Fabrication of Versatile Cyclodextrin-Functionalized Upconversion

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Fabrication of Versatile Cyclodextrin-Functionalized Upconversion Luminescence Nanoplatform for Biomedical Imaging Cheng Ma,† Tong Bian,‡ Sheng Yang,† Changhui Liu,† Tierui Zhang,‡ Jinfeng Yang,§ Yinhui Li,† Jishan Li,*,† Ronghua Yang,† and Weihong Tan† †

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, China ‡ Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China § Tumor Hospital, Xiangya School of Medicine, Central South University, Changsha, Hunan 410013, China S Supporting Information *

ABSTRACT: Lanthanide-based upconversion nanoparticles (UCNPs) are a new type of luminescent tags that show great application potential in biomedical fields. However, a major challenge when applying UCNPs in biomedical research is the lack of a versatile strategy to make water-dispersible and biocompatible UCNPs with high simplicity in functionalization. To address this problem, in the present work, we employed 6phosphate-6-deoxy-β-cyclodextrin (βPCD) as the novel ligand to fabricate a versatile upconversion luminescent nanoplatform. Using βPCD as the surface ligand not only enhances the stability and biocompatibility of the UCNPs under physiological conditions but also enables simple conjugation with various functional molecules, such as organic dyes and biomolecules, via the host−guest interaction between those molecules and the cyclodextrin cavity. The conjugated upconversion nanoprobe then displays excellent capability in labeling the cancer cells and tumor tissue slices for luminescent imaging. These results demonstrate that the versatile cyclodextrin-functionalized upconversion nanoplatform appears particularly flexible for further modifications, indicating great potential for applications in biosensing and bioimaging.

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been hindered, to a large extent, by the lag of advancement in lasers. This problem has been solved since the subpicosecond pulsed lasers became more readily available in the 1990s, though the price for such lasers is usually very high. The other roadblock for the wide application of the two-photon fluorecence imaging technology is the lack of suitable twophoton probes, since most of the readily available fluorescent probes have low two-photon cross sections.8 Photon upconversion, which emerges as an intense antiStokes photoluminescence via a two-photon or multiphoton mechanism,10−13 has been a fascinating technique for photoncontrolled drug release and biomedical imaging.14−19 Different from two-photon fluorescence, the upconversion process is based on sequential (not simultaneous) absorption of photons, which result in metastable excited states of long life times in the trivalent lanthanide cations.10 Besides, upconversion luminescence (UCL) possesses unique properties, such as sharp emission spectra, long lifetimes, and low photodamage.17

ore profound understandings on disease development, progression, and treatment have been achieved in the past decade, owing to the rapid advancement in bioluminescent imaging technologies.1 Imaging agents such as fluorescent dyes, quantum dots, gold nanocrystals, and fluorescent proteins have made tremendous contributions in understanding the origins and mechanisms of physiological processes.2−7 However, these general stokes-shifted fluorescent materials are excited by short wavelengths that would induce large photodamages, high tissue autofluorescence, big photon loss due to self-absorption, and scattering. Thus, these conventional probes only allow low tissue penetration depth and deliver unsatisfactory imaging effects. Recently, the emergence of the two-photon fluorescence imaging technology has stimulated the development of molecular probes with the two-photon absorption capability.8 In comparison with using a single photon of higher energy to excite the fluorophore, the two-photon absorption process converts two simultaneous absorbed photons to one photon of higher energy, providing the advantages of greater penetration depth (>500 μm), lower tissue autofluorescence, and localized excitation.9 Unfortunately, although simultaneous absorption of two photons by the same molecule was first demonstrated experimentally as early as 1961, the progress in this field has © 2014 American Chemical Society

Received: March 19, 2014 Accepted: May 21, 2014 Published: May 21, 2014 6508

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Therefore, various inorganic crystals doped with lanthanide ions have been synthesized to emit strong NIR-to-vis UCL. Among these lanthanide-doped nanocrystals, Yb/Er (or Yb/ Tm) codoped NaYF4 upconversion nanoparticles (UCNPs) have been reported as the most efficient NIR-to-vis UCL material.20,21 To date, there are many methods to directly make hydrophilic upconversion nanoparticles, including the polyol process, a one-pot synthesis assisted by hydrophilic ligands and ionic liquid-based synthesis, etc.22−24 However, it is hard to obtain uniform and hydrophilic UCNPs with small particle dimension by these methods.14 Alternatively, UCNPs synthesized with hydrophobic organic ligands easily control the shape, size, and crystallinity of the particles, but they have rather low solubility in water which restricts their development in the biomedical field.11,25−27 To solve this problem, several strategies via surface modification of hydrophobic UCNPs to transfer them to the hydrophilic phase have been reported, such as ligand exchange, ligand free, ligand oxidation and silanization, etc.28−35 However, to meet the needs of biomedical applications, an ideal method is needed to not only improve the solubility and biocampatibility of UCNPs under physiological conditions but also confer the capacity for further conjugation with expectant biomarkers, ranging from small organic molecules to proteins. Either the modification process or the surface ligand of the UCNPs is crucial for the production of biocompatible UCNPs. Generally, ligand linkers with terminal amino, maleimide, or carboxy groups are used for bioconjugation.28,36,37 However, these functional groups are also quite abundant in many biomolecules, such as folic acid, peptides, and proteins. As a result, the selectivity of bioconjugation is compromised. Alternatively, linkers with terminal azide or alkyne can facilitate the click reaction, one of the most popular reactions in biology sciences because azido and alkyne are hardly present in biomolecules including proteins and oligomers.38,39 However, click reaction needs to maintain a good concentration of Cu(I) and complete exclusion of oxygen throughout the reaction.40 Besides, the introduction of Cu(I) and auxiliary reaction ligands pose potential influence to some proteins, such as metalloenzymes and metalloproteins. Therefore, it is imperative to develop new surface ligands for UCNPs with special terminal groups to convert these particles into useful labeling agents for biomedical studies. β-Cyclodextrin (βCD), as a member of the CD family, is a widely used host molecule capable of internalizing guest molecules in water with binding constants in the 100.5−105 M−1 range.41 βCD and its assemblies have been extensively used for biomedical fields ranging from drug solubilization to being the building blocks for nonviral vector construction because of their high aqueous solubility and low toxicity, as well as their capabilities in destabilizing and permeating biological membranes and in obviating undesirable side effects.42−44 These features have triggered our interest in the combination of CD with UCNPs, which could lead to novel avenues in terms of sensing and labeling. In this work, we described the development of a versatile upconversion luminescent nanoplatform, UCNPs capped with 6-phosphate-6-deoxy-β-cyclodextrins (βPCD/UCNPs) (Scheme 1). Water-dispersible βPCD/UCNPs can be easily recovered from the hydrophobic organic phase through a two-step process, because the phosphonate groups of βPCDs have great affinity to the surface of lanthanide-based nanoparticles and thus displace the carboxylate groups in the oleic acid ligands. Compared to the

Scheme 1. Schematic Illustration of the Fabrication of the Versatile Cyclodextrin-Functionalized Upconversion Nanoplatform for Biomedical Imaging

previously reported methods of functionalizing the hydrophobic UCNPs using CDs,45−47 the peculiar hydrophobic cavities of βPCDs are still available on the surface of βPCD/ UCNPs. Therefore, the βPCDs can further serve as receptors for“arming” UCNPs with a series of functional molecules possessing hydrophobic moieties. Here, we employed the rhodamine B conjugated adamantane (denoted as Ad-RB) and a c(RGDyK) peptide conjugated with adamantane (denoted as Ad-RGD) as the typical models to demonstrate the versatile ability of the prepared βPCD/UCNPs platform in being further conjugated with desired functionalities. Particularly, the perfect spectral overlap between the green emission band of βPCD/ UCNPs and the absorption spectrum of Ad-RB can be used for fabrication of the FRET-based upconversion nanosensors, and the highly selective recognition property of c(RGDyK) to αvβ3 and αvβ5 integrin receptors can be used for actively targeting cells or tumor tissues expressing a high level of these two integrin receptors which promotes angiogenesis, an essential event in proliferation and metastasis of human tumors, regulates adhesion of cancer cells, and participates in the progression of osteoporosis.48 Finally, the βPCD/UCNPs capped with Ad-RGD (Ad-RGD/βPCD/UCNPs) was successfully used for imaging HeLa cells and human cervical cancer tissues.

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EXPERIMENTAL SECTION Chemicals and Instruments. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Solvents used were purified by standard methods prior to use. Twice-distilled water was used throughout all experiments. 2-Azidoethylphosphonic acid, 6propargylamino-6-deoxy-β-cyclodextrin, and rhodamine B conjugated adamantane (denoted as Ad-RB) were synthesized as described in the Supporting Information. MTT, CO-520, 6509

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collected and washed with ethanol three times. The isolated nanoparticles were redispersed in cyclohexane (10 mL) for further experiments. Preparation of βPCD-Coated Upconversion Nanoparticles (βPCD/UCNPs). Ligand Exchange on OA/UCNPs with 2-Azidoethylphosphonic Acid.39 Approximately 30 mg of precipitated OA/UCNPs was added to a 5 mL scintillation vial along with 2-azidoethylphosphonic acid (100 mg) and a mixture of CHCl3 (2 mL) and absolute EtOH (2 mL). The reaction mixture was stirred at room temperature for a minimum of 7 h but typically overnight. Then, hexanes were added to precipitate out the 2-azidoethylphosphonic acid coated UCNPs. The UCNPs were isolated via centrifugation at 2000 rpm. The upper phase was discarded. The precipitated UCNPs were redispersed in DMSO for further experiments. Procedure for Copper(I) Catalyzed Azide−alkyne Cycloaddition of 6-Propargylamino-6-deoxy-β-cyclodextrin (alkyne-cyclodextrin) onto the UCNPs. A 5 mL vial was charged with the azide-coated nanoparticles dispersed in degassed 1:1 DMSO/H2O (3 mL), alkyne-cyclodextrin (6.5 mg, 0.006 mmol), CuSO4·5H2O (0.5 mg, 0.002 mmol), sodium ascorbate (0.8 mg, 0.004 mmol), triethylamine (8 μL). The suspension was stirred under argon atmosphere at room temperature for 48 h. After this time, the mixture was centrifuged at 12 000 rpm for 15 min to isolate the UCNPs. Distilled water was then added to the centrifuge tube and agitated to disperse the UCNPs. The excess ligands and catalysts were removed by dialysis with a 14 kDa membrane against water. Preparation of Ad-RB/βPCD/UCNPs, Ad-polyD/βPCD/ UCNPs, and Ad-RGD/βPCD/UCNPs. For the preparation of Ad-RB/βPCD/UCNPs, excess Ad-RB (0.1 mg) was added into the βPCD/UCNPs solution (1 mg·mL−1, 1 mL) and incubated for 2 h at room temperature to guarantee the full anchoring of Ad-RB on the surface of βPCD/UCNPs. Then, the nanoparticles were centrifugally separated, and the products were collected and washed with aqueous solution three times. The obtained Ad-RB/βPCD/UCNPs complexes were redispersed in aqueous solution for further experiments. The processes in preparation of Ad-polyD/βPCD/UCNPs and Ad-RGD/ βPCD/UCNPs are same with that of Ad-RB/βPCD/UCNPs. Preparation of SiO2-Coated Core/Shell Upconversion Nanoparticles (SiO2/UCNPs).49 In a 5 mL glass vial, 20 μL of ammonia (28%) was injected into the mixture of 115 mg of CO-520, 2.25 mL of cyclohexane, and 37.1 μL of UCNPs/ cyclohexane (ca. 30 mg·mL−1). Keep stirring to obtain a clear solution. Then, 37.5 μL of TEOS was added, and the solution was kept stirring for longer than 48 h at 25 °C. The NPs are collected by adding ethanol and centrifugation at 12 000 rpm for 30 min. The obtained NPs were washed out with ethanol for three times to remove excess CO-520. Preparation of PAA-Coated Upconversion Nanoparticles (PAA/UCNPs).50 A DEG solution (8.0 mL) containing PAA (0.1 g) was heated to 80 °C with vigorous stirring under N2 flow. A cyclohenxane solution of NaYF4 nanocrystals (500 μL, ca. 30 mg·mL−1) was injected to the hot solution which became turbid immediately. The system was heated to 240 °C and kept at this temperature for 1 h until the solution became clear. After the solution was cooled down to room temperature, excess dilute hydrochloric aqueous solution was added, and white NPs were obtained by centrifuging. The NPs were washed three times with pure water. The washed NPs can be well dispersed in water by ionizing the carboxylic groups with a dilute NaOH solution.

ammonia, tetraethoxysilane (TEOS), diethylene glycol (DEG), and poly(acrylic acid) (PAA) were purchased from SigmaAldrich. Rare earth chlorides (LnCl3, Ln: Y, Yb, Er), oleic acid (OA) (>90%), 1-octadecane (ODE) (>90%), and NH4F were purchased from Alfa Aesar. β-Cyclodextrins, (+)-sodium Lascorbate, copper(II) sulfate pentahydrate, NaOH, ethanol, cyclohexane, acetone, and hydrochloric solution were purchased from Sinopharm Chemical Reagent Co. (China). Cyclo(Arg-Gly-Asp-Phe-Lys(mpa)) pepetide (c(RGDyK)) conjugated adamantane (denoted as Ad-RGD; purity: 98.5%), aspartate-riched peptide (GDDDDDDDDC, denoted as polyD; purity: 98.5%), and aspartate-riched peptide conjugated adamantane (Ad-GDDDDDDDDC, denoted as Ad-polyD; purity: 98.5%) were purchased from ChinaPeptides Co., Ltd. (Shanghai, China). The MCF-7 (human breast cancer, low αvβ3 integrin expression) and HeLa (cervical cancer, high integrin αvβ3 expression) cell lines were provided by the Biomedical Engineering Center of Hunan University (China). Cervical cancer tissue slices and normal cervical tissue slices were provided by the Hunan Provincial Tumor Hospital, Central South University (China). The study was approved by the Ethics Committee of Hunan Provincial Tumor Hospital. Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz on an Invoa+400 (Invoa 400) spectrometer. Chemical shifts (δ) are reported in parts per million relative to tetramethylsilane using the residual solvent peak as a reference standard. Coupling constants (J) are reported in hertz. Mass spectra (MS) were obtained on an LCQ/Advantage HPLC +Mass spectrotometer (Thermo Finnigan). Transmission electron microscopy (TEM), high-resolution TEM (HRTEM) images, and the energy-dispersive X-ray analyses (EDXA) were obtained using the TEM microscope (JEOL JEM-3010). Zeta potential and dynamic light scattering (Malvern Zetasizer 3000HS) were employed for characterization of modified processes of UCNPs. UV−vis absorption spectra were obtained on a Hitachi U-4100 UV/vis spectrometer (Kyoto, Japan) using a quartz cuvette having a 1 cm path length. Fluorescence measurements were recorded on a Hitachi F-7000 fluorescence spectrofluorometer. UCL spectra were obtained using an external 0−800 mW adjustable continuous wave laser (980 nm, Beijing Hi-Tech Optoelectronic Co., China) as the excitation source, instead of the Xenon source in the spectrophotometer. FT-IR spectra were acquired on a Bomem (Hartmann & Braun, MB-Series) spectrometer. UCL images of cells and tissues were obtained by using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Japan). The pH values were calibrated with a model 868 pH meter (Orion). Synthesis of OA-Capped NaYF4: 20%Yb, 2%Er Nanoparticles. OA/UCNPs were synthesized following a previously reported procedure.29 YCl3 (0.78 mmol), YbCl3 (0.18 mmol), and ErCl3 (0.02 mmol) were mixed with 6 mL of oleic acid and 15 mL of octadecene (ODE) in a 100 mL three-necked flask. The mixture was stirred magnetically and heated to 160 °C for 30 min to form a transparent solution and then cooled down to room temperature. Ten mL of methanol solution containing NaOH (2.5 mmol) and NH4F (4 mmol) was slowly dropped into the flask and stirred for 30 min at 50 °C and then heated slowly to 70 °C until all the methanol evaporated. Subsequently, the solution was heated to 300 °C for 1 h at argon atmosphere and then cooled down to room temperature. The nanoparticles were precipitated by adding ethanol, and the resultant mixture was centrifugally separated; the products were 6510

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UCNPs) utilized in this work were first studied by transmission electron microscopy (TEM). The TEM images of the nanoparticles show that the synthesized hexagonal OA/ UCNPs disperse well in cyclohexane and have a uniform size of ∼20 nm (Figure 1A). The different shades of gray of the

Cell Culture. 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 HeLa cells were grown in MEM supplemented with 10% FBS. Cultures were maintained at 37 °C under a humidified atmosphere containing 5% CO2. Cytotoxicity Assay. The cellular cytotoxicity was evaluated using the standard cell viability assay, the MTT assay. HeLa cells were seeded into a 96-well plate at a concentration of 4 × 103 cells/well in 100 μL of MEM medium with 10% FBS. Plates were maintained at 37 °C in a 5% CO2 incubator for 48 h. After the original medium was removed, the HeLa cells were incubated with βPCD/UCNPs, SiO2/UCNPs, and PAA/ UCNPs with different concentrations (100, 200, 300, 400, and 500 μg·mL−1) for 48 h. The cells incubated with the culture medium only served as the controls. Then, the cells were washed with PBS for three times, and 100 μL of MTT solution (0.5 mg·mL−1 in PBS) was added to each well for another 3 h. After addition of DMSO (150 μL/well), the assay plate was allowed to shake at room temperature for 10 min. A Tecan microplate reader was used to measure the OD570 (Absorbance value) of each well with the background subtraction at 690 nm. The following formula was used to calculate the viability of cell growth: cell viability (%) = (mean of absorbance value of treatment group/mean of absorbance value of control) × 100. Living Cell Imaging. For cell imaging experiments, cells were seeded in a 24-well culture plate and grown overnight on glass coverslips at the bottom of the plate. When the cells were ∼90% confluent, the coverslips were washed three times with PBS. Then, 50 μL of βPCD/UCNPs or Ad-RGD/βPCD/ UCNPs (1.0 mg·mL−1) was added into each well, followed by incubation for 60 min at 37 °C. For the blocking assay, the cells were first incubated with excess c(RGDyK) (10 μM) for 1 h at 37 °C prior to Ad-RGD/βPCD/UCNPs treatment. Finally, the treated cells were washed three times with PBS to remove unbounded nanoparticles, and UCL imaging of the cells was observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope. The UCL emission was collected at 490−540 nm. A 60× objective lens was used. Imaging of Tissue Slices. The cervical tumor tissues and the normal cervical tissues from a patient were cryosectioned at −20 °C into slices of 8 μM thickness, fixed in cold acetone for 5 min (−20 °C), blocked with 1:10 goat serum for 30 min, stained with 200 μg·mL−1 βPCD/UCNPs or Ad-RGD/βPCD/ UCNPs for 60 min at 37 °C, washed three times with PBS, and examined under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope. The UCL emission was collected at 490−540 nm. A 10× objective lens was used. Deep Tissue Imaging. A 2.0 mm-thick human cervical tumor tissue slice was incubated with 200 μg·mL−1 Ad-RGD/ βPCD/UCNPs in 10% goat serum-containing PBS for 60 min at 37 °C. After washing with PBS to remove the remaining nanoparticles, UCL imaging, Z-scan UCL imaging, and the 3D confocal image accumulated along the Z-direction at a depth of 0−700 μm of this treated tumor tissue were observed under an Olympus FV1000-MPE multiphoton laser scanning confocal microscope. The UCL emission was collected at 490−540 nm. A 10× objective lens was used.

Figure 1. (A) TEM image, (B) HRTEM image, and (C) selected-area electron diffraction (SAED) pattern of the OA/UCNPs dispersed in cyclohexane. (D) TEM image of the βPCD/UCNPs dispersed in water.

UCNPs come from the different diffraction contrast of crystalline materials observed in TEM images. The lattice fringes, with an interplanar distance of 0.51 nm, are shown in the high-resolution transmission electron microscopy (HRTEM) image (Figure 1B), corresponding to the (100) plane of the nanocrystals. The energy-dispersive X-ray analyses (EDXA) result for the hexagonal-phase nanocrystals confirms the presence of F, Y, Yb, and Er in the nanocrystals (Figure S1, Supporting Information). The selected-area electron diffraction (SAED) pattern shows spotty polycrystalline diffraction rings corresponding to the (100), (110), (111), (201), (311), and (321) planes of the hexagonal NaYF4 lattice (Figure 1C).26 Next, in order to obtain water-dispersible βPCD/UCNPs, we explored the use of 2-azidoethylphosphonic acid ligands to replace the original OA ligands on UCNPs based on a ligand exchange process.39 This process occurs through the high affinity between the negatively charged phosphates of the azidoethylphosphonate ligands and surface lanthanide ions of UCNPs. 34,51 On the other hand, the azides of the azidoethylphosphonate ligands provide a convenient way to conjugate the alkyne-functionalized βCDs through a coppercatalyzed cyclo-addition (CuAAC) “click” reaction. A representative TEM image (Figure 1D) of the obtained βPCD/ UCNPs shows that they are well-dispersed in water without change in shape and signs of aggregation. The two steps of the modification process are confirmed by the FT-IR spectrum. A strong absorbance peak at 2106 cm−1 attributed to the azide antisymmetric stretching vibration can clearly be seen in the spectrum of the azidoethylphosphonate ligands-capped UCNPs (Figure S2, Supporting Information). In addition, strong absorption bands at 1250 to 990 cm−1 are also found in the

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RESULTS AND DISCUSSION Preparation and Characterization of the βPCD/UCNPs Platform. OA-capped (β-NaYF4:Yb3+/Er3+) UCNPs (OA/ 6511

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agglomeration (Figure S3, Supporting Information) and photostability (Figure S4, Supporting Information), and the dynamic light scattering (DLS) measurements display no obvious difference (Figure S5, Supporting Information) in physiological buffer conditions over a wide range of pH (4.0− 8.0) and serum supplemented cell growth media over 48 h at 37 °C, indicating great potential applications in various physiological conditions. Conjugation between Organic Dyes/Biomolecules and βPCD/UCNPs. βPCDs on the surface of UCNPs not only afford an enhanced stability and biocompatibility for UCNPs in the physiological environment but also, more importantly, give the capability of further conjugation with various functional receptors via the host−guest interaction. To verify availability of the hydrophobic cavities of βPCDs on the surface of the prepared βPCD/UCNPs, Ad-RB was employed as a typical model based on the perfect spectra overlap between the green emission band of βPCD/UCNPs and the absorption spectrum of Ad-RB (Figure S6, Supporting Information). After incubating βPCD/UCNPs with Ad-RB in the buffer solution and then illuminating the obtained Ad-RB/βPCD/UCNPs complexes with a 980 nm laser, a broad emission band at 590 nm appears, which can be attributed to the emission of Ad-RB. The signal intensities of the green emissions at 520 and 540 nm decrease significantly while the red emission band at 650 nm remains largely unchanged (Figure 3A), thus indicating energy transfer from the green emission of UCNPs to Ad-RB. On the contrary, no substantial change of luminescence spectra is observed in the case of incubating βPCD/UCNPs with the same concentration of the original RB dyes without adamantane. The effective energy transfer implies that the resulting assembly brings the Ad-RB (energy acceptor) and the βPCD/UCNPs (energy donor) into close proximity via the host−guest interaction. These facts demonstrate the availability of hydrophobic cavities of βPCDs immobilized on the surface of UCNPs and the capability of this cyclodextrin-functionalized upconversion nanoplatform to conjugate with organic dyes. If the binding stoichiometry between βPCDs and Ad-RB is 1:1, by using the intensity of the absorption bands in the UV/vis absorption spectra corresponding to the free Ad-RB and the hybrid complex Ad-RB/βPCD/UCNPs and the results from the particle-size analysis of the decorated βPCD/UCNPs, the surface density of βPCDs can be estimated to be 390 βPCD molecules per UCNP according to a previous method (Figure

spectrum, associated with the P−O and P−O−M stretches of the phosphonate group, respectively. However, these features are completely absent in the case of the original OA/UCNPs. After the “click” reaction, the azide antisymmetric stretching vibration at 2106 cm−1 shows a clear decline when compared with the azide-functionalized UCNPs along with the appearance of a peak at 1554 cm−1, corresponding to the 1,2,3-triazole ring system. The absorption bands attributed to the phosphonate group (1250−990 cm−1) are also still present in the spectrum. On the basis of the above-described FT-IR results, it can be deduced that the βPCDs have been successfully immobilized on the surface of UCNPs and the βPCD/UCNPs were obtained successfully. The prepared βPCD/UCNPs colloids possess excellent water solubility and form a very stable solution without precipitation. Under the continuous-wave excitation at 980 nm, the luminescence appears predominantly green in color due to green emissions from the doped Er3+ ion in the UCNPs (inset of Figure 2). The upconversion emissions around 520, 540, and

Figure 2. Room-temperature upconversion luminescence spectrum of βPCD/UCNPs in water under excitation at 980 nm. Inset: photographs of the transparent solution of βPCD/UCNPs in water without laser illumination (left) and under 980 nm laser illumination (right).

650 nm in the corresponding upconversion spectrum of βPCD/UCNPs in water are the result of transitions from 2 H11/2, 4S3/2, and 4F9/2 to 4I15/2 of Er3+, respectively (Figure 2). In addition, the aqueous solution of βPCD/UCNPs exhibits good colloid stability with no discernible settling or

Figure 3. (A) Upconversion luminescence spectra of (a) βPCD/UCNPs and (b) Ad-RB/βPCD/UCNPs and (c) βPCD/UCNPs with RB without adamantane in water upon 980 nm excitation. Inset: the schematic illustration of the conjugation upconversion nanoplatform with Ad-RB. (B) The zeta potential of (a) βPCD/UCNPs, (b) Ad-polyD/βPCD/UCNPs, and (c) βPCD/UCNPs with polyD peptide chain without adamantane in water. Inset: the schematic illustration of the conjugation upconversion nanoplatform with Ad-polyD. 6512

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S7, Supporting Information).52 Then, to further expand the versatility of the upconversion nanoplatform, the aspartateenriched peptide chain conjugated adamantane (denoted as AdpolyD) is employed to be incubated with βPCD/UCNPs in an aqueous solution. With the conjugation of Ad-polyD via host− guest interaction to form Ad-polyD/βPCD/UCNPs complexes, the zeta potential of the nanoparticles changes from +29.8 mV of the βPCD/UCNPs to −26.0 mV of the Ad-polyD/βPCD/ UCNPs owing to the strong electronegativity of Ad-polyD in aqueous solutions. On the contrary, no substantial change (+20.3 mV) is observed in the case of incubating βPCD/ UCNPs with the same concentration of polyD peptide chain without adamantane (Figure 3B). These results further indicate the availability of hydrophobic cavities of βPCDs immobilized on the surface of UCNPs and the capability of this cyclodextrinfunctionalized upconversion nanoplatform to conjugate with biomolecules. Cytotoxicity Assays with the βPCD/UCNPs. For practical biomedical applications, a nanostructured probe should not interfere with the metabolism of the living system. Therefore, cytotoxicity of the βPCD/UCNPs toward HeLa cells as the model is evaluated using the standard cell viability assay, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.37 Here, we compared the biological toxicity of the βPCD/UCNPs with other two kinds of UCNPs using different coatings, namely, UCNPs coated by silicon dioxide (SiO2/UCNPs) (Figure S8, Supporting Information) and UCNPs coated by poly(acrylic acid) (PAA/UCNPs) (Figure S9, Supporting Information), respectively. After being cultured by the three kinds of UCNPs for 48 h, the cellular viability displays that the βPCD/UCNPs possesses a slightly higher cell viability compared with the others although the other two particles also show good biocompatibility.53 In addition, no significant difference in the proliferation of the HeLa cells is observed in the absence or presence of βPCD/ UCNPs even when the concentration of βPCD/UCNPs is increased up to 500 μg/mL (Figure S10, Supporting Information). The high biocompatibility of βCD and the efficient shielding of UCNPs by the βCD-based coating may be the causes of the considerably low cytotoxicity of βPCD/ UCNPs to cells. The MTT results show that βPCD/UCNPs have no obvious cytotoxic effect on the living system, indicating a great prospect for biomedical applications. βPCD/UCNPs for Imaging Diagnosis of Cervical Cancer. To demonstrate the practical applications of this prepared luminescent βPCD/UCNPs platform in biomedical imaging and clinical diagnostics, Ad-RGD peptide was employed to anchor on the surface of βPCD/UCNPs to form upconversion luminescent bionanoprobes (Ad-RGD/ βPCD/UCNPs) by the βPCD/Ad host−guest inclusion strategy. This strategy allows the exposed βPCD cavities on the surface of the βPCD/UCNPs platform to conjugate with the adamantane unit of Ad-RGD peptide. The cyclic RGD peptide selectively recognizes αvβ3 and αvβ5 integrin receptors, which play pivotal roles in angiogenesis, vascular intima thickening, and proliferation of malignant tumors.48,54−56 After conjugation of βPCD/UCNPs with Ad-RGD peptide, the zeta potential of the nanoprobes changes from +32 mV of βPCD/UCNPs to +8 mV of Ad-RGD/βPCD/UCNPs. The dynamic light scattering (DLS) analysis also indicates that the diameter of the obtained nanoprobes changed from ∼81 nm of βPCD/UCNPs to ∼118 nm of Ad-RGD/βPCD/UCNPs with excellent dispersion (Figure S11, Supporting Information). The

decrease in zeta potential and increase in hydrodynamic diameter are attributed to the anchoring of Ad-RGD on the surface of βPCD/UCNPs, indicating the successful conjugation of the cyclic RGD peptide with βPCD/UCNPs. Before the imaging diagnosis application, specificity of the Ad-RGD/βPCD/UCNPs probe should be first examined carefully. HeLa cells possessing αvβ3 and αvβ5 integrins and integrin-negative cells (MCF-7) were chosen as model cell lines to investigate the targeting effect. It can be seen clearly from Figure 4A that Ad-RGD/βPCD/UCNPs probe does not bind

Figure 4. (A) Confocal upconversion luminescence images of HeLa cells incubated with (a) Ad-RGD/βPCD/UCNPs and (b) βPCD/ UCNPs. Images of MCF-7 cells incubated with (c) Ad-RGD/βPCD/ UCNPs and (d) βPCD/UCNPs. (e) HeLa cells incubated with AdRGD/βPCD/UCNPs in the presence of unlabeled c(RGDyK). Scale bar is 5 μm. (B) Frozen cervical cancer tumor tissue stained by (a) AdRGD/βPCD/UCNPs and (b) βPCD/UCNPs. Frozen normal cervical tissues stained by (c) Ad-RGD/βPCD/UCNPs and (d) βPCD/ UCNPs. The tissue slices were blocked with 1:10 goat serum for 30 min before staining. Scale bar is 50 μm.

to integrin-negative cells (MCF-7) after a 60 min incubation because there is minimal luminescence signal observed, whereas the integrin-positive cells (HeLa) are clearly visualized and the staining can be blocked effectively by 10 μM cyclic RGD peptides. Additionally, the unconjugated βPCD/UCNPs do not exhibit any significant binding to any of the two cell lines under the same incubation time of 60 min. These results show that the cyclic RGD peptide can selectively bind to αvβ3 and αvβ5 integrins with high affinity, enabling the UCNPs to target integrin αvβ3/αvβ5-rich tumor cells specifically. Next, the AdRGD/βPCD/UCNPs probe is used for imaging diagnosis of human cervical cancer. Figure 4B shows the Ad-RGD/βPCD/ UCNPs or βPCD/UCNPs staining of frozen slices of the cervical tumor tissues and the normal cervical tissues, respectively. Remarkably, an intense luminescence signal is observed for the cervical tumor tissue slice stained with AdRGD/βPCD/UCNPs, while the normal cervical tissue slice shows virtually no luminescence signal under the same condition. Moreover, no obvious luminescence is noted for both the cervical tumor tissue slice and the normal cervical tissue slice stained with βPCD/UCNPs. The above confocal images clearly demonstrate the high integrins expression level 6513

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of the cervical cancer, and Ad-RGD/βPCD/UCNPs may be a potential tool for clinical analyses of biopsies and microbiopsies. Ad-RGD/βPCD/UCNPs for Deep Tissue Imaging of Cervical Cancer. As discussed earlier, NIR light is particularly useful for deep tissue imaging owing to its increased penetration depth and the capability of three-dimensional (3D) imaging with minimum interference from background emissions. Therefore, we further investigated the utility of this Ad-RGD/βPCD/UCNPs probe in a fresh thick cervical tumor tissue slice. UCL images were obtained from a 2.0 mm-thick tumor slice incubated with 200 μg·mL−1 Ad-RGD/βPCD/ UCNPs probe for 60 min at 37 °C. The bright field image of a part of a cervical tumor tissue slice reveals the complex surface morphology (Figure 5A). It is easy to distinguish an intense

Article

ASSOCIATED CONTENT

S Supporting Information *

Experimental details including synthesis of the 2-azidoethylphosphonic acid, 6-propargylamino-6-deoxy-β-cyclodextrin, and rhodamine B conjugated adamantane and their characterizations, as well as additional information as noted in text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-731-88822587. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (21135001, 21205143, 91127005, and 21305036), the Program for New Century Excellent Talents in University (NCET-13-0188), the “‘973’”National Key Basic Research Program (2011CB91100-0).

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Figure 5. (A) The bright field image, (B) UCL image, and (C) overlay image of a part of a fresh thick cervical tumor tissue slice stained by Ad-RGD/βPCD/UCNPs. (D) The 3D reconstitution of confocal XYZ scanning micrographs from 50 confocal Z-scan UCL imaging sections. (E) The confocal Z-scan UCL imaging sections at different penetration depths. Scale bar is 100 μm.

UCL signal in the UCNPs-incorporated tumor cells domain from the negligible UCL signal in the normal cells domain (Figure 5B,C). Besides, the Z-scanning confocal imaging shows that clear luminescence emissions are still present until the 700 μm of penetration depth, indicating that the prepared NPs have the excellent capacity of depth tissue imaging (Figure 5E). Then, the 3D reconstitution of confocal XYZ scanning micrographs is obtained from 50 confocal Z-scan UCL imaging sections (Figure 5D).

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REFERENCES

CONCLUSION

In summary, we have developed a unique and versatile upconversion luminescent nanoplatform by using βPCDs as the surface ligands. Initially, the βPCDs on the surface of UCNPs can dramatically improve the solubility, stability, and biocompatibility of UCNPs in the physiological environment. Additionally, the peculiar hydrophobic cavities of βPCDs on the surface of UCNPs can further serve as vehicles capable of “arming” UCNPs with a series of functional receptors, such as organic dyes and biomolecules, by host−guest interactions. Finally, the novel and general upconversion platform also affords active-targeting cancer cells and tumor slices imaging. The described host−guest approach of this nanoplatform for the rapid modular assembly of a potential nanosensor or nanotherapeutic will expand the scope toward construction of multifunctional nanoassembies and wider applications for UCNPs. 6514

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