Construction of the FRET Pairs for the visualization of mitochondria

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Construction of the FRET Pairs for the visualization of mitochondria membrane potential in dual emission colors Ruiqing Feng, Lifang Guo, Jinglong Fang, Yue Jia, Xueying Wang, Qin Wei, and Xiaoqiang Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05822 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 6, 2019

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Analytical Chemistry

Construction of the FRET Pairs for the visualization of mitochondria membrane potential in dual emission colors Ruiqing Fenga, Lifang Guob, Jinglong Fanga, Yue Jiaa, Xueying Wanga, Qin Weia, *, and Xiaoqiang Yua,b,* a

Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China. Email: [email protected]. b Center of Bio & Micro/Nano Functional Materials, Sate Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, P. R. China. Email: [email protected].

ABSTRACT: Mitochondria membrane potential (MMP) play significant roles during metabolism, signaling, and other important bioevents. Visualization of MMP levels is essential for many biological researches. However, fluorescent probes for monitoring MMP levels in dual emission colors are still deficient, which greatly limited the development of relative research areas. In this work, a pair of fluorescent probes have been designed and synthesized to monitor the MMP levels in dual emission colors based on Forster resonance energy transfer (FRET) mechanism. The FRET donor (FixD) is constructed by linking a benzyl chloride group to a fluorophore with bright green emission. The FixD could target mitochondria and be immobilized in mitochondria by linking to the thiol group of mitochondrial proteins. The FRET acceptor (LA) is designed with green absorption and deep-red emission. In live cells with high MMP levels, FixD and LA both target mitochondria, and deep-red (DR) emission could be detected with the excitation of 405 nm. Particularly, the spectral shift of fluorescence upon the decrease of MMP is up to 110 nm, which is greatly favorable for the clear observation of MMP levels. With the decrease of MMP, LA would be released from mitochondria while FixD be still immobilized in mitochondria, and decreased DR emission and increased green fluorescence could be detected due to the absence of FRET. In this manner, the MMP levels could be monitored in dual emission colors.

INTRODUCTION Mitochondria play crucial roles to supply the majority of energy for various biological activities.1-4 Aerobic respiration is the central part to generate the energy in the form of adenosine triphosphate (ATP).5-6 During an aerobic respiration cycle, mitochondria pumps two protons into the matrix while release four out to the cytoplasm, and therefore a negative mitochondrial membrane potential (up to -180 mV) is generated across the mitochondria inner membrane.1, 7-9 The mitochondria membrane potential (MMP) could promote the metabolism process, and is also involved in other biological events such as signaling.10-12 Particularly, MMP levels can sensitively and reversibly reflect the intracellular heathy status, which would be rapidly deceased during the apoptosis and necrosis processes.13-16 Therefore, visualization of MMP levels could promote the fundamental researches in biology and pathology. Analysis of MMP levels is always a difficult work, because the size of mitochondria is too small for the insertion of microelectrode.17 The accumulation of membrane-permeable cations into mitochondria provide possible methods for the monitoring of MMP.18-22 Up to now, fluorescent probes based on the organic cations serve as the most important tools for the in-situ and dynamic visualization of MMP levels. For example, tetramethyrhodamine (TMRM) with a positive charge has been used to quantify MMP via its distribution of fluorescence signals.23-24 Tang and co-workers have reported a fluorescent probe for the detection of decreased MMP based on AIEgens.25 Recently, Yoon et al. have also presented a fluorescent probe for MMP changes based on tetraphenyl ethylene.26 Our group has also constructed a fluorescent probe for MMP, based on its

localization change between mitochondria and nucleus.27 However, all of these probes response to MMP levels based on the fluorescence intensity, and their fluorescence wavelength never changes upon MMP variation. The inhomogeneous staining and the fluctuation of the power of excitation source would inevitably bring interferences. Up to now, very few fluorescent probes could detect MMP in a dual-color mode. JC-1 and its derivatives have been exploited to response to MMP in dual colors.28 JC-1 could form Jaggregates with red-shifted emission (595 nm) in mitochondria with high membrane potential, while presents as green monomers (525 nm) in mitochondria with decreased MMP.29 Therefore, JC-1 could be used to label mitochondria with high and low MMP in different colors. Moreover, commercial Mitotracker green (MTG, 510 nm) and TMRM (580 nm) have been utilized as the FRET pair to detect MMP levels.30 MTG could be immobilized into mitochondria independent on MMP,31 while TMRM could target mitochondria with high MMP and be released from mitochondria with decreased MMP.32 Therefore, MTG and TMRM could be used to detect mitochondrial membrane potential in dual emission colors. However, the emission shift of the JC-1 dyes and MTG-TMRM pair upon the MMP variation in not larger than 70 nm, which limited the clear imaging of MMP fluctuation under fluorescence microscopy. Consequently, construction of fluorescent probes responding to MMP with larger emission shift is greatly demanded. In this work, a FRET pair of fluorescent probes (FixD and LA) have been designed and synthesized, as shown in Scheme 1 (The synthetic details in Supporting Information). Both the two probes contain cationic groups to target mitochondria,

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while the FRET donor (FixD) contain additional phenyl chloride group to localize onto mitochondria. The FRET pair displayed deep-red emission (675 nm) in mitochondria with high MMP, while showed green fluorescence (565 nm) with decreased MMP. The emission shift is ~ 110 nm, larger than the presented probes (~ 70 nm). The FRET probes have been successfully utilized to image the decreased MMP of A549 cells treated with H2O2 and chloral hydrate.

Scheme 1. The design rationale and chemical structures of fluorescent probes for MMP detection.

EXPERIMENTAL SECTION Materials All chemicals used are of analytical grade, phosphorus oxychloride, carbazole and 4-methylpyridine were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). N,N-dimethylaniline, 5-methylquinoline, (chloromethyl)benzene and 1,4-bis(chloromethyl)benzene were purchased from J&K Chemical (Beijing, China). (1, 3dioxolan-2-yl) methyl)–triphenylphosphonium and piperidine were purchased from Aladdin Co (Shanghai, China). The solvents used in the synthesis and spectral measurement are of chromatographic grade. Ultrapure water was used throughout. TLC analyses were performed on silica gel plates and column chromatography was conducted over silica gel (mesh 200-300), both of which were obtained from Qingdao Ocean Chemicals. Apparatus and methods Nuclear magnetic resonance spectra (NMR) were obtained on a Bruker Avanace 400 spectrometer. The HRMS spectra were recorded on Agilent Technologies 6510 Q-TOF LC/MS. The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a HITACH U-2910 spectrophotometer using a quartz cuvette having 1 cm path length. Fluorescence spectra were obtained on a HITACH F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. PBS buffer solution: 10 mM NaCl, Na2HPO4·12H2O, NaH2PO4·2H2O, pH = 7.40. Confocal fluorescence imaging was obtained with LSM 780 (Zeiss) or IX83 (Olympus) confocal laser scanning microscope. Cell culture and staining A549 cells were grown in H-DMEM (Dulbecco’s Modified Eagle’s Medium, High Glucose) supplemented with 10% FBS (Fetal Bovine Serum) in a 5% CO2 incubator at 37 °C. Cells (1×105 / mL) were placed on glass coverslips and allowed to adhere for 24 h.

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For living cells imaging experiment of the probes, the culture medium surrounding the cells were firstly removed, and the cells were washed with PBS twice. Then the cells were incubated in 1 mL of PBS. On the other hand, 1 mM stock solutions of the two probes in DMSO were prepared. After that, 2 μL of stock solutions were mixed evenly with 1 mL PBS (pH 7.4) in a tube. The cells were incubated with the above mixed solutions for 30 min at 37 °C. After rinsing with PBS three times, cells were imaged immediately. The confocal microscopic image and differential interference contrast (DIC) image were taken with a 488 nm Argon laser in ZEISS LSM 780 or Olympus IX83 confocal microscope. Co-localization experiment First, A549 cells were incubated with 2 µM FixD or LA and 0.2 µM MTDR or MTG in culture medium for 30 min and then observed with fluorescent microscope. Then, incubated with 20 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 30 min, and were observed by fluorescent microscope finally. Changing process staining experiment Living A549 cells were incubated with FixD and LA (2 µM) for 30 min, and then these cells were treated by CCCP (5 µM) or H2O2 (50mM) or chloral hydrate (0.5 wt ‰) at different time. Before adding CCCP or H2O2 or chloral hydrate, the unbound probes were removed by washing. Spectroscopic Measurements The fluorescence quantum yields can be calculated by the following equation (1):

 A    c n 2 F  s   r  r r  r s2 s  As s   cs nr Fr

(1) where the subscripts s and r refer to the sample and the reference materials, respectively. Ф is the quantum yield, F is the integrated emission intensity, A stands for the absorbance, and n is the refractive index. In this paper, the quantum yields were calculated by using fluorescein in aqueous NaOH (pH = 13, Ф = 0.93) as a standard.33-34

RESULTS AND DISCUSSION Design of the fluorescent probes for MMP In this work, a FRET pair was designed for the visualization of MMP levels in dual emission colors, as shown in Scheme 1. Obviously, the FRET donor should target mitochondria, and could be immobilized into mitochondria irrespective of the MMP fluctuation. Moreover, the donor should exhibit bright fluorescence. Therefore, carbazole-pyridium system was used to construct the FRET donor (FixD), via the linkage to a benzyl chloride group. The system was reported to emit green fluorescence around 560 nm with the excitation of 405 nm. The FRET acceptor should also target mitochondria with high MMP, but it should be relocalized dependent on the MMP levels in order to detect the MMP. Consequently, the FRET acceptor should contain a cationic group while no mitochondria-immobilized groups. Moreover, the acceptor should exhibit absorption peak around 560 nm, and the Stokes shift should be as large as possible to enlarge the spectral separation from the FixD. Consequently, the FRET acceptor was designed as LA shown in Scheme 1. LA exhibits absorbance around 560 nm, and emission around 675 nm. Phenyl methyl group was used as the sidechain to increase its permeability to the membranes. Moreover, the D-π-A structure


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and the vinyl group could endow LA rotor properties, which only showed intense emission in high-viscosity environments and could image mitochondria with high-viscosity with highfidelity. With the FRET pair (FixD-LA), the mitochondria membrane potential could be visualized in dual emission colors. Optical properties of FixD and LA 1.2

FixD-Abs

LA-Abs

FixD-Em

LA-Em

intracellular fluorescent signals from probe FixD mainly distributed in the cytoplasm, and presented in filament morphology. The results indicated that the probe FixD may stain the mitochondria in live cells. To confirm the selectivity, the commercialized mitochondrial probe, mitotracher deep-red (MTDR), was used to perform the colocalization experiments with FixD. As shown in Figure 2B, the green signals from FixD overlapped well with the red signals from MTDR, and the colocalization coefficient was up to 0.85, demonstrating that FixD actually targets mitochondria in live cells.

Fluorescence

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Absorbance

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Analytical Chemistry

0.8 0.6 0.4 0.2 0.0 350

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550

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Wavelength [nm]

Figure 1. The normalized absorption and emission spectra of FixD (in ethanol) and LA (in glycerol).

The absorption and emission spectra of FixD and LA were initially acquired, as shown in Figure 1. The probe FixD showed absorption around 450 nm, and emission around 565 nm. FixD displayed intense emission in ethanol, and the quantum yield was measured to be around 0.16 (Фf = 0.93 for fluorescein in NaOH solutions as the standard, pH = 13). On the other hand, probe LA displayed absorption around 550 nm, and deep-red emission peaked around 675 nm. Moreover, as shown in Figure S1, the probe showed very weak fluorescence in lowviscosity medium such as the ethanol. The emission intensity gradually enhanced with the increase of solvent viscosity. Therefore, LA is potential to image the high-viscosity mitochondria with high fidelity. The FRET ability could be estimated between FixD and LA based on the emission spectra of FixD and the absorption spectra of LA. The Forster distance (R0) could be calculated by means of the spectra according to the following equation,35 (2)

(3) where is the factor to describe the relative orientation in space of the transition dipoles of the donor and acceptor, which is usually assumed to be 2/3; n is the refractive index of the medium, which is assigned as 1.33 here; QD is the quantum yield of the donor; FD is the fluorescence intensity of donor relative to λ; εA is molar extinction coefficient of the acceptor relative to λ. The R0 was calculated to be 36 Å, indicating that the FRET could easily occur between FixD and LA. As the qualified fluorescent probes for cell imaging applications, the interferences from various substrates should be excluded. As shown in Figure S2, with the addition of 1 mM substrates, the emission of FixD and LA would not be affected. These results indicate that the fluorescence of FixD and LA is hardly influenced by biological molecules. Intracellular localization of FixD and LA Live A549 cells were stained with FixD to check testify its localization in live cells. As shown in Figure 2A, the κ2

Figure 2. The DIC and fluorescent images of A549 cells. (A) Live A549 cells incubated with 5 μM FixD for 30 min; (B) live A549 cells co-stained with 5 μM FixD and 200 nM MTDR for 30 min; (C) live A549 cells incubated with 5 μM FixD and 200 nM MTDR for 30 min then treated with 20 μM CCCP for 30 min. Green Channel: λex = 405 nm, λem = 510-550 nm; DR Channel: λex = 647 nm, λem = 665-735 nm, Bar=20µm.

Considering that FixD should be retained in mitochondria independent on the decrease of MMP levels, the selectivity of FixD upon the decrease of MMP should be testified. The commercial probe MTDR is also equipped with phenyl chloride group, and could be immobilized in mitochondria, irrespective of the decrease of MMP. Therefore, live A549 cells were costained with FixD and MTDR, then treated with Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a protonophore that can efficiently decrease the MMP levels.36 As shown in Figure 2C, after the treatment with CCCP for 30 min, the fluorescent signals from FixD also overlapped well with that of MTDR. The colocalization coefficient is up to 0.89. These results demonstrated that FixD could still selectively stain the mitochondria with low MMP levels. Probe LA was then used to stain live A549, to confirm its subcellular selectivity. As shown in Figure 3, similar to FixD, the intracellular fluorescence signals of LA distributed in the cytoplasm, and presented in filament morphologies. The results implied that LA may target mitochondrial in live cells. The selectivity of probe LA in live cells was then confirmed with co-localization experiments with MTG, a commercialized fluorescent probe for mitochondria. As shown in Figure 3, the fluorescence from LA overlapped well with that of the MTG,



and the colocalization coefficient was 0.89, demonstrating that the probe LA actually target mitochondria in live cells.

Figure 3. The DIC and fluorescence images of A549 cells incubated with 2 μM LA for 30 min (top); the fluorescence images of A549 cells co-stained with 2 μM LA and 200 nM MTG (bottom). Green Channel: λex = 488 nm, λem = 510-550 nm; DR Channel: λex = 647 nm, λem = 665-735 nm, Bar=20µm.

Visualization of MMP decrease induced by CCCP Since FixD and LA could successfully target mitochondria in live cells, and FixD could be immobilized in mitochondria independent on the decrease of MMP levels, the FRET pair was then used to image the decrease of MMP levels. As shown in Figure 4, live cells incubated with FixD and LA display intense emission in both green and DR channels, with the excitation of 405 nm. In comparison, the cells solely stained with LA show no fluorescence under the same experimental conditions (Figure S3). Therefore, the fluorescence signals in the DR channel comes from the FRET process. With the addition of CCCP and the decrease of MMP levels, the fluorescence in the green channel obviously enhanced, and the fluorescence in the DR channel slightly weakened. These results indicate the decrease of FRET efficiency. The change could be clearly observed from the merged images of the two

channels. Consequently, the FRET pair could be used to detect the decrease of MMP levels.

Figure 4. The fluorescent images of live cells stained with FixD and LA for 30 min then treated with 5 μM CCCP for different time. λex = 405 nm, Green Channel: λem = 510-550 nm; DR Channel: λem = 665-735 nm, Bar=20µm. b): The time-dependent intensity ratio of green to deep-red channels.

Application in the detection of cell viability The MMP levels are extremely sensitive to the cellular healthy status, and thus the FRET pair should be able to detect the cell viability. The hydrogen peroxide (H2O2) is a byproduct of the aerobic respiration, and could in-return inhibit the metabolism and induce cell death.37 Based on these considerations, the live A549 cells were initially stained with the FRET pair, and then treated with H2O2. As shown in Figure 5 and video S1, the live A549 cells stained with the FRET pair show weak fluorescence in green channel, and strong emission in the DR channel. After the addition of H2O2, the cells were damaged, and the MMP levels were decreased. In response, the fluorescence signals of the green channel gradually and obviously enhanced, while the emission intensity in the DR channel steadily decreased. The variation of the fluorescence signals could be clearly observed via the merged images of the green and DR channels. These results demonstrated that the FixD-LA pair could be used to clearly observe the cell viability change induced by the H2O2.

Figure 5. The fluorescence images of A549 cells pre-stained with FixD and LA, then treated with 50 mM H2O2 for different time. λex = 405 nm, Green Channel: λem = 510-550 nm; DR Channel: λem = 665-735 nm, Bar=20µm. b): The time-dependent intensity ratio of green to deepred channels.


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Analytical Chemistry Since the FixD-LA pair could detect the decrease of MMP levels and the decrease of cell viability, the pair was applied to study the effect of chloral hydrate on cell viability. Chloral hydrate is widely used as a general anesthetic in veterinary medicine, and also used for the short-term treatment of insomnia.38 Live A549 cells were subsequently prestained with the FixD-LA pair and then treated with chloral hydrate. As shown in Figure 6, the live A659 cells stained with the FRET pair shown weak emission in the green channel, and strong emission in the DR channel. After the addition of chloral hydrate, the fluorescence in the green channel obviously enhanced, and that in the DR channel kept almost unchanged. The emission color change could be clearly observed via the merged channel of green and DR ones. Therefore, chloral hydrate could induce the decrease of MMP levels, and may cause the damage of live A549 cells.

Figure 6. The fluorescence images of A549 cells pre-stained with FixD and LA, and the treated with 0.5 ‰ chloral hydrate for different time, λex = 405 nm, Green Channel: λem = 510-550 nm; DR Channel: λem = 665-735 nm, Bar=20µm. b): The timedependent intensity ratio of green to deep-red channels.

CONCLUSION In summary, a FRET pair (FixD and LA) were rationally designed and synthesized for the visualization of MMP levels. A FRET donor bright emission and a FRET acceptor with large Stokes shift were used to construct the probes, and the emission spectra of the FRET donor showed large overlap with the absorption spectra of the acceptor. The donor was linked with a phenyl chloride group, and could be immobilized in mitochondria. As a result, both FixD and LA can target the mitochondria in healthy cells with high MMP, and FixD can still target mitochondria after the decrease of MMP. The FixD-LA pair could detect the decrease of MMP levels in live cells, and the emission spectral shift is up to 110 nm, larger than the current ones. The probes were successfully used to visualize the cell damage and MMP decrease induced by hydrogen peroxide and chloral hydrate. We believe that the probes can serve as powerful tools to promote the research areas relative to mitochondria membrane potential and cell viability.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis of details, spectra, imaging assays, NMR(1H) and HRMS spectra, etc. were placed in it. Video S1. The live A549 cells were initially stained with the FRET pair (FixD-LA), and then treated with H2O2. Real-time tracking confocal fluorescence imaging for 11 minutes were shown in Video S1 (λex = 405 nm).

AUTHOR INFORMATION

Corresponding Author * E-mail: [email protected].

* E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was supported by the National Key Scientific Instrument and Equipment Development Project of China (No.21627809), National Natural Science Foundation of China (Nos. 21575050, 21777056, 51773111) and the startup fund of University of Jinan (301- 1003849).

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Construction of the FRET Pairs for the visualization of mitochondria

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