Gadolinium Oxide Nanoparticles and Aptamer-Functionalized Silver

Oct 22, 2014 - Department of Radiology, Affiliated Hospital of Xuzhou Medical College, ... School of Medical Imaging, Xuzhou Medical College, Xuzhou, ...
3 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Gadolinium Oxide Nanoparticles and Aptamer-Functionalized Silver Nanoclusters-Based Multimodal Molecular Imaging Nanoprobe for Optical/Magnetic Resonance Cancer Cell Imaging Jingjing Li,†,‡ Jia You,† Yue Dai,† Meilin Shi,‡ Cuiping Han,†,‡ and Kai Xu*,†,‡ †

Department of Radiology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu 221006, China School of Medical Imaging, Xuzhou Medical College, Xuzhou, Jiangsu 221004, China

‡

S Supporting Information *

ABSTRACT: Multimodal molecular imaging has attracted more and more interest from researchers due to its combination of the strengths of each imaging modality. The development of specific and multifunctional molecular imaging probes is the key for this method. In this study, we fabricated an optical/magnetic resonance (MR) dual-modality molecular imaging nanoprobe, polyethylene glycol-coated ultrasmall gadolinium oxide (PEGGd2O3)/aptamer-Ag nanoclusters (NCs), for tracking cancer cells. To achieve this aim, PEG-Gd2O3 nanoparticles (NPs) as magnetic resonance imaging (MRI) contrast agent and aptamer functionalized silver nanoclusters (aptamer-Ag NCs) as fluorescence reporter were first synthesized by a one-pot approach, respectively. They were then conjugated by the covalent coupling reaction between the carboxyl group on the surface of PEG-Gd2O3 NPs and amino group modified on the 5′end of AS1411 aptamer. With a suitable ratio, the fluorescence intensity of aptamer-Ag NCs and MR signal of PEG-Gd2O3 nanoparticles could both be enhanced after the formation of PEG-Gd2O3/aptamer-Ag NCs nanoprobe, which favored their application for multimodal molecular imaging. With this nanoprobe, MCF-7 tumor cells could be specifically tracked by both fluorescence imaging and magnetic resonance imaging in vitro.

W

MRI and optical imaging. MR imaging can offer high spatial resolution and the capacity to simultaneously obtain physiological and anatomical information, whereas optical imaging allows for high sensitivity. The signal for MRI is produced either by T1 or T2 contrast agents. Iron oxide NPs as T2 contrast agents have been involved in the fabrication of the MR/optical imaging probe.6 However, one remaining issue should be noted: due to the huge light absorption cross-section of IO, the fluorescence energy transfer between IO NPs and optical signals significantly decreased the fluorescence signal. To solve this problem, silica was usually introduced to enlarge the distance between IO and fluorescence materials to preserve the optical signal as much as possible.7 To further explore a simpler and easier approach for the preparation of the MR/ optical imaging probe, gadolinium(III) as T1 contrast agent has gradually attracted more and more attention. Paramagnetic Gd (III)-chelate is prevailing in clinical use. Yan and co-workers proposed a multimodal imaging probe by simply conjugating Gd-DTPA with NIR-emitting persistent luminescent nanoparticles (PLNPs) in the presence of 3-aminopropyltriethox-

ith the requirement of noninvasive imaging of cancer to present both the tumor anatomical structure information and its metabolism as well as biochemistry information, multimodal molecular imaging has been more developed in recent years.1 Current clinical imaging modalities commonly include nuclear imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), conventional radiological modalities such as magnetic resonance imaging (MRI), computed tomography (CT), and ultrasonography (USG), as well as optical imaging. However, each imaging modality has its own pros and cons. The integration of them can provide more structural and functional information, which can not be realized only by one imaging modality independently.2,3 Nowadays, the achievement of multimodal molecular imaging mainly relies on two aspects: hardware- or software-based image fusion and the development of multifunctional molecular imaging probes. The latter has attracted a lot of attention from the researchers due to its easy achievement. Especially, with the innovations in chemistry and nanotechnology,4,5 inorganic nanoparticles exhibiting intrinsic imaging abilities have been integrated and used as multimodal molecular imaging probes, for example, iron oxide nanoparticles (IO NPs) for MRI, gold NPs for CT, and quantum dots (QDs) for optical imaging. Among them, most popularly reported multimodal probes are combining © 2014 American Chemical Society

Received: August 14, 2014 Accepted: October 22, 2014 Published: October 22, 2014 11306

dx.doi.org/10.1021/ac503026d | Anal. Chem. 2014, 86, 11306−11311

Analytical Chemistry

Article

(Optima 5300DV, PerkinElmer, USA). Fluorescence imaging was conducted with a confocal microscope (TCS SP5, Leica, Germany). MRI scanning was performed on 3.0 T human magnetic resonance scanner (Signa, USA). Cells and Cell Culture Conditions. Human breast adenocarcinoma cell line (MCF-7) and NIH-3T3 mouse fibroblast cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and seeded in RPMI 1640 and HDMEM medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco, Grand Island, NY), penicillin (100 μg/mL), and streptomycin (100 μg/mL) with incubation under 5% CO2, 37 °C. Preparation and Characterization of PEG-Gd 2 O 3 Nanoparticles. Polyethylene glycol-coated ultrasmall gadolinium oxide nanoparticles (PEG-Gd2O3 NPs) were prepared according to the literature with some modification.15 0.4062 g of Gd(NO3)·6H2O (99.9%, Sinopharm Chemical Reagent Co., Ltd.) was dissolved in 10 mL of poly(ethylene glycol) bis(carboxymethyl) ether 600 (Sigma-Aldrich), which served as both solvent and particle surfactant. The solution was heated to 90−100 °C and then to 140 °C for 1 h and to 180 °C for 4 h. After being cooled to room temperature, the solution was dialyzed against ultrapure water (1:1000, v/v) to remove free Gd3+ and excess PEG. The membrane pore size was 3500 MW. The size distribution of PEG-Gd2O3 NPs was assessed by high-resolution transmission electron microscopy (JEOL JEM 200CX, Japan). The sample was prepared by depositing a drop of PEG-Gd2O3 colloidal solution onto a copper grid and allowing the liquid to dry in air at room temperature. The hydrodynamic diameter of dialyzed PEG-Gd2O3 NPs was measured by dynamic light scattering (NiComp380ZLS, USA). The hydrodynamic diameter was calculated from the average of three measurements. In order to further confirm the grafting of PEG, the FT-IR spectrum was introduced. PEG-Gd 2O3 colloidal solution was precipitated by ethanol and then dried in a vacuum oven at 40 °C for 2 days. The obtained sample was mixed with KBr for FT-IR scanning. Inductively Coupled Plasma-Mass Spectrometry (ICPMS) Analysis. The concentration of gadolinium in PEGGd2O3 NPs was determined by ICPMS analysis (Optima 5300DV, PerkinElmer, USA). 500 μL of 10× concentrated PEG-Gd2O3 nanoparticles was mixed with 500 μL of 14 M HNO3 and 25 mL of 15% HNO3. After being heated for 30 min at 80 °C, 3 mL of the above solution was diluted with 22 mL of ultrapure water for ICPMS analysis. The sample preparation of PEG-Gd2O3/aptamer-Ag NCs nanoprobes for ICPMS analysis was similar to PEG-Gd2O3 NPs. Preparation of Aptamer-Ag NCs. Aptamer-Ag NCs were synthesized according to the procedure in our previous report.16 Briefly, 30 μL of 250 μM NC-AS1411-L5T was mixed with 24 μL of 20 mM phosphate (pH 7.0) buffer, and then, 16 μL of 10 mM AgNO3 solution was added to reach a nucleobase to Ag+ molar ratio of 2:1. After being chilled on ice for 15 min, the mixture was reduced by quickly adding 80 μL of 2 mM NaBH4, followed by vigorous shaking for 1 min. The reaction was kept at 4 °C for at least 5 h before use. Fabrication and Characterization of Multimodal Molecular Imaging Nanoprobe, PEG-Gd2O3/AptamerAg NCs. PEG-Gd2O3 nanoparticles were conjugated with aptamer-Ag NCs to obtain the specific multimodal molecular imaging probe through a covalent link between the carboxyl group (−COOH) of PEG and the amino group (−NH2) modified on the 5′-end of DNA scaffold. Briefly, 200 μL of 1.2

ysilane (APTES). The obtained Gd (III)-PLNPs multimodal nanoprobe displayed potential for MRI/optical imaging in vivo.8 Gd-DTPA-phospholipid stabilized QD,9 Gd-DTTA-mesoporous silica nanorod-FITC,10 and Gd-DTTA-mesoporous silica nanoparticle-RITC11 as multimodal imaging probes have also been developed. However, Gd(III)-chelates which were prepared to reduce the toxicity of Gd3+ displayed lower relaxivity than Gd3+. Furthermore, the short blood circulation time and nonspecific biodistribution also hampered their wider applications.12 Thus, researchers developed high-Gd density paramagnetic ultrasmall gadolinium oxide nanoparticles (Gd2O3 NPs) to improve these shortcomings.13,14 Gd2O3 NPs not only display higher relaxivities than commercial Gdchelates but also could more easily connect with other imaging agents. On the basis of these, we hypothesize that Gd2O3 NPs as MRI contrast agent would possess a bright promise in multimodal molecular imaging. Herein, we prepared a MR/ optical imaging probe simply by the conjugation of PEG-Gd2O3 NPs with aptamer-functionalized Ag nanoclusters (NCs) via the EDC and NHS coupling reaction. Aptamer-functionalized Ag NCs which were synthesized by a one-pot approach not only possess excellent fluorescence properties but also preserve the specific targeting ability of aptamer, which further simplified the procedure for the production of the multimodal molecular imaging probe. Unexpectedly, with a suitable ratio between PEG-Gd2O3 NPs and aptamer-Ag NCs, the fluorescence emissions of Ag NCs and the MRI signal of PEG-Gd2O3 were both enhanced after the formation of the nanoprobe, which both favored the application of PEG-Gd2O3/aptamer-Ag NCs as a multimodal molecular imaging probe. MCF-7 human breast tumor cells could be specifically tracked by fluorescence imaging and MR imaging in vitro.

■

EXPERIMENTAL SECTION Materials and Reagents. Gadolinium(III) nitrate hexahydrate, silver nitrate (99+%), sodium borohydride (NaBH4, powder, 98%), disodium hydrogen phosphate, sodium chloride, and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Poly(ethylene glycol) bis(carboxymethyl) ether 600 was obtained from SigmaAldrich. Cell cultures including HDMEM, RPMI 1640, and fetal calf serum were obtained from Kangmei Biotechnology Co., Ltd. (Xuzhou, China). DNA oligos were synthesized and purified by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). All chemicals involved in this work were analytical grade. All aqueous solutions were prepared with ultrapure water (≥18MΩ, Milli-Q, Millipore). The DNA sequence was listed as follows.

Apparatus and Characterization. Fluorescence measurements were carried out using a LS-45/55 Fluorescence/ Phosphorescence Spectrometer (PerkinElmer, USA). The excitation/emission wavelengths were set at 585/645 nm. FTIR spectra were obtained from the infrared absorption spectroscopy (Bruker, Germany). The size and morphology of PEG-Gd2O3 NPs and PEG-Gd2O3/aptamer-Ag NCs nanoprobes were observed by the high-resolution transmission electron microscope (HRTEM) (JEOL JEM 200CX, Japan) and dynamic light scattering (DLS) (NiComp380ZLS, USA). The determination of gadolinium content was performed with inductively coupled plasma-mass spectrometry (ICPMS) 11307

dx.doi.org/10.1021/ac503026d | Anal. Chem. 2014, 86, 11306−11311

Analytical Chemistry

Article

washed three times with PBS. Then, cells were fixed for 20 min in 200 μL of 3.7% paraformaldehyde, and nucleus staining was performed with 4′6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were further washed for three times with PBS buffer for confocal laser scanning after 10 min. MR Imaging of Cell Pellets. MCF-7 cells (5 × 104 cells) were seeded in each well of the 6-well plate for 12 h. After incubation with PBS, PEG-Gd2O3 NPs, or PEG-Gd2O3/ aptamer-Ag NCs nanoprobes for 1 h, the labeled cells were washed with PBS and trypsinized, centrifuged, and dispersed in 300 μL of 3.7% paraformaldehyde for coronal MRI scanning. The scanning parameters were the same as those set in the relaxivity calculation part except for the different slice thickness (1.0 mm) and spacing (0.5 mm). NIH-3T3 cells were treated the same with MCF-7 cells as the control. For quantitative determination of MCF-7 cells, the PEGGd2O3/aptamer-Ag NCs nanoprobe was treated with different amounts of MCF-7 cells. After being washed with PBS, the labled cells were dispersed in 300 μL of 1% agarose for axital MRI scanning. The scanning parameters were set as the same in the relaxivity calculation part.

μM PEG-Gd2O3 nanoparticles was mixed with 1 mg of EDC for 15 min at 37 °C to activate the carboxyl group. Then, 32 μL of 37.5 μM aptamer-Ag NCs and 1 mg of NHS were added into the reaction system for a 2 h incubation. Finally, PEG-Gd2O3/ aptamer-Ag NCs conjugates were obtained by ultracentrifugation to remove unreated reagents at 10 000 rpm for 30 min, and the final volume was 200 μL. The obtained PEG-Gd2O3/ aptamer-Ag NCs nanoprobes were stored at 4 °C before use. For confirmation of such successful conjugation, changes in the fluorescence spectrum of aptamer-Ag NCs, changes in FTIR spectroscopy, and MRI signal of PEG-Gd2O3 were studied, respectively. HRTEM and DLS characterization of PEGGd2O3/aptamer-Ag NCs were performed similarly to that of PEG-Gd2O3 NPs. Relaxivity Calculation of PEG-Gd2O3 Nanoparticles, Gd-DTPA, and PEG-Gd2O3/Aptamer-Ag NCs Nanoprobe. The MRI behavior test of PEG-Gd2O3 was performed with a 3.0 T human magnetic resonance scanner (Signa, USA). Various concentrations of PEG-Gd2O3 solution were prepared before MRI scanning, which varied from 1.5 μM to 0.3 mM with a volume of 600 μL. The following parameters were adopted in data acquisition. ① T1 weighted images: echo time (TE) = 16.5 ms, repetition time (TR) = 425 ms, field of view (FOV) = 14 cm × 14 cm, matrix = 384 × 256, slice thickness = 2.0 mm, and spacing = 1.5 mm; ② T1 map images: TE = 7.4 ms, TR = 200−800 ms, FOV = 14 cm × 14 cm, matrix = 384 × 256, slice thickness = 2.0 mm, and spacing = 1.5 mm. Quantitative T1 relaxation maps were reconstructed from data sets using function software at a workstation (ADW 4.2). The signal intensity of the samples was measured, and the T1 values were calculated accordingly. MRI scannings of Gd-DTPA and PEG-Gd2O3/aptamer-Ag NCs nanoprobe with different amounts were carried out in the same way. The relaxivity values of PEG-Gd2O3, Gd-DTPA, and PEG-Gd2O3/aptamerAg NCs nanoprobe were determined by measuring longitudinal proton relaxation time (T1) as a function of Gd concentration. MTT Assay. MCF-7 cells or NIH-3T3 cells (1 × 104) were first seeded in each well of a 96-well plate for 24 h, respectively. After incubation with PEG-Gd2O3 nanoparticles or PEGGd2O3/aptamer-Ag NCs nanoprobes of various concentrations for 24 h, cells were washed twice with PBS buffer before the addition of RPMI 1640 or DMEM containing MTT (5 mg/ mL) and further incubated at 5% CO2, 37 °C for another 4 h. Then, the medium containing MTT was replaced by 100 μL of dimethyl sulfoxide (DMSO) to solubilize the formazan crystals. The absorbance for each sample was determined by a microplate reader (Multiskon MK3, USA) at 490 nm. Confocal Laser Microscopy Assay. The specific optical imaging of PEG-Gd2O3/aptamer-Ag NCs multimodal molecular imaging nanoprobe to MCF-7 cells was examined with confocal laser microscopy by the fluorescence emissions of aptamer-Ag NCs. MCF-7 cells (5 × 104 cells) were seeded in special Petri dish. After 24 h, cells were incubated with PEGGd2O3/aptamer-Ag NCs conjugates at 5% CO2, 37 °C for 1 h. The cells were then washed with PBS for three times and placed above a 20× objective on the confocal microscope. The aptamer-Ag NCs were excited with 543 nm. NIH-3T3 cells were cultured in a special Petri dish and treated with the same concentration of PEG-Gd2O3/aptamer-Ag NCs conjugates as the control. For nucleus staining, MCF-7 and NIH-3T3 cells were seeded in a specific Petri dish, respectively. After 24 h, cells were exposed to PEG-Gd2O3/aptamer-Ag NCs for 1 h at 37 °C and

■

RESULTS AND DISCUSSION Characterization of PEG-Gd2O3 Nanoparticles. PEGGd2O3 nanoparticles were prepared with a polyol-like method, where poly(ethylene glycol) bis (carboxymethyl) ether 600 was employed as both a solvent and surfactant. The HRTEM image in Figure 1a revealed that PEG-Gd2O3 nanoparticles were

Figure 1. Characterization of PEG-Gd2O3 nanoparticles. (a) HRTEM image of PEG-Gd2O3 NPs and the distribution of particle size. (b) Dynamic light scattering (DLS) profiles of PEG-Gd2O3 NPs. (c) FTIR spectrum of PEG-Gd2O3 NPs.

monodispersed, and the average size in the distribution was 1.3 nm. The lattice fringe of d222 = 0.31 nm in HRTEM micrographs is in agreement with that of 3.13 Å of cubic Gd2O3.17,18 The hydrodynamic diameter (DLS) was measured to be around 20 nm (Figure 1b). Such an increase might come from the hydration corona formed by the PEG coating around the nanoparticles.15 The coating of nanoparticles with PEG was further characterized by the FT-IR absorption spectrum. The PEG backbone chain gives rise to numerous peaks in the fingerprint area. As shown in Figure 1c, the observed absorption frequencies characteristic of PEG(COOH)2 in11308

dx.doi.org/10.1021/ac503026d | Anal. Chem. 2014, 86, 11306−11311

Analytical Chemistry

Article

cluded the CO stretch at 1616 cm−1, the PEG chain C−O− C vibrations at 1100 cm−1, and the C−H stretch at 2867 cm−1.17 Peaks at around 1451, 1349, and 1250 cm−1 were arising from scissoring, wagging, and twisting CH2 vibrational modes.19 The successful grafting of PEG on the nanoparticle provided the basis for the following fabrication of the multimodal molecular imaging probe. Relaxivity Calculation of PEG-Gd2O3 Nanoparticles. Gd-related nanoparticles were reported to possess higher relaxivity compared with commercial MR imaging contrast agent Gd-DTPA, which was the most important property for their applications as contrast agent in disease diagnosis.11 Thus, the relaxivity values of PEG-Gd2O3 nanoparticles and GdDTPA were determined and compared by measuring longitudinal proton relaxation time (T1) as a function of Gd concentration. The Gd concentration in the dialyzed PEGGd2O3 was detected to be 0.3 mM with ICPMS. As shown in Figure 2, the r1 value of the obtained PEG-Gd2O3 nanoparticles

Figure 3. Interaction effect between PEG-Gd2O3 NPs and aptamer-Ag NCs. (a) Fluorescence spectrum of aptamer-Ag NCs before and after the conjugation with PEG-Gd2O3 NPS. (b) T1 weighted and T1 map MR images of PEG-Gd2O3 NPs, aptamer-Ag NCs, and a PEG-Gd2O3/ aptamer-Ag NCs nanoprobe. (c) FT-IR characterization of the formation of PEG-Gd2O3/aptamer-Ag NCs nanoprobes.

the aptamer-Ag NCs.11 To testify our hypothesis, the fluorescence influence of Gd3+ on aptamer-Ag NCs was studied, but no fluorescence changes of aptamer-Ag NCs could be observed in the presence of Gd3+ (Figure S1b in the Supporting Information). The reason is still unclear to us now. The 5 nm red-shift emission of aptamer-Ag NCs after the nanoprobe formation might come from their conjugation with PEG-Gd2O3 NPs, which resulted in a slight difference in the environment around aptamer-Ag NCs.20−23 Additionally, compared with PEG-Gd2O3 nanoparticles alone, PEG-Gd2O3/ aptamer-Ag NCs nanoprobe showed a stronger MR signal, which might come from the larger molecular weight of aptamer-Ag NCs attaching to PEG-Gd2O3 nanoparticles (Figure 3b). It was reported that larger molecules with higher rotational correlation time had higher relaxivity under MRI scanning.24 T1 relaxation times for PEG-Gd2O3 NPs, aptamerAg NCs, and PEG-Gd2O3/aptamer-Ag NCs nanoprobes were 326, 1979, and 146 ms, respectively, and the relaxivity value of PEG-Gd2O3/aptamer-Ag NCs nanoprobes was increased to be 46.4 s−1 mM−1 Gd (Figure S2 in the Supporting Information). The successful conjugation was further confirmed by the emerging absorption bands of acylamide vibration and carboxyl stretching vibration at 1641 and 1710 cm−1, respectively (Figure 3c).25 By HRTEM characterization, the average size of PEG-Gd2O3/aptamer-Ag NCs nanoprobes was 3.2 nm and DLS was about 41.9 nm (Figure S3 in the Supporting Information). Furthermore, the good fluorescence stability of aptamer-Ag NCs and MR signal stability of PEG-Gd2O3 NPs in PEG-Gd2O3/aptamer-Ag NCs nanoprobes (Figure S4 in the Supporting Information) also indicated their promising potentials in multimodal molecular imaging applications. MTT Assay. The biocompatibility of the nanoprobes is an important point of concern and should be evaluated before their applications both in vitro and in vivo. With concerns for this issue, we examined the cytotoxicity of PEG-Gd2O3 nanoparticles and PEG-Gd2O3/aptamer-Ag NCs nanoprobes using the MTT ((3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and looked for the potentially safe concentrations for the following targeting experiments. MCF-7 and NIH-3T3 cells were exposed to PEG-Gd2O3 nanoparticles and PEG-Gd2O3/aptamer-Ag NCs nanoprobes ranging from 0.02 to 0.3 mM Gd. As shown in Figure 4, no significant cellular toxicities of both PEG-Gd2O3 nanoparticles and PEGGd2O3/aptamer-Ag NCs nanoprobes were observed below 0.25 mM Gd. Thus, the concentration of 0.25 mM Gd was chosen for the following fluorescence imaging and MR imaging experiments. Optical/MR Dual-Modality Molecular Imaging of PEGGd2O3/Aptamer-Ag NCs Nanoprobe to MCF-7 Cells. The specific cellular-targeting of the PEG-Gd2O3/aptamer-Ag NCs

Figure 2. r1 relaxivity curves and T1 weighted MR images of PEGGd2O3 NPs (a) and Gd-DTPA (b) with various Gd concentrations.

was 29.0 s−1 mM−1 Gd, almost seven times that of Gd-DTPA (4.2 s−1 mM−1 Gd). To further compare the T1 weighted MR images, PEG-Gd2O3 nanoparticles at lower Gd concentration displayed even much stronger MR signals than that of GdDTPA at high Gd concentration. The high r1 value and excellent MR imaging ability made PEG-Gd2O3 nanoparticles a promising MR imaging contrast agent, and they could be used for the fabrication of a multimodal molecular imaging nanoprobe. Preparation and Characterization of PEG-Gd2O3/ Aptamer-Ag NCs Nanoprobe. To produce a multimodal molecular imaging nanoprobe, the interaction effect between them should be considered. Herein, PEG-Gd2O3 nanoparticles were conjugated with aptamer-Ag NCs through the covalent coupling between the carboxyl group of PEG and amino group of the aptamer. In this multimodal molecular imaging nanoprobe, PEG-Gd2O3 nanoparticles were used as MR contrast agent and aptamer-Ag NCs were the fluorescent reporter. Thus, the influence of PEG-Gd2O3 nanoparticles on the fluorescence emissions of aptamer-Ag NCs as well as the influence of aptamer-Ag NCs on the MR signal of PEG-Gd2O3 nanoparticles should be studied. To our surprise, the presence of PEG-Gd2O3 nanoparticles could enhance the fluorescence intensity of aptamer-Ag NCs and the fluorescence emission wavelength presented a slight red-shift (Figure 3a). It was found that such fluorescence enhancement could only be observed in a suitable ratio between aptamer-Ag NCs and PEGGd2O3 nanoparticles (Figure S1a in the Supporting Information). We ascribed such fluorescence enhancement to the electron transfer from Gd3+ existing in the crystalline core to 11309

dx.doi.org/10.1021/ac503026d | Anal. Chem. 2014, 86, 11306−11311

Analytical Chemistry

Article

Figure 4. MCF-7 human breast cancer cell viability and NIH-3T3 mouse fibroblast cell viability after a 24 h exposure to PEG-Gd2O3 NPs (a) or PEG-Gd2O3/aptamer-Ag NCs nanoprobes (b), ranging from 0.02 to 0.3 mM Gd.

nanoprobe was first tracked by the fluorescence emission of aptamer-Ag NCs. MCF-7 cells expressing nucleolin were incubated with PEG-Gd2O3/aptamer-Ag NCs nanoprobes. NIH-3T3 cells were treated the same way as the control. As shown in Figure 5, PEG-Gd2O3/aptamer-Ag NCs nanoprobes Figure 6. T1 weighted MR images of MCF-7 human breast cancer cells (up) and NIH-3T3 mouse fibroblast cells (bottom) treated with PBS (1), PEG-Gd2O3 NPs (2), or PEG-Gd2O3/aptamer-Ag NCs nanoprobe (3).

Figure 5. Confocal laser scanning microscopic images of MCF-7 human breast cancer cells (a) and NIH-3T3 mouse fibroblast cells (b) incubated with PEG-Gd2O3/aptamer-Ag NCs nanoprobes. (1) brightfield images; (2) Ag NCs fluorescence images (red); (3) fluorescence images with DAPI nuclear staining (blue); (4) overlap of corresponding fluorescence image and bright-field image. The Ag NCs were excited with 543 nm and DAPI with UV. Scale bar, 20 μm. Figure 7. (a) T1 weighted and T1 map MR images of different amounts of MCF-7 cells treated with the PEG-Gd2O3/aptamer-Ag NCs nanoprobe. The numbers of cells were 4 × 104, 2 × 105, 4 × 105, 7.5 × 105, and 1.35 × 106, respectively (from the top to the bottom). (b) The corresponding linear calibration plot between the cell number and T1 relaxation rate.

could recognize MCF-7 cells easily with the help of AS1411 aptamer while no fluorescence signal was observed in the case of NIH-3T3 cells, indicating the cell specificity of the multimodal molecular imaging nanoprobe. With further comparisons using nuclear DAPI staining, we found that the intracellular Ag NCs were mainly located in the nucleus of MCF-7 cells. The specific targeting of PEG-Gd2O3/aptamer-Ag NCs nanoprobes to MCF-7 cells was further monitored by T1 weighted MRI. MCF-7 cells were cocultured with PBS buffer, PEG-Gd2O3 nanoparticles, or PEG-Gd2O3/aptamer-Ag NCs nanoprobes, respectively. As shown in Figure 6, aptamer recognized cells could be identified on the basis of bright signal at the bottom of the tube. Although nonspecific adsorption of PEG-Gd2O3 nanoparticles existed, an obvious difference could still be distinguished in the presence of the aptamer. For further quantitative analysis of MCF-7 cells with different amounts, the PEG-Gd2O3/aptamer-Ag NCs nanoprobe labeled cells were fixed in 1% agarose for axital MRI scanning. As shown in Figure 7, the T1 relaxation rate (1/T1) of PEG-Gd2O3/aptamer-Ag NCs nanoprobe was proportional to the amount of MCF-7 cells. These results indicated that MCF-7 cells could be recognized specifically and efficiently at the potentially safe concentrations both by optical imaging and MR imaging.

■

CONCLUSIONS Facile fabrication of a specific and sensitive multifunctional probe is crucial for the multimodal molecular imaging of cancer. In this study, a nucleolin-targeted optical/MR dual imaging nanoprobe was simply fabricated by a covalent coupling reaction between PEG-Gd2O3 NPs and aptamer-Ag NCs. The excellent fluorescence property and MRI behavior after the formation of the PEG-Gd2O3/aptamer-Ag NCs nanoprobes ensured their application in multimodal molecular imaging. With the help of aptamer specific targeting to nucleolin, MCF-7 cells were specifically tracked by fluorescence imaging and magnetic resonance imaging in vitro.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text (Figures S1−S4). This material is available free of charge via the Internet at http://pubs.acs.org. 11310

dx.doi.org/10.1021/ac503026d | Anal. Chem. 2014, 86, 11306−11311

Analytical Chemistry

■

Article

AUTHOR INFORMATION

Corresponding Author

*Fax: +86-516-83262158. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21305120), Natural Science Foundation of Jiangsu Province (BK20130211, BK2010178), and Natural Science Fund for Colleges and Universities in Jiangsu Province (13KJB150036).

■

REFERENCES

(1) Zhou, T.; Wu, B. Y.; Xing, D. J. Mater. Chem. 2012, 22, 470−476. (2) Louie, A. Chem. Rev. 2010, 110, 3146−3195. (3) Lee, D.-E.; Koo, H.; Sun, I.-C.; Byu, J. H.; Kim, K.; Kwon, I. C. Chem. Soc. Rev. 2012, 41, 2656−2672. (4) Anderson, C. J.; Bulte, J. W.; Chen, K.; Khaw, B. A.; Shokeen, M.; Wooley, K. L.; VanBrocklin, H. F. J. Nucl. Med. 2010, 51, 3S−17S. (5) Chen, K.; Chen, X. Curr. Top. Med. Chem. 2010, 10, 1227−1236. (6) Poselt, E.; Schmidtke, C.; Fischer, S.; Peldschus, K.; Salamon, J.; Kloust, H.; Tran, H.; Pietsch, A.; Heine, M.; Adam, G.; Schumacher, U.; Wagener, C.; Forster, S.; Weller, H. ACS Nano 2012, 6 (4), 3346− 3355. (7) Lee, J.-H.; Kim, J.; Cheon, J. Mol. Cells 2013, 35, 274−284. (8) Abdukayum, A.; Yang, C.-X.; Zhao, Q.; Chen, J.-T.; Dong, L.-X.; Yan, X.-P. Anal. Chem. 2014, 86, 4096−4101. (9) Mulder, W. J. M.; Koole, R.; Brandwijk, R. J.; Storm, G.; Chin, P. T. K.; Strijkers, G. J.; de Mello Donegá, C.; Nicolay, K.; Griffioen, A. W. Nano Lett. 2006, 6, 1−6. (10) Tsai, C.-P.; Hung, Y.; Chou, Y.-H.; Huang, D.-M.; Hsiao, J.-K.; Chang, C.; Chen, Y.-C.; Mou, C.-Y. Small 2008, 4, 186−191. (11) Taylor, K. M. L.; Kim, J. S.; Rieter, W. J.; An, H. Y.; Lin, W. L.; Lin, W. B. J. Am. Chem. Soc. 2008, 130, 2154−2155. (12) Kim, J.; Piao, Y. Z.; Hyeon, T. Chem. Soc. Rev. 2009, 38, 372− 390. (13) Bridot, J.-L.; Faure, A.-C.; Laurent, S.; Riviere, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Coll, J.-L.; Elst, L. V.; Muller, R.; Roux, S.; Perriat, P.; Tillement, O. J. Am. Chem. Soc. 2007, 129, 5076− 5084. (14) Sun, S.-K.; Dong, L.-X.; Cao, Y.; Sun, H.-R.; Yan, X.-P. Anal. Chem. 2013, 85, 8436−8441. (15) Faucher, L.; Tremblay, M.; Lagueux, J.; Gossuin, Y.; Fortin, M.A. ACS Appl. Mater. Interfaces 2012, 4, 4506−4515. (16) Li, J. J.; Zhong, X. Q.; Cheng, F. F.; Zhang, J.-R.; Jiang, L.-P.; Zhu, J.-J. Anal. Chem. 2012, 84, 4140−4146. (17) Park, J. Y.; Baek, M. J.; Choi, E. S.; Woo, S.; Kim, J. H.; Kim, T. J.; Jung, J. C.; Chae, K. S.; Chang, Y. M.; Lee, G. H. ACS Nano 2009, 3, 3663−3669. (18) Curtis, C. E.; Johnson, J. R. J. Am. Chem. Soc. 1957, 40, 15−19. (19) Ahren, M.; Selegard, L.; Klasson, A.; Soderlind, F.; Abrikossova, N.; Skoglund, C.; Bengtsson, T.; Engstrom, M.; Kall, P.-O.; Uvdal, K. Langmuir 2010, 26, 5753−5762. (20) Chen, Z.; Lu, D. T.; Cai, Z. W.; Dong, C.; Shuang, S. M. Luminescence 2014, 29, 722−727. (21) Zhang, Y. D.; Cai, Y. N.; Qi, Z. L.; Lu, L.; Qian, Y. X. Anal. Chem. 2013, 85, 8455−8461. (22) Gwinn, E. G.; O’Neill, P.; Guerrero, A. J.; Bouwmeester, D.; Fygenson, D. K. Adv. Mater. 2008, 20, 279−283. (23) Robin, I.; Schulze, W.; Ertl, G.; Felix, C.; Sicber, C.; Harbich, W.; Buttet, J. Chem. Phys. Lett. 2000, 320, 59−64. (24) Xu, W. C.; Lu, Y. Chem. Commun. 2011, 47, 4998−5000. (25) Zhang, P. H.; Cheng, F. F.; Zhou, R.; Cao, J. T.; Li, J. J.; Burda, C.; Min, Q. H.; Zhu, J.-J. Angew. Chem., Int. Ed. 2014, 53, 2371−2375.

11311

dx.doi.org/10.1021/ac503026d | Anal. Chem. 2014, 86, 11306−11311