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A Single fluorescent probe for Dual-imaging Viscosity and H2O2 in Mitochondria with Different Fluorescence Signals in Living Cells Mingguang Ren, Beibei Deng, Kai Zhou, Xiuqi Kong, Jian-Yong Wang, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04385 • Publication Date (Web): 07 Dec 2016 Downloaded from http://pubs.acs.org on December 9, 2016

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Analytical Chemistry

A Single fluorescent probe for Dual-imaging Viscosity and H2O2 in Mitochondria with Different Fluorescence Signals in Living Cells Mingguang Ren, Beibei Deng, Kai Zhou, Xiuqi Kong, Jian-Yong Wang and Weiying Lin * Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P. R. China. Fax: (+) 86-531-82769031, E-mail: [email protected]. ABSTRACT: Mitochondria, as essential and interesting organelles within the eukaryotic cells, play key roles in a variety of pathologies, and its abnormalities are closely associated with Alzheimer's disease (AD) and other diseases. Studies have shown that the abnormal of viscosity and concentration of hydrogen peroxide in mitochondria were all associated with AD. Accordingly, the detection of viscosity and hydrogen peroxide in mitochondria has attracted great attention. However, it remains great challenge to explore a single probe, which can dual-detect the viscosity and H2O2 in mitochondria. Herein, in two ways to prevent the twisted internal charge transfer (TICT) process, we designed and sythesized the first dual-detection fluorescent probe Mito-VH that can visualize viscosity and H2O2 in mitochondria with different fluorescence signals in living cells.

Cellular viscosity, as one of the major parameters, strongly influences intracellular biomolecules interact, chemical signals transportation as well as diffusion of reactive metabolites within live cells. Changes of cellular viscosity have been linked to disease and malfunction at the cellular level.1 Mitochondrion is the energy-producing compartments in cells. Changes in mitochondrial matrix viscosity may modulate metabolite diffusion and mitochondrial metabolism by decreasing the mitochondrial membrane fluidity, reducing electron transport chain (ETC) activating.2 Hydrogen peroxide (H2O2) plays important roles in cell growth, proliferation, host defense, immune responses and signalling pathway under physiological conditions.3-5 In living cells, H2O2 is generated from activation of NADPH oxidase complexes.6 At the cell organelles level, mitochondria is a major source of H2O2 and a primary cellular compartment of oxygen consumption.7,8 Mitochondrial H2O2 has both beneficial and detrimental side for organisms. On one hand, mitochondrial H2O2 can also serve beneficial roles for cell survival, growth, differentiation, and maintenance.9-12 However, on the other hand, over production of H2O2 can cause many diseases, such as inflammatory disease, cardiovascular disease, Alzheimer's disease and cancer.13-17 Notably, studies have indicated that both the viscosity of the mitochondrial membrane and the abnormality of H2O2 production in the mitochondria all related to the amyloid betapeptide (Aβ) accumulation and Aβ accumulation is considered to be a key pathogenic factor in sporadic Alzheimer’s disease (AD).18 Mecocci and coworker had found that membrane fluidity of mitochondria extracted from AD brains was significantly reduced in compared to controls, and the reduction of membrane fluidity in mitochondrial may be secondary to lipid

peroxidation caused by ROS.19 Therefore, there is a substantial need for simple but reliable and precise techniques to dualdetect viscosity and H2O2 production in mitochondria. Compared with other analytical techniques methods, fluorescence imaging has become a powerful tool to monitor biomolecules and biological parameters in living systems due to its high sensitivity, noinvasive detection, high selectivity, as well as real-time imaging. Recently, there have been many outstanding outcomes for imaging viscosity and H2O2 in mitochondria individually.20,21 However, to the best of our knowledge, a single fluorescent probe which capable of dualsensing of viscosity and H2O2 in mitochondria has never been reported. Compared with combination of several fluorescent probes in one system, a single fluorescent probe with the ability of sensing multiple targets have several advantages, such as it can avoid cross-talk, different localizations, and different metabolisms, which induced by the combination use of several fluorescent probes. Therefore, some research groups have made significant achievements in the identification of multiple analytes using a single fluorescent probe.22-24 Howerver, it is still interesting to develop a probe which could image mitochondrial H2O2 and also could provide the information of the viscosity in mitochondria.

Scheme 1. Rational design of dual-detection fluorescent probe Mito-VH

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Herein, we report the first dual-detection fluorescent probe (Mito-VH) which not only can detect the hydrogen peroxide in mitochondria but also can respond to the change of viscosity in mitochondria with different imaging channels. The probe has high selectivity of H2O2 over other reactive oxygen, nitrogen, sulfur species and highly sensitivity (around 67-fold). In addition, with the change of solution viscosity, the spectra of the probe have obvious changes (around 16-fold fluorescence enhanced). Besides, the cell imaging confirmed that the probe Mito-VH can be used to monitor the level of H2O2 and viscosity changes in mitochondria with a high colocalization coefficient compared with mitochondrial location dye. Intramolecular rotation was a kind of characteristic of TICT molecule. In addition, the emission from the zero vibrational level of the TICT state is forbidden, which make TICT emission typically weak and low background signal, thus benefiting design off-on response fluorescent probe with high detection sensitivity. TICT molecules usually have “D-π-A” molecular configuration with strong electron withdraw group (Acceptor) and strong electron donor group (Donor). Pyridinium cation act as strong electron withdraw group widely used to synthesis of dyes and fluorescent probe.25, 26 In addition, pyridinium cation as mitochondrial targeting group possesses superior membrane permeability so they could rapidly enter the mitochondria in cells in a short period of time.20 N,N-dimethylamino act as strong electron donor group be used in the designing of probe Mito-VH, which also applied in many TICT molecules,2730 including the first case TICT molecule DMABN reported by Lippert et al.31 Double bond and benzene used as a connector to connect the electron donor and acceptor, which can make the geometry of the probe molecular free rotation in the excited state. As a reaction site of H2O2, boronate was widely used in fluorescent H2O2 probe because of its good selectivity. Combined with the above elements, we rationally designed and synthesized the first dual-detection fluorescent probe which can be used to monitor the level of H2O2 and viscosity changes in mitochondria. The design and synthesis of probe Mito-VH was outlined in Scheme 1 and Scheme S1. Compound 1 was prepared according to a reported literature.32 The synthesis of target compound Mito-VH was relatively simple only one step reaction. The structure of the target compound synthesized was characterized unambiguously by standard 1HNMR, 13C-NMR and HRMS in supporting information. With the probe Mito-VH in hand, we first evaluated its optical response to viscosity. To find out whether the restriction of intramolecular rotation by increasing the viscosity of the solvent will enhance its fluorescence, we studied fluorescence behaviour of probe Mito-VH in different EtOH with glycerol fractions, and found that Mito-VH exhibits weak emission band (Ф=0.023), when excited at 500 nm in EtOH (Fig. 1A). However, with increase the proportion of glycerol, affording a higher viscosity, the corresponding emission intensity of Mito-VH was significantly enhanced and showed a quantum yield of 0.27 in the solution of 95 % glycerol, approximately 16-fold enhancement of intensity from pure ethanol to 95 % glycerol. A good linear relationship existed between log (I607) and log (viscosity) with a correlation coefficient of 0.99 (Fig. S1, ESI†), indicating the probe Mito-VH can be used for quantitative determination of viscosity.

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Moreover, we also utilized density functional theory (DFT) calculations to prove the existence of intramolecular rotation of the probe in the excited state (Fig. S2, ESI†). DFT calculations show that the probe Mito-VH in the ground state, N,Ndimethylamino benzene and pyridine moieties are almost planar leading to efficient electron conjugation between the two moieties. However, in excited state, the N,N-dimethylamino benzene group against the pyridine plane until it is twisted about 90o and conjugation between the two parts is lost, producing the TICT excited state. Since the rotation of the N,Ndimethylamino benzene group in formation of TICT state is necessary, the probe Mito-VH is very unique sensitive to microenvironments, especially solution viscosity. Whereas the rotation is restricted in highly viscous media, which will prevent the formation of TICT states, and the excited energy, is reserved for emission without nonradiative energy dissipation throughout TICT states. The results indicated that based on TICT mechanism the probe Mito-VH could be used as an efficient fluorescent viscosity probe.

Figure 1. (A) The fluorescence spectra of Mito-VH (5 µM) in different ratios of ethanol (E)–glycerol (G) mixtures, (λex = 500 nm); (B) Fluorescence spectra of Mito-VH (5 μM) treated with H2O2 (0–50 μM) at 37 oC (λex = 400 nm).

After proving the probe Mito-VH could use as a viscosity fluorescent probe, we studied another function of the probe in recognition of H2O2. The matrix in mitochondria sustains an alkaline pH under physiological conditions.33 In order to simulate the environment in mitochondria, we tested the optical properties of the probe in the absence or presence of H2O2 in PBS buffer pH 8.4 at 37oC. Through investigated the effect of pH on the fluorescence intensity of compound 1 and probe Mito-VH, weak alkaline conditions were favorable for the imaging of hydrogen peroxide (Fig. S3, ESI†). Mito-VH has strong absorption in the visible region (Fig. S4, ESI†). However, upon addition of increasing concentrations of H2O2 (0–50 µM), the absorption at 483 nm was gradually decreased, and the absorption peak at 378 nm increased. At the same time, there is an equal absorption point at 410 nm. In good agreement with the absorption spectra, as shown in Fig. 2A, upon excitation at 400 nm, the free probe Mito-VH has a weak fluorescent emission due to TICT process. However, upon addition of H2O2, the fluorescence of Mito-VH at 510 nm increased dramatically. Notably, the probe has a large emission shift about 100 nm between the emission peak of the response of viscosity and the emission peak of the response of H2O2, which is desirable for the detection of the two peaks intensities due to the small spectral overlap after the probes responding to different analytes. At the same time, it is also beneficial for the probe for fluorescence imaging the H2O2 and viscosity in different channels. The emission intensity and the concentrations of H2O2 in range of 0-30 µM shows a good linear relationship, which is indicated that the probe Mito-VH is suitable for quantitative determination of H2O2 (Fig S5, ESI†). The probe

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showed relatively high sensitivity to H2O2 with a detection limit of 2.1×10-6 M under the experimental conditions, which is much lower than the physiological H2O2 concentration in inflammation of the organization (10-100 µM),34, 35 indicating that the probe Mito-VH is sensitive enough to determine the lesion site H2O2. In addition, the probe Mito-VH has highly selective for H2O2 over other reference species (Fig. S6, ESI†). To confirm the sensing mechanism, the product of reaction of Mito-VH and H2O2 was isolated and confirmed to be compound 1 by 1H-NMR (Fig. S9, ESI†). By DFT theoretical calculation, the mechanism of the “Turn-On” fluorescent response of probe Mito-VH to H2O2 could be easy explained (Fig. S2, ESI†). Due to the ability of the electron withdraw group has a significant change in the molecule before and after reacted with H2O2, in excited state of the reaction product cannot form the TICT state, which forms the ICT state and makes the product have a strong fluorescence emission. The standard MTT assay indicates that the probe Mito-VH has no marked cytotoxicity to the cells after a long period at low micromolar concentrations (Fig. S7, ESI†), implying that the probe Mito-VH less toxic to cells can be used for cell imaging. To examine the subcellular location of Mito-VH, the commercially available mitochondrial dye, Mito Tracker Deep Red, was employed for a colocalization study. Colocalization results obtained using a confocal laser microscope with MitoVH and Mito Tracker Deep Red are shown Fig. S8. The experiments showed that Mito-VH could readily enter HeLa cells and give clear fluorescence image in TRITC channel (λex= 561 nm; λem =570-620 nm). Mitochondrial Tracker imaging of mitochondria was shown in the Cy5 channel (λex= 640 nm; λem = 663-738 nm). The merged image (Fig. S8d) indicated that the staining of Mito-VH fits well with that of mitochondrial tracker. Moreover, the intensity scatter plot of TRITC channel and Cy5 channel is high correlationship with a high overlap Pearson´s coefficient of 0.93. As expected, the experiment indicates that the probe Mito-VH mainly localized in the mitochondria.

Figure 2. (A) Confocal laser fluorescence imaging of Mito-VH (5.0 µM) in HeLa cells. (a) Brightfield image of HeLa cells costained only with Mito-VH; (b) Fluorescence images of (a) from TRITC channel; (c) overlay of (a) and (b); (d) Brightfield image of MitoVH in HeLa cells upon treatment with nystatin (10 µM); (e) Fluorescence images of (d) from TRITC channel; (f) overlay of the brightfield image (d) and TRITC channels (e). (B) Quantified relative fluorescence intensities for (A). Statistical analyses were performed with Student’s t-test (n = 3) and the error bars represent standard deviation (±S.D.).

To validate whether probe Mito-VH could detect mitochondrial viscosity changing by fluorescence imaging, we use nystatin as ionophore to change the viscosity in mitochondria. nystatin is well-known to induce mitochondrial malfunction

caused by structural changes or swelling of mitochondria through interruption of the ionic balance.36, 37 As shown in Fig. 2, the HeLa cells incubated with only the probe (5 µM) show a weak fluorescence. However, when the cells were pre-treated with nystatin (10 µM) for 30 min and then incubated with Mito-VH (5 µM) solution for another 30 min, displayed about 1.5-fold turn-on in the integrated intracellular fluorescence of the sensor from the TRITC channel at the same test conditions. The increased viscosity in ionophore-treated mitochondria agrees with previous findings, indicating that these ionophores can induce ultrastructural changes or swelling.36, 38 Furthermore, the cuvette experiment with the addition of nystatin to probe Mito-VH in PBS did not produce an increase in fluorescence (Fig. S9, ESI†), indicating that intracellular fluorescence increase is attributed to viscosity increase in mitochondria and the probe Mito-VH could be used as a mitochondrial viscosity probe in the context of metabolic changes in mitochondria. We examined whether Mito-VH could be used as a probe to image of H2O2 in living cells. As shown in Fig. S10, Mito-VH (5 µM) was initially incubated with HeLa cells for 20 min, and then washed three times by PBS buffer (pH 7.4). Results show that the HeLa cells incubated with only the probe Mito-VH exhibited almost no fluorescence in FITC channel (Fig. S10b; λex = 488 nm; λem = 500−550 nm). In contract, , HeLa cells, which incubated with 5.0 μM probe Mito-VH for 20 min and then treated with 30 μM H2O2 for another 30 min, display strong fluorescence at the same test conditions. Colocation experiment showed that the product, which generated by the reaction of probe Mito-VH with H2O2, was mainly distributed in the mitochondria, and the Pearson´s coefficient is 0.92 compared with mitochondrial location dye (Fig. S11, ESI†). These data established that the probe Mito-VH is cell membrane permeable and could be used as a fluorescence probe to image exogenous H2O2 in mitochondria.

Figure 3. Imaging of endogenous H2O2 in RAW 264.7 cells stained with the probe Mito-VH (a) Brightfield image of RAW 264.7 macrophages cells costained with Mito-VH; (b) Fluorescence images of (a) from FITC channel; (c) from TRITC channel (d) overlay of (a), (b) and (c); (e) Brightfield image of RAW 264.7 macrophages cells stimulated with PMA (3.0 μg/mL) and costained with Mito-VH, (f) Fluorescence images of (e) from FITC channel; (g) from TRITC channel; (h) overlay of (e), (f) and (g).

We further examine the feasibility of the probe Mito-VH to detect endogenous produced H2O2 in living macrophage cells， and whether the production of hydrogen peroxide will affect the viscosity of mitochondria (Fig. 3). When stimulated by phorbol myristate acetate (PMA), macrophage cells could produce endogenous H2O2.39 The living RAW 264.7 macro-

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phage cells loaded with only the sensor Mito-VH (5.0 µM) displayed almost no fluorescence in the FITC channel, but in TRITC channel a clear fluorescence image was presented (Fig. 3c). However, the macrophage cells co-treated with PMA (3.0 μg/mL) and the sensor Mito-VH (5.0 µM) for 6 h exhibit a dramatic enhancement in the FITC channel emission (Fig. 3f). At the same time, the fluorescence intensity from the TRITC channel enhanced 1.2 times (Fig. 3g), meaning that the generation of H2O2 may lead to viscosity increased in mitochondria. These data indicated that the sensor Mito-VH is capable dualimaging viscosity and endogenous H2O2 in the living RAW264.7 macrophage cells. In summary, the probe Mito-VH was developed as the first dual-detection fluorescent probe which could respond to viscosity and H2O2 with large turn-on fluorescence signal around 607 nm and 510 nm respectively by two ways to prevent the TICT process of the probe. Fluorescence imaging shows that Mito-VH was membrane-permeable and could be applied for sensing of both viscosity and H2O2 in mitochondria in living cells with two channels imaging.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis of the probes, absorption and fluorescence spectra, imaging assays, 1H NMR and 13 C NMR spectra. (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by NSFC (21472067, 21502067, 21672083), the Natural Science Foundation of Shandong Province, China (ZR2014BP001), Taishan Scholar Foundation (TS 201511041), and the startup fund of University of Jinan (309-10004, 160082101).

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