL imaging achieved a high signal-to-noise ratio (∼24) between the tumor and the background. These results demonstrate that the UCL imaging technique appears particularly suited for applications in tracking and labeling components of complex biological systems. Fluorescence imaging has subcellular resolution; it, thus, offers a unique approach for visualizing morphological details in tissue that cannot be resolved by other medical imaging techniques including nuclear, ultrasound, and magnetic resonance imaging. Recently, to obtain anatomical and physiological details in vivo, more attention has been paid to fluorescence imaging of specific target structures from in vitro to in vivo. In the process of fluorescence imaging in vivo, however, a high autofluorescence background could mask the signals from the fluorescent probes, owing to the existence of ubiquitous endogenous components in biological samples.1-3 Therefore, the key consideration for the fluorescent probes for in vivo imaging is to reduce autofluorescence. One recent strategy is to develop new luminescent labels that are excited in the near-infrared (NIR) range, including NIR * To whom correspondence should be addressed. Fax: 86-21-55664621. Tel: 86-21-55664185. E-mail: [email protected]. † Department of Chemistry. ‡ The Obstetrics and Gynecology Hospital. (1) Billinton, N.; Knight, A. W. Anal. Biochem. 2001, 291, 175–197. (2) Yu, M. X.; Zhao, Q.; Shi, L. X.; Li, F. Y.; Zhou, Z. G.; Yang, H.; Yi, T.; Huang, C. H. Chem. Commun. 2008, 2115–2117. (3) Mansfield, J. R.; Gossage, K. W.; Hoyt, C. C.; Levenson, R. M. J. Biomed. Opt. 2005, 10, 41207. 10.1021/ac901960d CCC: $40.75  2009 American Chemical Society Published on Web 10/09/2009

organic probes4,5 and semiconductor quantum dots (QDs)6-10 for in vivo imaging. These NIR probes belong to downconversion luminescence materials. However, owing to the similar downconversion luminescence processes of these NIR labels and ubiquitous endogenous components in biological samples, background autofluorescence cannot be completely suppressed (especially at high penetration depth), which limits their application in fluorescence imaging in vivo. Upconversion luminescence (UCL) is a process in which lowenergy light, usually near-infrared (NIR), is converted to higherenergy light (visible) through sequential absorption of multiple photons or energy transfers.11,12 By virtue of the f-f transition under continuous-wave (CW) excitation at 980 nm, some rareearth nanophosphors exhibit unique UCL properties (such as sharp emission lines and high photostability).13-26 In particular, rare-earth upconversion nanophosphors (UCNPs) have attractive features as biomarkers for cell imaging with no background fluorescence,17 because neither endogenous components in samples nor conventional fluorescent probes are excited by a CW laser at 980 nm. Consequently, UCNPs have become one of the most promising classes of luminescent materials for bioimaging.17-24 Recently, Zhang’s group and our group have developed some biocompatible UCNPs as luminescent labels for in vitro imaging,17-22 and Prasad et al. have for the first time reported in vivo Maestro whole-body images of a Balb-c mouse injected with the UCNPs.23 (4) Chen, X. Y.; Conti, P. S.; Moats, R. A. Cancer Res. 2004, 64, 8009–8014. (5) Leevy, W. M.; Gammon, S. T.; Jiang, H.; Johnson, J. R.; Maxwell, D. J.; Jackson, E. N.; Marquez, M.; Piwnica-Worms, D.; Smith, B. D. J. Am. Chem. Soc. 2006, 128, 16476–16477. (6) Cai, W. B.; Shin, D. W.; Chen, K.; Gheysens, O.; Cao, Q. Z.; Wang, S. X.; Gambhir, S. S.; Chen, X. Y. Nano Lett. 2006, 6, 669–676. (7) Smith, B. R.; Cheng, Z.; De, A.; Koh, A. L.; Sinclair, R.; Gambhir, S. S. Nano Lett. 2008, 8, 2599–2606. (8) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759–1762. (9) Smith, A. M.; Duan, H. W.; Mohs, A. M.; Nie, S. M. Adv. Drug Delivery Rev. 2008, 60, 1226–1240. (10) Ute, R. G.; Markus, G.; Sara, C. J.; Roland, N.; Thomas, N. Nat. Methods 2008, 5, 763–775. (11) Auzel, F. Chem. Rev. 2004, 104, 139–173. (12) Wang, F.; Liu, X. G. Chem. Soc. Rev. 2009, 38, 976–989. (13) Heer, S.; Kompe, K.; Gudel, H. U.; Haase, M. Adv. Mater. 2004, 16, 2102– 2105.

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Scheme 1. Synthesis of UCNP-RGDa

a a: cyclohexane, tert-butanol, water, 5 wt % K2CO3 aqueous solution, and Lemieux-von Rudloff reagent. b: EDC, sulfo-NHS, MES buffer, and O,O′-bis(3-aminopropyl)polyethylene glycol (MW 1500). c: 6-maleimidohexanoic acid N-hydroxysuccinimide ester, HEPES buffer, and c(RGDFK).

In the present study, we developed a high contrast UCL imaging protocol based on UCNPs as luminescent labels for targeted imaging of tumors in vivo and ex vivo. Our design strategy for targeted imaging of tumors is based on the high affinity of the arginine-glycine-aspartic peptide c(RGDFK) (Scheme 1) to the Rvβ3 integrin receptor.4,6,7,24,27-32 Moreover, to meet (14) Mai, H. X.; Zhang, Y. W.; Si, R.; Yan, Z. G.; Sun, L. D.; You, L. P.; Yan, C. H. J. Am. Chem. Soc. 2006, 128, 6426–6436. (15) Li, P.; Peng, Q.; Li, Y. D. Adv. Mater. 2009, 21, 1945–1948. (16) Chen, Z. G.; Chen, H. L.; Hu, H.; Yu, M. X.; Li, F. Y.; Zhang, Q.; Zhou, Z. G.; Yi, T.; Huang, C. H. J. Am. Chem. Soc. 2008, 130, 3023–3029. (17) Yu, M. X.; Li, F. Y.; Chen, Z. G.; Hu, H.; Zhan, C.; Huang, C. H. Anal. Chem. 2009, 81, 930–935. (18) Hu, H.; Xiong, L. Q.; Zhou, J.; Li, F. Y.; Cao, T. Y.; Huang, C. H. Chem.sEur. J. 2009, 15, 3577–3584. (19) Hu, H.; Yu, M. X.; Li, F. Y.; Chen, Z. G.; Gao, X.; Xiong, L. Q.; Huang, C. H. Chem. Mater. 2008, 20, 7003–7009. (20) Xiong, L. Q.; Chen, Z. G.; Yu, M. X.; Li, F. Y.; Liu, C.; Huang, C. H. Biomaterials 2009, 30, 5592–5600. (21) Chatterjee, D. K.; Rufaihah, A. J.; Zhang, Y. Biomaterials 2008, 29, 937– 943. (22) Jalil, R. A.; Zhang, Y. Biomaterials 2008, 29, 4122–4128. (23) Nyk, M.; Kumar, R.; Ohulchanskyy, T. Y.; Bergey, E. J.; Prasad, P. N. Nano Lett. 2008, 8, 3834–3838. (24) Zako, T.; Nagata, H.; Terada, N.; Utsumi, A.; Sakono, M.; Yohda, M.; Ueda, H.; Soga, K.; Maeda, M. Biochem. Biophys. Res. Commun. 2009, 381, 54– 58. (25) Yi, G.; Chow, G. Chem. Mater. 2007, 19, 341–343. (26) Li, Z.; Zhang, Y. Angew. Chem., Int. Ed. 2006, 45, 7732–7735. (27) Sun, Y.; Cressman, S.; Fang, N.; Cullis, P. R.; Chen, D. D. Y. Anal. Chem. 2008, 80, 3105–3111. (28) Xiong, L. Q.; Yu, M. X.; Cheng, M. J.; Zhang, M.; Zhang, X. Y.; Xu, C. J.; Li, F. Y. Mol. BioSyst. 2009, 5, 241–243.

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the needs of combined application in cells, tissues and in vivo UCL imaging with different penetration depth, multicolor UCNPs (NaYF4: 20%Yb, 1.8%Er, 0.2%Tm) with green, red, and NIR upconversion luminescence (UCL) were designed and synthesized. To improve the blood circulation of the UCNPs, a polyethylene glycol (PEG) linkage (MW 1500) was introduced to link the multicolor UCNPs and the RGD peptide, because PEGylated amphiphilicpolymers are proven to have very high stability and low nonspecific absorption levels.33 Interestingly, the experimental results revealed that upconversion luminescence imaging showed no autofluorescence signal even at a penetration depth as high as ∼600 µm and was suitable for use in targeted imaging in vivo. EXPERIMENTAL SECTION Materials. All of the chemicals used were of analytical grade and were used without further purification. Deionized water was used throughout. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (29) Mulder, W. J. M.; Strijkers, G. J.; Habets, J. W.; Bleeker, E. J. W.; van der Schaft, D. W. J.; Storm, G.; Koning, G. A.; Griffioen, A. W.; Nicolay, K. FASEB J. 2005, 19, 2008–2010. (30) Wang, Q.; Jakubowski, J. A.; Sweedler, J. V.; Bohn, P. W. Anal. Chem. 2004, 76, 1–8. (31) Lo ¨ssner, D.; Kessler, H.; Thumshirn, G.; Dahmen, C.; Wiltschi, B.; Tanaka, M.; Knoll, W.; Sinner, E. K.; Reuning, U. Anal. Chem. 2006, 78, 4524– 4533. (32) Liu, S. Mol. Pharm. 2006, 3, 472–487. (33) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. M. Nat. Biotechnol. 2004, 22, 969–976.

hydrochloride (abbreviated as EDC) and N-hydroxysulfosuccinimide sodium salt (abbreviated as sulfo-NHS) were purchased from Sinopharm Chemical Reagent Co. (China). 9-Fluorenylmethyl chloroformate (abbreviated as FmocCl) was obtained from Acros. 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, oleic acid, and O,O’-bis(3-aminopropyl)polyethylene glycol (MW 1500) were obtained from Alfa Aesar Ltd. Cyclo(Arg-Gly-Asp-Phe-Lys(mpa)) was kindly provided by GL Biochem (Shanghai) Ltd. (purity: 98.5%). Rare earth chlorides (LnCl3, Ln: Y, Yb, Er, Tm) were purchased from Beijing Lansu Co. China. Synthesis of Oleic Acid-Capped UCNPs. UCNPs (NaYF4: 20%Yb, 1.8%Er, 0.2%Tm) were prepared by a modified hydrothermal process.16,34 Synthesis of Carboxylic Acid-Capped UCNPs. Hydrophilic UCNPs were prepared according to the method of directly oxidizing oleic acid ligands to azelaic acid (HOOC(CH2)7COOH) with the Lemieux-von Rudloff reagent.16 Synthesis of PEG-NH2-Modified UCNPs. To activate the surface -COOH groups, EDC (20 mg, 1.0 × 10-5 mol) and sulfoNHS (20 mg, 9.2 × 10-5 mol) were added to MES (2-(Nmorpholino)ethanesulphonic acid) buffer (0.1 M, 10 mL, pH 6.0) containing the oxidized UCNPs (40 mg). The mixture was then stirred overnight at room temperature. After centrifugation and washing twice with water, the precipitate was slowly added to 10 mL of water solution of PEG-NH2 (60 mg, 4 × 10-5 mol). The reaction was allowed to stir gently at room temperature for 24 h. The PEG-NH2-modified UCNPs were obtained by centrifugation and washing three times with water. To quantitatively analyze amine moieties on UCNP-PEG-NH2 samples, FmocCl (50 mg, 1.9 × 10-4 mol) was added to 3 mL of anhydrous DMF (N,N-dimethylformamide) solution containing the dried UCNPs (21 mg). The reaction mixture was stirred overnight at room temperature. The reaction mixture was centrifugated, followed by washing with methanol. Subsequently, Fmoc (9-fluorenylmethoxycarbonyl) protected UCNPs were precisely weighed in an Eppendorf tube and resuspended in 2.5 mL of DMF. Piperidine (0.5 mL) was added into the above solution for Fmoc cleavage. Amine quantification was performed using the standard Fmoc quantification protocol by detection of the supernatant dibenzofulvene solution with UV absorption at λ ) 300 nm. For greater accuracy, the sample was tested three times with UV absorbance between about 0.3 and 1.2 absorbance units. Synthesis of RGD-Labeled UCNPs. To modify the surfaceNH2 groups, 6-maleimidohexanoic acid N-hydroxysuccinimide ester (30 mg, 9.7 × 10-4 mol) was added to HEPES (4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (0.1 M, 5 mL, pH 8.0) containing the PEG-NH2-modified UCNPs. The mixture was then stirred 8 h at room temperature. After centrifugation and washing twice with water, the precipitate was added to HEPES buffer (0.1 M, 5 mL, pH 7.3) of the thiolated cyclo(Arg-Gly-Asp-Phe-Lys(mpa)) (c(RGDFK)) (69 mg, 1 × 10-3 mol). The reaction was allowed to stir gently overnight at room temperature. After centrifugation and washing twice with water, RGD-activated UCNPs were resuspended in HEPES buffer (0.1 M, 5 mL, pH 7.3) and stored at 4 °C. (34) Wang, L. Y.; Li, Y. D. Chem. Mater. 2007, 19, 727–734.

Characterization. Sizes and morphologies of UCNPs were determined at 200 kV using a JEOL JEM-2010F high-resolution transmission electron microscope (HR-TEM). Energy-dispersive X-ray analysis (EDXA) of the samples was performed on a SUPERSCAN SSX-550 scanning electron microscope. X-ray diffraction (XRD) measurements were made with a Bruker D4 X-ray diffractometer using Cu KR radiation (λ ) 0.15418 nm). Dynamic light scattering (DLS) experiments were carried out on an ALV5000 spectrometer-goniometer equipped with an ALV/LSE-5004 light scattering electronic and multiple tau digital correlator and a JDS Uniphase He-Ne laser (632.8 nm) with an output power of 22 mW. UCL spectra were measured with an Edinburgh LFS920 fluorescence spectrometer, by the use of an external 0-800 mW adjustable laser (980 nm, Beijing Hi-Tech Optoelectronic Co., China) as the excitation source. Cell Culture. The MCF-7 (human breast cancer, low integrin Rvβ3 expression) and U87MG (human glioblastoma, high integrin Rvβ3 expression) cell lines were provided by the Institute of Basic Medical Sciences Chinese Academy of Medical Sciences. The MCF-7 cells were grown in MEM (modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) and 1% insulin (10 mL: 400 U), and the U87MG cells were grown in MEM supplemented with 10% FBS. Cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2. For use in experiments, 5 × 105 cells/well were seeded in 18 mm glass coverslips and allowed to attach for 24 h prior to the assay. Cytotoxicity Assay. Similar to the method reported in the literature,18-20 the in vitro cytotoxicity was measured using the methyl thiazolyl tetrazolium (MTT) assay in human glioblastoma cell line U87MG (see Supporting Information for details). Tumor Xenografts. Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee. Tumor cells were harvested when they reached near confluence by incubation with 0.05% trypsin-EDTA. Cells were pelleted by centrifugation and resuspended in sterile PBS. U87MG cells (2 × 106 cells/site) and MCF-7 cells (5 × 106 cells/site) were implanted subcutaneously into the left and right hind leg of four- to five-week-old female athymic nude mice, respectively. When the tumors reached 0.4-0.6 cm in diameter (14-21 days after implant), the tumor-bearing mice were subjected to biodistribution and imaging studies. Confocal Microscopy Studies. Confocal imaging of cells and tissues, including XY scanning and XYZ scanning imaging, were performed on our laser scanning upconversion luminescence microscope (LSUCLM) equipped with a CW laser emitting at 980 nm.17 A 60× objective lens (1.35 numerical apertures) was used for XY scanning imaging and a 10× objective lens (0.4 numerical apertures) was used for XYZ scanning imaging. For cell staining assays, cells were incubated with the UCNPs for 30 min at 37 °C in HEPES. For confocal microscopic analysis of tissues, we excised tumors and organs after tail vein injection of 50 µg of UCNP or UCNP-RGD for 24 h. The tissues were cryosectioned at -20 °C into slices of 5 µm or ∼1.1 mm thickness (LEICA CM1900, Leica Company). Excitation of the cells and tissue slices was provided by the CW laser at 980 nm; green UCL emission of UCNPs was collected at 530 ± 30 nm, and red UCL emission of UCNPs was collected at 650 ± 50 nm. Quantization Analytical Chemistry, Vol. 81, No. 21, November 1, 2009

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Figure 1. Diagram depicts the experimental setup for the upconversion luminescence in vivo imaging system designed by our group. Two external 0-5 W adjustable CW 980 nm lasers were used as the excitation sources, and an Andor DU897 EMCCD was used as the signal collector.

by line plots and regions were analyzed using the software package provided by OLYMPUS instrument. Vivo Imaging of Tumors. In vivo and ex vivo upconversion luminescence imaging was performed with a modified upconversion luminescence in vivo imaging system designed by our group. Figure 1 shows the diagram of this upconversion luminescence in vivo imaging system. In this system, two external 0-5 W adjustable CW 980 nm lasers (Shanghai Connet Fiber Optics Co., China) were used as the excitation sources and an Andor DU897 EMCCD as the signal collector. Images of luminescent signals were analyzed with Kodak Molecular Imaging Software. UCL signals were collected at 800 ± 12 nm. RESULTS AND DISCUSSION Synthesis and Characterization of UCNPs. The synthetic protocol is presented in Scheme 1. Oleic acid (OA)-capped NaYF4: 20% Yb, 1.8% Er, and 0.2% Tm (UCNP-OA) was synthesized by a modified hydrothermal route.16,33 Due to the presence of oleic acid on the surface of UCNPs, the UCNP-OA sample was welldispersed in nonpolar solvent such as cyclohexane, chloroform, and dichloromethane. As shown in Figure 2A, the transmission electron microscopy (TEM) image shows that the UCNP-OA sample was dispersed with an average diameter of ∼14 nm. The energy-dispersive X-ray analysis (EDXA) patterns (Figure S1 in the Supporting Information) confirmed the presence of Na, Y, F, Yb, and Er in the as-synthesized samples. A peak due to the minordoped Tm ion cannot usually be discerned due to its minute amount (only 0.2 mol % Tm in the rare-earth elements). The powder X-ray diffraction (XRD) patterns (Figure S2 in the Supporting Information) were in good agreement with the data for pure cubic NaYF4 nanocrystals, as reported in the JCPDS card (No. 77-2042, a ) 5.470 Å). This indicates a high purity of the NaYF4 nanocrystals obtained with good crystallinity, which is very beneficial for obtaining bright luminescence. As shown in Figure 2C, the UCL spectrum of the UCNP-OA sample exhibited both three distinct Er3+ emission bands and three distinct Tm3+ emission bands. The UCL bands at 522, 543, and 654 nm were consistent with 2H11/2 f 4I15/2, 4S3/2 f 4I15/2, and 4 F9/2 f 4I15/2 transitions of Er3+, respectively; while the UCL bands at 490, 695, and 800 nm originated from 1G4 f 3H6, 3F3 f 3H6 and 3H4 f 3H6 transitions of Tm3+, respectively. 8690

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We then obtained the hydrophilic and carboxylic acid-functionalized nanophosphors (UCNP-COOH) by oxidizing oleic acid ligands to azelaic acid with the Lemieux-von Rudloff reagent.16 By the use of the coupling reagents ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydrocylsulfo-succinimide (SulfoNHS), PEG groups were linked to the UCNPs surface.16,35 This successful conjugation was confirmed by the measurement of surface amine moieties of UCNPs. Through a standard Fmoc quantification protocol,36 the amine content of the amine-functionalized nanophosphors (UCNP-PEG-NH2) was found to be about 3 × 10-5 mol/g. UCNP-PEG-NH2 were further conjugated to a heterobifunctional cross-linker, 6-maleimidohexanoic acid N-hydroxysuccinimide ester,6 yielding a maleimide-functionalized nanophosphors surface. These were finally coupled with the thiolated c(RGDFK) to yield the RGD-conjugated UCNPs (UCNP-RGD). Owing to the presence of PEG linkage (MW 1500), the UCNP-RGD samples could be well dispersed in both polar solvents (such as ethanol or water) and solvents of low polarity (such as CHCl3). This conversion process of UCNPOA into UCNP-RGD was investigated by XRD, TEM, and photoluminescence spectra. As shown in Figure S2 in the Supporting Information, little variation in XRD was detected after these treatments of UCNP samples. Similarly, no obvious effect on morphology, diameter, and luminescence spectra of UCNP samples was observed as shown in Figure 2 and Figure S3 in the Supporting Information. The dynamic light scattering (DLS) measurement showed that the effective hydrodynamic diameter of UCNP-RGD was 25.8 nm. The increase in hydrodynamic diameter is attributed to the linkage of PEG to the surface of UCNPs. We further tested the UCL spectra of UCNP-RGD in fetal bovine serum; data showed that UCNP-RGD was stable in serum for over 24 h at 37 °C (Figure S4 in the Supporting Information). Furthermore, the novel multicolor upconversion luminescence (UCL) was also successfully realized by doping with Yb3+/Tm3+/ Er3+ ions. Liu et al.37 and Yan et al.38 have recently reported multicolor UCNPs by modifying the relative intensity ratio of the blue/green/red emissions (490, 543, and 654 nm) or green/ red emissions (543 and 654 nm). Herein, we realized the relative intensity ratio of the green/red/NIR emissions (543, 654, and 800 nm) based on tuning the tridoped Yb3+/Tm3+/ Er3+ ions. In our case, the 800 nm emission was preferable for in vivo imaging in light of the high penetration depth of the NIR light. To investigate the cytotoxicity of UCNP-RGD, an MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with the U87MG cells was used to determine the effect of UCNP-RGD on cell proliferation after 24 h. No significant differences in the proliferation of the cells were observed in the absence or presence of 0.1-1 mg/mL UCNP-RGD (Figure S5 in the Supporting Information). The cellular viabilities were estimated to be greater than 85% after 24 h. These data show that UCNP-RGD (e1 mg/ mL) can be considered to have low cytotoxicity. (35) Van Delden, C. J.; Bezemer, J. M.; Engbers, G. H. M.; Feijen, J. J. Biomater. Sci., Polym. Ed. 1996, 8, 251–268. (36) Yoon, T. J.; Yu, K. N.; Kim, E.; Kim, J. S.; Kim, B. G.; Yun, S. H.; Sohn, B. H.; Cho, M. H.; Lee, J. K.; Park, S. B. Small 2006, 2, 209–215. (37) Wang, F.; Liu, X. G. J. Am. Chem. Soc. 2008, 130, 5642–5643. (38) Mai, H. X.; Zhang, Y. W.; Sun, L. D.; Yan, C. H. J. Phys. Chem. C 2007, 111, 13721–13729.

Figure 2. TEM images of UCNP-OA (A) and UCNP-RGD (B) samples. (C) Room temperature upconversion luminescence spectrum of the 1 mg/mL UNCP-OA and UCNP-RGD samples in HEPES buffer under CW excitation at 980 nm (power ≈ 800 mW). NaYF4: 20%Yb, 1.8%Er, 0.2%Tm.

Targeted Imaging of Cancer Cells with RGD-Coupled UCNPs in Vitro. Integrin Rvβ327 plays a pivotal role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed RGD tripeptide sequence. To demonstrate the Rvβ3 integrin specificity of the probe in vitro, the human glioblastoma U87MG cells (expressing high levels of integrin Rvβ3, Figure S6 in the Supporting Information) were chosen for target-specific imaging, whereas the human breast cancer cell line MCF-7 (expressing low levels of integrin Rvβ3) was used in the control experiments. The living cells were incubated with UCNPs (∼10 µg/mL, 200 µL) for 30 min at 37 °C in HEPES. Cell imaging was then performed by confocal upconversion luminescence microscopy as reported previously by our group (Figure S7 in the Supporting Information).17 As shown in Figure 3, strong green UCL and red UCL signal were observed within the U87MG cells after 30 min of incubation with UCNP-RGD at 37 °C (Figure 3A). Bright-field measurements after treatment with UCNP-RGD confirmed that the cells were viable throughout the imaging experiments. Meanwhile, the observed yellow signal by overlaying the green and red UCL signal further established that the emission signals came from the intracellular UCNPs. In contrast, cell controls (MCF-7 cells, Figure 3B) and

probe controls (UCNP-PEG-NH2, Figure 3C) showed very weak UCL emission. Integrin receptor specificity of UCNP-RGD was further demonstrated by a competition assay. The U87MG cells were preincubated with a 10-fold excess of unlabeled c(RGDFK) peptide at 37 °C for 30 min and then incubated with UCNP-RGD at 37 °C for 30 min. Only weak UCL signal was observed in the competition experiment (Figure 3D). In particular, it should be noted that no autofluorescence signal was measured in the UCL signal of cell images. As demonstrated in Figure 3E, qualification analysis of the UCL signal (λem ) 650 ± 50 nm) across the line reveals strong UCL intensity (counts >4095, region-1 and region-3) and nearly no background fluorescence (counts ∼ 0, region-2). This feature cannot be obtained in conventional fluorescence bioimaging. Targeted Imaging of Tumors With UCNPs-RGD in Vivo and Ex Vivo. After successfully imaging the U87MG cells (integrin Rvβ3 overexpressing) in vitro using peptide-conjugated UCNPs (UCNP-RGD), we then performed in vivo and ex vivo imaging using UCNP-RGD to specifically target tumors with integrin Rvβ3 overexpression. Herein, athymic nude mice simultaneously bearing a U87MG tumor on the left hind leg for targeted imaging and a MCF-7 tumor on the right hind leg Analytical Chemistry, Vol. 81, No. 21, November 1, 2009

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Figure 3. Confocal upconversion luminescence images of UCNP-RGD incubated with U87MG cells (A) and MCF-7 cells (B) for 30 min at 37 °C. U87MG cells were incubated with UCNP-PEG-NH2 at 37 °C (C), and U87MG cells were incubated with UCNP-RGD in the presence of unlabeled c(RGDFK) at 37 °C (D). UCL intensity along the line shown in UCL image (inset) of U87MG cells labeled with UCNP-RGD (E). The green and red UCL emission was collected at 530 ( 30 nm and 650 ( 50 nm, respectively. A 60× objective lens (1.35 numerical apertures) was used. Scale bar is 20 µm.

(control) were administered UCNP-RGD (∼50 µg per animal) through tail vein injection. The mice were imaged using our modified upconversion luminescence in vivo imaging system. 8692

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Figure 1 shows the setup for the upconversion luminescence in vivo imaging system. In this system, two CW lasers emitting at 980 nm (0-5 W) passed the optical fiber into the light-tight

Figure 4. Time-dependent in vivo upconversion luminescence imaging of subcutaneous U87MG tumor (left hind leg, indicated by short arrows) and MCF-7 tumor (right hind leg, indicated by long arrows) borne by athymic nude mice after intravenous injection of UCNP-RGD over a 24 h period. All images were acquired under the same instrumental conditions (power ≈ 80 mW/cm2 and temperature ≈ 21.5 °C on the surface of the mouse). (H) The in vivo signal-to-noise ratio (SNR) calculation. Region of interest (ROI) 1, specific uptake; ROI 2, nonspecific uptake; ROI 3, background. SNR ) [(mean fluorescence intensity of the specific uptake) - (mean fluorescence intensity of background)] / [(mean fluorescence intensity of the nonspecific uptake) - (mean fluorescence intensity of background)].

housing and then passed through beam expanders and the lens to be converted into two large bundles of beams, which then homogeneously illuminated the mouse. Light emitted from the mouse passed through the emission filter (800 ± 12 nm) and lens and finally was recorded with a cooled EMCCD camera (Andor DU897). Under excitation with the CW 980 nm laser, intense UCL signal was observed in the U87MG tumor of the mice injected with UCNPRGD whereas no significant UCL signal was seen in the MCF-7 tumor (Figure 4). Furthermore, the time-course series of in vivo imaging of the tumor-bearing mice injected with UCNP-RGD from 1 to 24 h was investigated. As shown in Figure 4A, the UCL signal observed in the U87MG tumor at 1 h postinjection was low, while high UCL signal was seen in the liver (Figure S8 in the Supporting Information). The maximum U87MG tumor binding for the UCNPRGD appeared at about 4 h postinjection and was retained after 24 h owing to the PEG linkage, while the intensity of the UCL signal observed in the liver weakened after 24 h of injection. These facts suggest that tumor imaging in vivo using UCNP-RGD as a label was successful. In particular, region of interest (ROI) analysis of the UCL signal (λem ) 800 ± 12 nm) reveals a high signal-to-noise ratio (∼24) between the tumor and the background (Figure 4H and Figure S9 in the Supporting Information). The specific targeting of integrin Rvβ3-overexpressing tumors was further confirmed by ex vivo UCL imaging of organs, confocal UCL imaging of frozen slices of tissues, and the measurement of the Y3+ concentration in organs by inductively coupled plasma atomic emission spectroscopy (ICP-AES).23

Figure 5A shows ex vivo UCL images of dissected organs of a tumor-bearing mouse sacrificed at 24 h postinjection of UCNPRGD. Integrin receptor specificity of the probe was established by the clearly visible UCL signal in the U87MG tumor, while no passive tumor targeting was observed; virtually no UCNP-RGD signal was seen in the MCF-7 tumor (Figure 5A). Similarly, in the U87MG tumor slices of tumor-bearing mice sacrificed after 24 h of injection, the UCL signal was organized in longitudinal patterns (Figure S10 in the Supporting Information), whereas no UCL signal was observed in the MCF-7 tumor slices (control), indicating a specific association with integrin Rvβ3 expressed in the angiogenic endothelium.29 Moreover, the data from ICPAES analysis of the Y3+ concentration within the tissues indicated that UCNP-RGD uptake for the U87MG tumor was much higher than that for the MCF-7 tumor, further demonstrating that the accumulation of UCNP-RGD was mediated by integrin Rvβ3 binding (Figure 5B). In addition, ex vivo UCL imaging, confocal UCL imaging of slices, and ICP analysis showed that nonspecific UCNP uptake and retention took place primarily in the liver and the spleen, with little UCNP accumulation in the heart, the kidney, or the lung. This pattern of organ uptake and distribution was similar to that of multifunctional nanoparticle probes based on QDs.33 In light of the high penetration depth of 980 nm light, UCL imaging using the CW 980 nm laser for excitation is expected to make possible the visualization of biological samples at high penetration depth. Herein, 1.1 mm-thin-spleen slices of tumorbearing mice sacrificed at 24 h after tail vein injection of UCNPAnalytical Chemistry, Vol. 81, No. 21, November 1, 2009

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as the output signal, even deeper tissue imaging depth (>600 µm) could be achieved. (2) The emissive UCL signal can be completely separated from biological background fluorescence (autofluorescence) by the use of an upconversion luminescence technique. These important features of high penetration depth and no autofluorescence signal in UCL imaging based on UCNPs as labels cannot be obtained in single-photon or twophoton fluorescence imaging. This fact can be attributed to two factors: (I) the very weak single-photon absorption of dyes and biological samples at 980 nm and (II) the very low likelihood of these dyes and biological samples absorbing two photons simultaneously under low-power CW excitation at 980 nm.

Figure 5. (A) Representative images of dissected organs of a tumorbearing mouse sacrificed at the 24 h time point after intravenous injection of UCNP-RGD (power ≈ 50 mW/cm2 and temperature ≈ 21.5 °C on the surface of the mouse). 1, U87MG tumor; 2, MCF-7 tumor; 3, spleen; 4, heart; 5, lung; 6, liver; 7, kidney. (B) Biodistribution of particles in organs of tumor-bearing mice sacrificed at 24 h postinjection of UCNP-RGD. Error bars were based on triplet samples.

RGD were selected as an example for confocal Z-scan UCL imaging. As shown in Figures S11 and S12 and the movie in the Supporting Information, the imaging of the spleen slices still measurably highlighted the UCNPs in the deep layer (penetration depth of ∼600 µm), suggesting that confocal UCL imaging had a high penetration depth (∼600 µm). Moreover, quantification of the UCL signal (λem ) 600 ± 50 nm) of the spleen tissue slices at different penetration depths (150, 300, 450, and 600 µm) by line plots revealed an intense UCL signal (counts >4095) in the UCNPs-incorporated domain and zero UCL counts from non-UCNPs-incorporated tissue slices, showing no autofluorescence signal in the UCL image even for high penetration depth. Compared with the values of tissue imaging depths described in the literature, including imaging depths reported for confocal microscopy of ∼80 µm and two-photon microscopy of ∼500 µm,39 the imaging depths achieved by UCL imaging present some significant advantages: (1) UCL imaging can reach an imaging depth of ∼600 µm. If UCL at 800 nm is used (39) Cahalan, M. D.; Parker, I.; Wei, S. H.; Miller, M. J. Nat. Rev. Immunol. 2002, 2, 872–880.

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CONCLUSIONS In conclusion, we have demonstrated a high contrast upconversion luminescence (UCL) imaging protocol for in vitro, ex vivo, and in vivo target-specific imaging of tumors based on the high affinity of the Rvβ3 integrin receptor to the RGD-labeled multicolor upconversion nanophosphors (UCNPs). Multicolor UCNPs tridoped with Yb3+/Tm3+/Er3+ ions whose ratio can be fine-tuned to favor the green, red, or NIR emissions were synthesized. They were further used as a novel kind of biolabel for combined application in confocal UCL microscopy of cells/ tissues and/or whole-body animal imaging with different penetration depth. The results of the tissue slices imaging revealed no autofluorescence signal in UCL imaging even at high penetration depth (∼600 µm). In particular, region of interest (ROI) analysis of the UCL signal in vivo revealed a high signal-to-noise ratio (∼24) between the tumor and the background. The important features of high tissue imaging depth and no autofluorescence signal in UCL imaging cannot be obtained in single-photon or two-photon fluorescence imaging. In combination with the UCL imaging techniques, these high contrast, highly intense, and multicolor UCNPs appear particularly suited for applications in tracking and labeling components of complex biological systems in vitro and in vivo. Our studies here open up new perspectives for cell recognition and targeted imaging-guided cancer diagnosis with the UCL imaging technique. ACKNOWLEDGMENT The authors thank the financial support from NSFC (20825101 and 20775017), NCET-06-0353, and Shanghai Leading Academic Discipline Project (B108). SUPPORTING INFORMATION AVAILABLE Additional figures and a movie. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 11, 2009. Accepted September 28, 2009. AC901960D