A Bimodal Phosphorescence-Magnetic Resonance Imaging

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Biological and Medical Applications of Materials and Interfaces

A Bimodal Phosphorescence-Magnetic Resonance Imaging Nanoprobe for Glutathione Based on MnO Nanosheet-Ru(II) Complex Nanoarchitecture 2

Wenbo Shi, Bo Song, Wenjing Shi, Xiaodan Qin, Zhiwei Liu, Mingqian Tan, Liu Wang, Fengling Song, and Jingli Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08872 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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ACS Applied Materials & Interfaces

A Bimodal Phosphorescence-Magnetic Resonance Imaging Nanoprobe for Glutathione Based on MnO2 Nanosheet-Ru(II) Complex Nanoarchitecture Wenbo Shi,a Bo Song,a,* Wenjing Shi,a Xiaodan Qin,a Zhiwei Liu,a Mingqian Tan,b Liu Wang,a Fengling Song,a Jingli Yuana a

State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology,

Dalian 116024, China b

School of Food Science and Technology, National Engineering Research Center of Seafood,

Dalian Polytechnic University, Qinggongyuan1, Ganjingzi District, Dalian 116034, China

*

Corresponding author.

Tel./Fax: +86-411-84986042; E-mail: [email protected]

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ABSTRACT Bimodal fluorescence-magnetic resonance imaging (MRI) technique has showed great utilities in bioassays since it combines both advantages of optical imaging and MRI to provide more sufficient information over any modality alone. In this work, based on a MnO2 nanosheet-Ru(II) complex nanoarchitecture, a bimodal phosphorescence-MRI nanoprobe for glutathione (GSH) has been constructed. The nanoprobe, Ru(BPY)3@MnO2, was constructed by integrating MnO2 nanosheets with a phosphorescent Ru(II) complex [Ru(BPY)3](PF6)2 (BPY = 2,2'-bipyridine), which resulted in the complete phosphorescence quenching of the Ru(II) complex, accompanied by the very low longitudinal and transverse relaxivity. Upon exposure to GSH, the reduction of MnO2 nanosheets by GSH triggers a recovery of phosphorescence, and simultaneously produces a number of Mn2+ ions, a perfect MRI contrast agent. The as-prepared nanoprobe showed good water-disperse and biocompatibility, and rapid, selective and sensitive response towards GSH in phosphorescence and MR detection modes. The practicability of the nanoprobe was proved by time-gated luminescence (TGL) assay of GSH in human serum, phosphorescent imaging of endogenous GSH in living cells, zebrafish and tumor-bearing mice, as well as the MRI of GSH in tumor-bearing mice. The research outcomes suggested the potential of Ru(BPY)3@MnO2 for the bimodal phosphorescence-MRI sensing of GSH in vitro and in vivo. KEYWORDS: glutathione, manganese dioxide nanosheet, ruthenium complex, phosphorescent imaging, magnetic resonance imaging


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INTRODUCTION Molecular imaging, a non-invasive detection technology for studying biological events in vivo at cellular and molecular levels, is one of the most inspiring and fast-growing areas of science.1-2 It makes molecular events in biosystem to be visible, traceable and quantifiable over time, targeting to monitor biomolecular abnormalities that are the basis of diseases.3-5 Various imaging modalities (MRI, SPECT, PET, CT, ultrasound imaging, optical imaging and photoacoustic tomography) have been developed and used to assay specific biomolecular targets.6-8 A certain modality is well applicable for some applications, but very poorly applicable for other applications. Considering any modality is not perfect enough to provide sufficient information on structure and function of objective samples owing to their inherent limitation, detection of a subject using multiple imaging modalities in tandem is obviously appealing.9-11 For example, optical imaging (OI) is regarded as one of the most powerful imaging techniques in terms of its high sensitivity and selectivity, submicron resolution and fast response times, but it has encountered problems for in vivo applications due to the lack of optical transparency.12,13 While MRI provides high quality 3D images of soft tissues with sub-millimeter spatial resolution and no depth limit.14,15 Thus, a variety of MRI-OI bimodal strategies combined the high sensitivity of OI and the high spatial resolution and no penetration depth limit of MRI, have been developed for biomedical researches and clinical practices.16-21 The use of dual-modal MRI-OI could minimize artifacts, to enable significant improvement in diagnostic accuracy and expand practical applications. In the past few decades, luminescent d6 transition-metal complexes, particularly the Ru(II) complexes, have attracted numerous attention in the area of luminescence bioassay and imaging owing to their good biocompatibility and extraordinary photophysical properties, such as strong red emission under the excitation of visible-light, large Stokes shift, and high thermal, chemical and photo-stabilities. Furthermore, the optical properties and biological activities of Ru(II) complexes can be tuned by the variation of ligands, which enables the complexes to be functionalized for developing new classes of luminescent probes, and diagnostic and therapeutic agents.22,23 In addition, the long-lived phosphorescence of transition-metal complexes enables their emissions to be distinguished from scattering lights and short-lived autofluorescence by employing TGL mode, thus to dramatically improve the signal-to-noise contrast ratio and 3 ACS Paragon Plus Environment


sensitivity.24,25 Therefore, transition-metal complex-based bimodal OI-MRI probes have drawn great attention.26-29 However, the transition-metal complex-based bimodal MRI-OI probe that can respond to specific biological stimuli has not been reported. It is still a great challenge to integrate a responsive MR contrast agent with an activatable phosphorescence probe in one system. In recent years, two-dimensional (2D) layered nanosheets and their biological applications have attracted a lot of attention due to their unique characteristics.30,31 As one kind of two-dimensional transition metal dichalcogenides (2D TMDCs) nanomaterials, manganese dioxide (MnO2) nanosheets with ultrahigh specific surface area, fast electron transfer rate and excellent light absorption capability, are proposed to be a promising fluorescence quencher for using to develop ―turn-on‖ fluorescence sensing systems.32,33 Moreover, the MnO2 nanosheets are a very poor T1- or T2-weighted MRI contrast agent since the manganese atoms in MnO2 nanosheets are restricted in MnO6 octahedra and shielded from water. And the redoxable MnO2 nanosheets could be rapidly reduced by GSH, which converts MnO2 to Mn2+ ions, a perfect MRI contrast agent.34 These characteristics of MnO2 nanosheets make it possible to construct fluorometric/magnetic dual-model nanoprobes for GSH detection with enhanced fluorescence and magnetic resonance signals. Recently, some dual-model MRI-OI nanoprobes for GSH and H2O2 detection based on MnO2 nanosheets have been developed, but no their application for dual-model MR-fluorescence imaging of target analytes in vivo was reported. Furthermore, the sensitivity and accuracy of optical determination using these reported nanoplatforms could be suffered from the effects of background autofluorescence and scattering lights.35-37 Inspired by the preceding works, we designed a bimodal phosphorescence-MR nanoprobe, Ru(BPY)3@MnO2, for the determination of GSH in vitro and in vivo, by combining MnO2 nanosheets with a phosphorescent Ru(II) complex, [Ru(BPY)3](PF6)2. In the proposed nanoprobe, the [Ru(BPY)3](PF6)2 molecules are absorbed onto the surface of MnO2 nanosheets, which leads to the efficient phosphorescence quenching of the Ru(II) complex. Meanwhile, the nanoprobe exhibits very low T1- and T2-weight MR signals since Mn atoms in MnO2 nanosheets are completely insulated from water. Upon exposed to GSH, MnO2 nanosheets can be rapidly reduced to Mn2+ ions, resulting in a recovery of the Ru(II) complex’s phosphorescence and remarkable enhancements of T1- and T2-weighted MR signals, to allow the GSH concentration to 4 ACS Paragon Plus Environment

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be quantified with phosphorescence and MR dual-modes. Scheme 1 illustrates the activation mechanism of the nanoprobe Ru(BPY)3@MnO2 for bimodal phosphorescence-MR imaging of GSH in vitro and in vivo.

Scheme 1. Schematic illustration of activation mechanism of the nanoprobe Ru(BPY)3@MnO2 for bimodal imaging of GSH in vitro and in vivo.

EXPERIMENTAL SECTION Materials

and

instruments.

tetramethylammonium

hydroxide

Manganese

chloride

(TMAH),

tetrahydrate

(MnCl2·4H2O),

Ruthenium(II)-tris(2,2´-bipyridyl)

dihexafluorophosphate ([Ru(BPY)3](PF6)2, 97%), hydrogen peroxide (H2O2, 30 wt%), L-glutathione (GSH), and 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from SIGMA-ALDRICH LLC. Cultured HepG2 cells, HeLa cells, human sera and tumor-bearing BALB mice were provide from Dalian Medical University. Zebrafish were purchased from Huante Biological Technology Co. Ltd., Hangzhou, China. Milli-Q water was used throughout the whole experiments. All other reagents were purchased from commercial sources and used directly. The morphology of nanocomposites was investigated using a JEOL JEM-2000EX transmission electron microscope (TEM). Zeta potential and dynamic light scattering (DLS) were examined on the Malvern Nano-Zetasizer ZSE (Malvern Instruments, Ltd., Worcestershire, UK). The contents 5 ACS Paragon Plus Environment


of Mn were measured on a PE Optima 2000DV ICP Optical Emission Spectrometer (ICP-OES). Atomic force microscopy (AFM) measurements were carried out on a Pico Scan-2500 microscope (MI, USA). UV−Vis spectra were measured on a PE Lambda 35 UV-Vis spectrometer. Phosphorescence spectra, TGL spectra and emission lifetimes were measured on a FS5 spectrofluorometer of Edinburgh Instruments. All bright-field and phosphorescence imaging measurements were performed with an Olympus FV1000 confocal laser scanning microscope (CLSM). The luminescence imaging of BALB mice was carried out on a NightOWL II LB983 luminescent imaging system (Berthold Technologies, Germany) for living small animals. The measurements of longitudinal and transverse relaxation times and MRI were performed on a 0.5 T NM12 MR analyzer and a NMI20-030H-I MR imager (NIUMAG Corporation, Shanghai, China), respectively. Preparation of MnO2 nanosheets. The MnO2 nanosheets were synthetized according to the literature method.35 Typically, 18 mL of aqueous TMAH solution (0.66 M) was mixed with 2.0 mL H2O2 solution (30 wt%). Then the mixture was added into 10 mL of the water solution of MnCl2 (0.3 M) within 25 s. The resulting dark-brown suspension was stirred vigorously at room temperature (RT) for 10 h. The as-prepared bulk MnO2 was separated by centrifugation and washed with Milli-Q water and methanol five times. After dispersing the obtained MnO2 precipitate in 20 mL water, the resulting dark-brown suspension was ultrasonicated for 10 h to form a colloidal solution of layered MnO2 nanosheets. The content of Mn in the solution was examined by ICP-OES analysis. Dual-modal detection of GSH in aqueous buffer. For phosphorescence detection of GSH in buffer, the Ru(BPY)3@MnO2 nanoprobe was prepared by mixing [Ru(BPY)3](PF6)2 (1.0 M) with MnO2 nanosheets (120 M) for 2 min at RT in 10 mM HEPES buffer (pH 7.4). The obtained nanoprobe solution was incubated with different concentrations of GSH for 2 min, and the mixtures were subjected to the phosphorescence and TGL measurements. For quantifying the amount of Ru(BPY)32+ on the surface of MnO2 nanosheet, the as-prepared Ru(BPY)3@MnO2 nanoprobe solution was separated by centrifugation using an Amicon Ultra centrifugal filter unit (pore size 10 kDa MWCO), and the content of free Ru(BPY)32+ in filtrate was evaluated by the fluorescence assay. For MR detection of GSH in buffer, the Ru(BPY)3@MnO2 nanoprobe was prepared by mixing 6 ACS Paragon Plus Environment

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[Ru(BPY)3](PF6)2 (5.0 M) with MnO2 nanosheets (600 M) for 2 min at RT in 10 mM HEPES buffer (pH 7.4). The obtained nanoprobe solution was incubated with various concentrations of GSH for 2 min, and then the longitudinal and transverse relaxation times and MR images of the mixtures were measured on the MR analyzer and imager, respectively. For investigating the response specificity of the nanoprobe to GSH, Ru(BPY)3@MnO2 (1.0 M [Ru(BPY)3](PF6)2 mixed with 120 M MnO2 nanosheets) was incubated with GSH (0.8 mM), Cys (1.0 mM), Hcy (1.0 mM), NADH (1.0 mM) and other interferents (10 mM), respectively. Then the phosphorescence intensities of the mixtures were recorded. Furthermore, after the nanoprobe prepared by mixing 5.0 μM [Ru(BPY)3](PF6)2 with 600 μM MnO2 nanosheets was incubated with GSH (2.0 mM), Cys (2.0 mM), Hcy (2.0 mM), NADH (2.0 mM) and other interferents (10 mM), respectively, the transverse relaxation times of the mixtures were determined. Quantification of GSH in human sera. To the quantitative detection of GSH in human sera, the fresh human serum samples were 30-fold diluted with 10 mM HEPES buffer (pH 7.4), and then incubated with Ru(BPY)3@MnO2 (1.0 μM [Ru(BPY)3](PF6)2 mixed with 120 μM MnO2 nanosheets) for 2 min at RT in 10 mM HEPES buffer (pH 7.4). The TGL emission spectra of the mixtures were measured with the setting of 100 ns delay time. After that, the GSH-added human serum samples were measured under the same conditions. Phosphorescence imaging of GSH in vitro and in vivo. HeLa and HepG2 cells were cultured on a glass-bottom culture dish in cell culture medium (Gibco DMEM) containing 10% fetal bovine serum (GIBCO) at 37 oC in a 5% CO2/95% air incubator. For imaging GSH in the cells, the cultured cells were rinsed with PBS buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4 and 10.1 mM Na2HPO4, pH 7.4), and then incubated with Ru(BPY)3@MnO2 (1.0 μM [Ru(BPY)3](PF6)2 mixed with 60 μM MnO2 nanosheets) in PBS buffer for 2 h. After rinsing three times with PBS buffer, the cells were subjected to the phosphorescence imaging measurements on a CLSM (ex = 488 nm, em = 610-660 nm). For the control group, the cells were pretreated with 0.5 mM N-ethylmaleimide (NEM) for 0.5 h, and then incubated with Ru(BPY)3@MnO2 for 2 h. After rinsing, the cells were imaged using a CLSM. Zebrafish were cultured in an E3 embryo medium without methylene blue at 26 oC with a 12 h light/12 h dark cycle. For imaging GSH in the fish, the zebrafish were incubated with 7 ACS Paragon Plus Environment


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Ru(BPY)3@MnO2 (1.0 μM [Ru(BPY)3](PF6)2 mixed with 60 μM MnO2 nanosheets) for 45 min at 26 oC in culture medium. The zebrafish were washed with fresh culture medium and imaged using a CLSM (ex = 488 nm, em = 610-660 nm). For the control group, the zebrafish were pretreated with 0.2 mM NEM for 0.5 h, and then loaded with Ru(BPY)3@MnO2 for 45 min before use for phosphorescence imaging. To evaluate the performance of the nanoprobe for phosphorescence imaging in vivo, the tumor xenograft models of four BALB mice (female, ~20 g bodyweight) were established by implanting H22 cells (mice hepatoma) in subcutaneous tissue. After the tumor size reached 1~1.5 cm in diameter, they were randomly divided into two groups. The mice in experimental group were subcutaneously injected with 100 μL physiological saline solution containing Ru(BPY)3@MnO2 nanoprobe (1.0 mM [Ru(BPY)3](PF6)2 mixed with 60 mM MnO2 nanosheets) into the tumor and the opposite normal tissue. The mice in control group were peritumorally injected with 100 μL physiological saline solution containing 2.0 mM NEM. After 0.5 h, a physiological saline solution of Ru(BPY)3@MnO2 was further peritumorally injected. The two groups of mice were subjected to the phosphorescence imaging measurements on an in vivo luminescence imaging system (excitation filter, 465 nm and emission filter, 610 nm), after the final injection of the nanoprobe for 15 min. Bimodal imaging of GSH in vivo. Four tumor-bearing BALB mice were divided into two groups randomly and treated as described above (phosphorescence imaging of GSH in mice). The MR imaging was carried out on a 0.5 T MR imager, after the final injection of the nanoprobe for 15 min. In addition, after Ru(BPY)3@MnO2 was peritumorally injected into the mice for 15 min, the mice were killed, and the tumor was excised and stored at -20 oC for 24 h. The frozen tumor tissue was cryosectioned by microtome at -20 oC into slices of 50 μm thicknesses for the phosphorescence imaging measurements on a CLSM.

RESULTS AND DISCUSSION Design,

preparation

and

characterization

of

the

bimodal

nanoprobe

for

phosphorescence-MR detection of GSH To date, several MnO2 nanosheet-based biosensing methods have been reported, and in which ultrathin MnO2 nanosheets were employed as the fluorescence quencher and activatable MRI 8 ACS Paragon Plus Environment

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contrast agent to construct the bimodal fluorescence-MR sensing systems.35,36 It is worthy being noticed that although a lot of phosphorescent Ru(II) complexes have been designed for biosensing applications,22-25 the construction of a bimodal phosphorescence-MR biosensing probe using the Ru(II) complex-MnO2 nanosheets system has not been explored. Considering salient optical properties of Ru(II) complexes, especially their long emission lifetimes that benefit for background-free TGL detection, in this work we constructed the nanoprobe, Ru(BPY)3@MnO2, for the bimodal phosphorescence-MR detection of GSH. After positively charged Ru(BPY)32+ ions are adsorbed on the surface of negatively charged MnO2 nanosheets by electrostatic attraction, the nanocomposite, Ru(BPY)3@MnO2, is formed, which could cause the phosphorescence of Ru(BPY)32+ to be quenched.38 Upon exposure to GSH, accompanied by the reduction of MnO2 nanosheets, a number of Mn2+ ions are produced, and Ru(BPY)32+ molecules are released into the solution. This reaction can result in the remarkable enhancements both of phosphorescence and MR signals. Thus, the designed nanoprobe, Ru(BPY)3@MnO2, is proposed for the bimodal phosphorescence-MR detection of GSH. To fabricate the proposed nanoprobe, MnCl2 and H2O2 were mixed with TMAH aqueous solution under vigorous stirring, and then the layered MnO2 nanosheets were obtained by simply exfoliating the resulted bulk MnO2 under sonication. The MnO2 nanosheets were characterized by several microscopic and spectroscopic methods. As shown in Figure 1a, the as-prepared MnO2 nanosheets exhibited typical two-dimensional and layered nanosheet morphology with occasional wrinkles and folds. AFM analysis results indicate that the thickness of the nanosheets is around 1 nm and the slice layer size of them is about 50-100 nm (Figure 1c and 1d). The dimensions of MnO2 nanosheets and their size distribution were further investigated by the dynamic light scattering (DLS) measurements. As shown in Figure S1, the dimensions of most MnO2 nanosheets are in the range of 40 to 90 nm with a centered size of 50 nm. The -potential of MnO2 nanosheets in HEPES buffer (pH 7.4) was determined to be -47.8 mV (Figure S1a), showing the negatively charged surface of MnO2 nanosheets for absorbing positively charged Ru(BPY)32+ ions. The UV-Vis absorption spectrum of MnO2 nanosheets showed a broad band ranged from 240 nm to 700 nm with an absorption maximum at 376 nm (Figure 1b), which is consistent with the reported result.32 The UV-Vis absorption and phosphorescence spectra of [Ru(BPY)3](PF6)2 were recorded in 10 9 ACS Paragon Plus Environment


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mM HEPES buffer (pH 7.4). As shown in Figure S2a, Ru(BPY)32+ showed a strong absorption peak below 300 nm dominated by the spin-allowed ligand →* transition, and an intense absorption peak at 450 nm attributed to the metal-to-ligand charge transfer (MLCT) transition of the Ru(II) complex.22,23 After excitation at 450 nm, Ru(BPY)32+ exhibited the typical 3

MLCT-based phosphorescence emission with the maximum emission at 620 nm and a large

Stokes shift of 170 nm (Figure S2b). The emission lifetime of Ru(BPY)32+ was determined to be 389 ns (Figure S3a), which is much longer than the lifetimes of typical organic fluorophores (several nanoseconds) to enable the Ru(II) complex to be used for the TGL detection.

Figure 1. (a) TEM image of MnO2 nanosheets. (b) UV-Vis absorption spectrum of MnO2 nanosheets. (c) AFM image of MnO2 nanosheets. (d) The corresponding height profile of MnO2 nanosheets in the interest linear across image (c). Scale bars: 100 nm.

Before preparing the nanoprobe Ru(BPY)3@MnO2, the quenching ability of MnO2 nanosheets to the phosphorescence of [Ru(BPY)3](PF6)2 was evaluated. It was found that after mixing Ru(BPY)32+ (1.0 M) with MnO2 nanosheets (120 M) for 2 min at RT, 99.3% of Ru(BPY)32+ in the system was absorbed on the MnO2 nanosheet. This result reveals that 0.993 mol Ru(BPY)32+ can be absorbed on the surface of 120 mol MnO2 nanosheets in the experimental condition. As shown in Figure 2a and 2b, the phosphorescence of the Ru(II) complex was gradually decreased 10 ACS Paragon Plus Environment

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with the increasing concentration of MnO2 nanosheets in aqueous buffer. The emission of Ru(BPY)32+ (1.0 μM) was almost quenched in the presence of 120 μM MnO2 nanosheets (quenching efficiency is 85%). The quenching dynamics of the system indicates that the interaction between Ru(BPY)32+ and MnO2 nanosheets is a fast process, which can reach equilibrium within 20 s (Figure 2c). As the result obtained above, 1.0 μM Ru(BPY)32+, 120 μM MnO2 nanosheets and a 2 min quenching time were used for constructing the nanoprobe, Ru(BPY)3@MnO2, for the detection of GSH (for phosphorescence detection of GSH in cells and zebrafish, 1.0 μM Ru(BPY)32+ and 60 μM MnO2 nanosheets were used to prevent the aggregation of the nanosheets in culture media). The influence of MnO2 nanosheets on the emission lifetime of Ru(BPY)32+ was investigated to clarify the phosphorescence quenching mechanism. As shown in Figure S3, in the absence and presence of MnO2 nanosheets, the emission lifetimes of Ru(BPY)32+ were determined to be 393 ns and 389 ns, respectively. These results indicate that the phosphorescence quenching of Ru(BPY)32+ by MnO2 nanosheets is not caused by the energy transfer from Ru(BPY)32+ to MnO2 nanosheets. Considering the overlap of the absorption spectrum of MnO2 nanosheets with the excitation and emission spectra of Ru(BPY)32+, as well as the electrostatic interaction between Ru(BPY)32+ cations and negatively charged MnO2 nanosheets, it can be concluded that the quenching of MnO2 nanosheets to the phosphorescence of Ru(BPY)32+ is attributed to the association of the inner filter effect (IFE) and the static quenching effect (SQE).38

Figure 2. (a) Emission spectra (ex = 450 nm) of [Ru(BPY)3](PF6)2 (1.0 μM) in the presence of different concentrations of MnO2 nanosheets. (b) The correlation between the phosphorescence intensity of [Ru(BPY)3](PF6)2 and the concentration of MnO2 nanosheets. (c) Time course of phosphorescence intensity response of [Ru(BPY)3](PF6)2 (1.0 μM) at 620 nm to the addition of 120 μM MnO2 nanosheets. 11 ACS Paragon Plus Environment


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In this nanosystem, MnO2 nanosheets were also employed as a GSH recognition unit. In order to verify GSH-triggered reduction of MnO2 nanosheets to Mn2+, the electrochemical behaviors of MnO2 nanosheets in the presence and absence of GSH were investigated by employing a cyclic voltammetry (CV) method. In the absence of GSH, MnO2 nanosheets exhibited only one reduction peak (Epc = -0.519V) in the potential range of -1.5 to 1.0 V because of its high oxidation state (Figure S4). Upon exposure to GSH, the characteristic reduction peak of MnO2 nanosheet obviously disappeared (Figure S4), and the CV curve of MnO2-GSH showed a different reduction peak (Epc = -0.738 V) and a new oxidation peak (Epa = 0.767 V) in the same potential range, which is consistent with the characteristic redox peaks of Mn2+ (Epc = -0.749 V, Epa = 0.775 V). The above result proved that MnO2 nanosheets could indeed be reduced by GSH to afford Mn2+ ions. Phosphorescence and MR responses of the nanoprobe towards GSH To employ the nanoprobe for GSH detection, at first, the phosphorescence response dynamics of Ru(BPY)3@MnO2 nanoprobe towards GSH was investigated by monitoring the phosphorescence increase (at 620 nm) of the nanoprobe upon the addition of GSH. As shown in Figure 3c, upon exposure to GSH, the phosphorescence intensity of Ru(BPY)3@MnO2 was rapidly increased, and reached a stable value within 30 s. The result reveals that the reaction between GSH and Ru(BPY)3@MnO2 is quite fast. On the basis of this result, an equilibrium time of 2 min was used for the reaction of GSH with the nanoprobe in the experiments of GSH detection. To investigate the capability of the nanoprobe for quantitative phosphorescence detection of GSH, the emission spectra of Ru(BPY)3@MnO2 exposed to different concentrations of GSH were examined. As shown in Figure 3, upon reaction with increased concentration of GSH, the phosphorescence of Ru(BPY)3@MnO2 was gradually increased (Figure 3a), and the dose-dependent phosphorescence enhancement displayed a linear correlation to the GSH concentration ranged from 0 to 300 M with an excellent correlation coefficient of 0.9978 (Figure 3b). The detection limit for GSH is calculated to be 420 nM according to the 3σ (triple standard deviations of the background signal) criteria, which indicates that Ru(BPY)3@MnO2 could truly be employed as a sensitive phosphorescence probe for quantitative assay of GSH at physiological conditions. The moderate detection limit of fabricated sensing nanosystem could be attributed to the quenching-recovery efficiency of Ru(BPY)3@MnO2-GSH system, which might 12 ACS Paragon Plus Environment

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be improved by modifying the structure of the phosphorescent Ru(II) complex.

Figure 3. (a) Emission spectra (ex = 450 nm) of Ru(BPY)3@MnO2 exposed to different concentration of GSH. (b) Calibration curve for the quantitative phosphorescence detection of GSH using Ru(BPY)3@MnO2 as a probe. (c) Time course of phosphorescence intensity response of Ru(BPY)3@MnO2 at 620 nm to the addition of 1.0 mM GSH.

After the nanoprobe is reacted with GSH, since MnO2 in the probe is reduced to Mn2+, an effective MRI contrast agent, the nanoprobe possesses also unique properties for the MR detection of GSH.35,36 To assess the validity of MnO2 nanosheets as a GSH-responsive MRI contrast agent, the longitudinal relaxivity (r1) and transverse relaxivity (r2) of Ru(BPY)3@MnO2 before and after reaction with GSH were recorded. As shown in Figure S5, the longitudinal (T1) and transverse (T2) relaxation times of protons in aqueous media were dramatically shortened after Ru(BPY)3@MnO2 was reacted with GSH, and correspondingly, the longitudinal and transverse relaxivities were remarkably increased (r1 increased from 0.11 to 9.33 mM-1s-1, and r2 increased from 0.16 to 48.77 mM-1s-1 ). These 85- and 305-fold enhancements of r1 and r2 provide a wide signal-to-noise contrast window when Ru(BPY)3@MnO2 is used for the MR detection of GSH. It is noteworthy that the enhancement of r2 is 2.6-fold higher than that of r1, which means that a higher signal-to-noise contrast can be obtained when the nanoprobe is used as a T2 MRI contrast agent. So the T2-weighted MRI was used for the imaging of GSH in vivo. Figure 4 shows the correlations between longitudinal/transverse relaxation rates of Ru(BPY)3@MnO2 and the concentration of GSH. It is clear that the longitudinal and transverse relaxation rates (R1=1/T1, R2=1/T2) of Ru(BPY)3@MnO2 are gradually increased upon reaction with increased concentration of GSH with a linear correlation to the GSH concentration ranged from 0.0 to 1500 μM (Figure 4a and 4b). These results proved the practicability of 13 ACS Paragon Plus Environment


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Ru(BPY)3@MnO2 for the quantitative MR detection of GSH in aqueous media. According to the 3 criteria, the detection limits for GSH using R1 and R2 as signals were calculated to be 26.1 μM and 6.3 μM, respectively. Although the detection limit of MR detection is higher than that of phosphorescence detection, it is believed that the MR detection is more suitable to be used for the detection of GSH in vivo. To demonstrate the capability of Ru(BPY)3@MnO2 nanoprobe for MRI detection of GSH, the T1- and T2-weighted MR images of Ru(BPY)3@MnO2 reacted with various concentrations of GSH were collected. As shown in Figure 4c and 4d, upon reaction with increased concentration of GSH, the brightness of T1-weighted MRI images was gradually enhanced, and that of T2-weighted MRI images was gradually decreased. All of these results proved the potential of the nanoprobe for the quantitative detection of GSH with MR mode.

Figure

4.

Correlations

between

longitudinal/transverse

relaxation

rates

(a,

b)

of

Ru(BPY)3@MnO2 and the concentration of GSH. The (c) and (d) showed T1- and T2-weighted MR images of Ru(BPY)3@MnO2 reacted with various concentrations of GSH, respectively.

The reactions of Ru(BPY)3@MnO2 with common metal ions, biomolecules and amino acids were examined in 10 mM HEPES buffer (pH 7.4) to assess the specificity of phosphorescence and MR responses of the nanoprobe to GSH. As shown in Figure 5a, a remarkable increase in phosphorescence intensity was observed after Ru(BPY)3@MnO2 was reacted with GSH only. For 14 ACS Paragon Plus Environment

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MR tests, the GSH-induced MR enhancement was superior and apparently greater than that induced by other interferents (Figure 5b). Although high concentrations of Hcy and Cys can cause to small phosphorescence enhancements, and high concentrations of NADH, Hcy and Cys also induce an apparent MR signal recovery, the concentration of GSH in mammalian cells is in the range of 0.5 - 10 mM (especially high content of GSH was found in cancerous cells and tumors), 39,40 which is much higher than that of Cys (30-200 M) 41, Hcy (5-15 M)41 and NADH (0.4 - 200 M),42,43 the three main interferents do not interfere with the detection of intracellular GSH. In human sera, the concentration of GSH was found to be in the range of 18-492 M,44,45 which is similar to the concentration of Cys (165-335 M),46 but much higher than that of Hcy (11-15 M)47 and NADH (less than 0.02 M)42. The impact of Cys on the detection of GSH in human sera should be noticed. All of these results indicate that Ru(BPY)3@MnO2 exhibits good selectivity responding towards GSH, and it can be used as a bimodal phosphorescence-MR probe to discriminate GSH without remarkable disturbance of other biomolecules.

Figure 5. (a) Phosphorescence and (b) MR responses of Ru(BPY)3@MnO2 towards GSH and other interferents (F0: phosphorescence intensity of Ru(BPY)3@MnO2; F: phosphorescence intensity of Ru(BPY)3@MnO2 reacted with GSH or interferents; R0: transverse relaxation rate of Ru(BPY)3@MnO2; R: transverse relaxation rate of Ru(BPY)3@MnO2 reacted with GSH or interferents). Determined interferents: 1: control; 2: Na+; 3: K+; 4. Ca2+; 5: Mg2+; 6: Zn2+; 7: NADH; 8; Cu2+; 9: Arg; 10: Thr; 11: Gly; 12: Glu; 13: Trp; 14: Ser; 15: Pro; 16; His; 17: Val; 18: Tyr; 19: Ala; 20: Leu; 21;Asp; 22: Cys; 23: Hcy; 24: GSH.

TGL detection of GSH in human sera using Ru(BPY)3@MnO2 as a probe 15 ACS Paragon Plus Environment


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TGL detection technique based on long-lived luminescent probes has achieved great successes for improving precision and accuracy of bioassays.48,49 Herein taking advantages of long-lived emissions of Ru(II) complexes, TGL detection of GSH in human serum samples was performed using Ru(BPY)3@MnO2 as a probe. It should be noticed that the serum sample itself displayed strong autofluorescence ranged from 500 nm to 700 nm under excitation at 450 nm (Figure 6a), which extremely inhibited the quantitative detection of GSH in the sample with steady-state luminescence mode. However, when TGL mode was employed, the autofluorescence of serum sample has been completely suppressed, moreover remarkable luminescence enhancement was observed after the sample was incubated with Ru(BPY)3@MnO2 or Ru(BPY)3@MnO2 and GSH (Figure 6b). Using this mode, the concentration level of GSH in human serum was determined to be 219.6 ± 10.2 μM (the calibration curve for TGL detection of GSH showed in Figure S6 was applied for calculation of GSH concentration), which is in agreement with the reported result.50 Moreover, upon addition of exogenous GSH into serum samples, the recoveries were examined to be in the range of 93.2% to 98.6% (Table S1), demonstrating that the TGL detection of GSH using Ru(BPY)3@MnO2 as a probe is highly precise and accurate.

Figure 6. Steady-state (a) and TGL (b) spectra of 30-fold diluted human serum samples before and after incubation with Ru(BPY)3@MnO2 or Ru(BPY)3@MnO2 and GSH (20 μM), ex = 450 nm. Phosphorescence imaging of GSH in vitro and in vivo Based on outstanding sensing performance of Ru(BPY)3@MnO2 to GSH, its applicability for imaging GSH in living cells was investigated. Before use for cell imaging, the influence of 16 ACS Paragon Plus Environment

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Ru(BPY)3@MnO2 on cell proliferation and viability was determined using the MTT assay method.51 As shown in Figure S7, after incubation with the nanoprobe for 24 h, the viabilities of cells remained still higher than 87%, and no obvious cytotoxicity of Ru(BPY)3@MnO2 to HeLa cells was noticed. Furthermore, considering that free Mn2+ ions at high concentration have some toxicity problems (such as cardiotoxicity and neurodegenerative effects),52,53 the intracellular concentration of Mn2+ and the toxicity of Mn2+ to HeLa cells were evaluated. After HeLa cells were incubated with the MnO2 nanosheets in described condition, the amount of Mn2+ ions per cell was determined by the differential pulse voltammetric (DPV) measurement (experimental details, see Supporting Information). From this experiment, the average amount of Mn2+ ions in a single HeLa cell was found to be 1.31×10-16 mol. Assuming that the volume of a HeLa cell is around 4.2×103 m3,54 the intracellular concentration of Mn2+ ions in a HeLa cell can be deduced to be about 31.2 M. The cytotoxicity of Mn2+ was assessed by MTT assay. As shown in Figure S9, when the concentration of Mn2+ is less than 160 M, no obvious cytotoxicity of Mn2+ ions to HeLa cells was noticed. So in our experimental condition, the Ru(BPY)3@MnO2 nanoprobe and its reduced product, Mn2+ ions, did not show notable cytotoxicity due to their low concentrations. Based on above results, a proof-of-concept experiment was performed by imaging endogenous GSH in living HepG2 and HeLa cells. As shown in Figure 7a and S10a, after HeLa and HepG2 cells were incubated with Ru(BPY)3@MnO2 for 2 h, the nanoprobe-loaded cells presented intense red phosphorescence signals, which demonstrated that Ru(BPY)3@MnO2 had been loaded into living cells, and internalized nanoprobe was reduced to Mn2+ ions by intracellular GSH, leading to the phosphorescence recovery. For the control group, HeLa and HepG2 cells were firstly incubated with 0.5 mM NEM for 30 min to remove intracellular GSH,55 and further incubated with Ru(BPY)3@MnO2 for 2 h. In this case, only weak red phosphorescence signals could be observed from the cells (Figure 7b and S10b), demonstrating that the red phosphorescence signals observed in Figure 7a and S10a were truly triggered by intracellular GSH. These results corroborate that Ru(BPY)3@MnO2 can be employed as a probe for the phosphorescence imaging of GSH in living cells.



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Figure 7. Phosphorescence images of (a) HeLa cells and (b) NEM-pretreated HeLa cells incubated with Ru(BPY)3@MnO2 for 2 h (1: bright field images; 2: phosphorescence images; 3: merged image of 1 and 2). Scale bars: 20 μm.

The feasibility of Ru(BPY)3@MnO2 for the in vivo phosphorescence imaging was then assessed by the imaging of GSH in living animal models. At first, zebrafish were selected as an animal model for investigating the distribution of endogenous GSH using Ru(BPY)3@MnO2 as a probe. Apparent red phosphorescence signals were found in the intestinal tract of larval zebrafish that was incubated with Ru(BPY)3@MnO2 in culture medium for 45 min at 26 oC (Figure 8a). In the control experiments, larval zebrafish were preloaded with 200 μM NEM for 30 min and further incubated with Ru(BPY)3@MnO2. No observable red phosphorescence signals were found from the NEM-preloaded fish (Figure 8b). These results reveal that the nanoprobe Ru(BPY)3@MnO2 can be taken into the body of zebrafish and then reacted with local endogenous GSH to give red phosphorescence signals.


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Figure 8. Phosphorescence images of (a) zebrafish and (b) NEM-pretreated zebrafish incubated with Ru(BPY)3@MnO2 for 45 min (1: bright field images; 2: phosphorescence images; 3: merged image of 1 and 2). Scale bars: 500 μm.

The BALB mice with grafted subcutaneously tumor was used to further evaluate the performance of Ru(BPY)3@MnO2 for phosphorescence imaging GSH in vivo. A physiological saline solution (100 μL) containing Ru(BPY)3@MnO2 was peritumorally injected into the tumor-bearing mice (n = 2). And as a control, the same Ru(BPY)3@MnO2 solution was injected into the opposite normal subcutaneous tissue of the same mouse. Then the in vivo phosphorescence images of the mice were recorded on a small animal luminescence imager. It was reported that the GSH levels in tumors were significantly higher than that in normal tissues due to the over-expressed of redox stress,56,57 so it was anticipated that GSH-induced phosphorescence enhancement of the nanoprobe should be higher in the tumors than that in normal tissues. As shown in Figure 9a and 9c, the tumor injected with the nanoprobe presented strong phosphorescence signals, while the normal tissue injected with the nanoprobe only exhibited weaker phosphorescence, which revealed higher concentration level of GSH in the tumor. Importantly, when the tumor-bearing mice were pretreated with NEM (peritumorally injection), and then loaded with the nanoprobe (Figure 9b and 9c), relatively weaker phosphorescence signals were observed in the tumor, suggesting that the observed phosphorescence enhancement was indeed induced by the reaction of the nanoprobe with GSH. 19 ACS Paragon Plus Environment


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All of above results demonstrate the feasibility of Ru(BPY)3@MnO2 as a GSH-responsive nanoprobe for in vivo phosphorescence imaging applications.

Figure 9. Phosphorescence images of (a) a tumor-bearing mouse and (b) a NEM-pretreated tumor-bearing mouse loaded with Ru(BPY)3@MnO2 for 15 min. (c) The corresponding phosphorescence intensities of tumor, normal tissue and NEM-pretreated tumor obtained from the in vivo phosphorescence images.

In vivo MR imaging of GSH using Ru(BPY)3@MnO2 as a contrast agent The applicability of Ru(BPY)3@MnO2 for T2-weighted MRI of GSH in vivo was evaluated by employing tumor-bearing mice as an animal model. After Ru(BPY)3@MnO2 was injected into the tumor (peritumoral) and the opposite normal tissues (subcutaneous) of tumor-bearing BALB mouse, respectively, the T2-weighted MR images of the mouse were collected on a magnetic resonance imager. As shown in Figure 10a and 10c, due to the presence of higher concentration of GSH in the tumor, the nanoprobe injected into tumor region was reacted with GSH to generate higher concentration of Mn2+ ions, thus to result in an significant MRI contrast enhancement (darkening) on the tumor area in the T2-weighted MR image. In contrast with the tumor region, the opposite normal tissue region (indicated by the red circle) loaded with Ru(BPY)3@MnO2 did not show any enhancement in T2 MRI signals. As a control, a tumor-bearing mouse was peritumorally pre-injected with NEM to eliminate the GSH in the tumor before injection of the nanoprobe. As expected, no obvious changes of T2-weighted MRI contrast values in the tumor area were observed after the injection of Ru(BPY)3@MnO2 (Figure 10b and 10c), which further testified that the enhancement in T2 MR signal was caused by the GSH-induced reduction of the 20 ACS Paragon Plus Environment

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nanoprobe.

Figure 10. T2-weighted MR images of (a) a tumor-bearing mouse and (b) a NEM-pretreated tumor-bearing mouse injected with Ru(BPY)3@MnO2 (the tumor was highlighted with yellow dashed circle, and normal tissues were highlighted with red dashed circle). (c) The corresponding MR contrast values of tumor, normal tissue and NEM-pretreated tumor obtained from the in vivo MR images.

To further confirm the precision and accuracy of T2-weighted MR imaging of GSH in vivo, the phosphorescence imaging of cryosectioned tumor tissue was carried out (the frozen tumor tissues were cryosectioned into slices of 50 μm thicknesses for phosphorescence imaging). As shown in Figure 11a, the slice of the tumor injected with Ru(BPY)3@MnO2 exhibited strong red phosphorescence signals. However, the control slice of the tumor injected with NEM and Ru(BPY)3@MnO2 exhibited rather weak phosphorescence (Figure 11b). These results verified the accuracy of the MRI results, supported the potential of Ru(BPY)3@MnO2 as a MRI contrast agent for the MRI detection of GSH in vivo.



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Figure 11. Phosphorescence images of the cryosectioned tumor from (a) a tumor-bearing mouse and (b) a NEM-pretreated tumor-bearing mouse injected with Ru(BPY)3@MnO2 (1: bright field images; 2: phosphorescence images; 3: merged image of 1 and 2). Scale bars: 10 μm.

CONCLUSIONS In summary, a GSH-responsive biomodal phosphorescence-MR imaging nanoprobe, Ru(BPY)3@MnO2, has been successfully prepared by integrating MnO2 nanosheets with a phosphorescent Ru(II) complex, [Ru(BPY)3](PF6)2. The design of this nanoprobe is based on the outstanding luminescence quenching capability of MnO2 nanosheets and the GSH-induced reduction of MnO2 nanosheets into Mn2+ ions. Besides the merits of common bimodal fluorescence-MRI

approach,

the

nanoprobe

Ru(BPY)3@MnO2

possesses

long-lived

phosphorescence benefitting for the background-free TGL detection. Furthermore, the as-prepared nanoprobe displayed rapid response towards GSH with high sensitivity and selectivity in phosphorescence and MR detection modes. The practicability of the nanoprobe in bioassays has been roundly evaluated by TGL assay of GSH in human sera, and by the visualization of endogenous GSH in living cells, zebrafish and tumor-bearing mice in both phosphorescence and MR imaging modes. We believe that this cost-effective, easy-to-handle and simple bimodal phosphorescence-MRI nanoprobe could be a powerful tool for studying the roles of GSH in physiology and relative diseases, as well as for the early diagnosis of cancers.


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ASSOCIATED CONTENT Supporting

Information.

Additional

experimental

details,

CV

and

DPV

analysis,

characterization of the nanoprobe Ru(BPY)3@MnO2, TGL assay of GSH in human sera, cytotoxicity of the nanoprobe and Mn2+, phosphorescence imaging of GSH in HepG2 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments. Financial supports from the National Natural Science Foundation of China (Grant Nos. 21475016, 21477011) and the Fundamental Research Funds for the Central Universities (Grant No. DUT16LAB10) are gratefully acknowledged.

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Biomedical Applications. Biomaterials 2009, 30, 2180-2198.


A Bimodal Phosphorescence-Magnetic Resonance Imaging

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