Aggregation-Induced Fluorescence Probe for Monitoring Membrane

Nov 20, 2017 - Fluorescent probe 2 was designed for selectively determining mitochondria membrane potential changes. The probe selectively detects cha...
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Aggregation-Induced Fluorescence Probe for Monitoring Membrane Potential Changes in Mitochondria Jun Li,†,‡ Nahyun Kwon,† Yerin Jeong,† Songyi Lee,† Gyoungmi Kim,† and Juyoung Yoon*,† †

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea Department of Chemistry, College of Science, Huazhong Agriculture University, Wuhan 430070, China

‡

S Supporting Information *

ABSTRACT: Fluorescent probe 2 was designed for selectively determining mitochondria membrane potential changes. The probe selectively detects changes in the mitochondria membrane potential in a manner that is more sensitive than that of the commercially available indicator, Rodamine 123. As a result, the probe 2 is ideal for future studies designed to assess the functions of mitochondria in cells. KEYWORDS: AIE, fluorescent probe, mitochondria, membrane potential, cell apoptosis

1. INTRODUCTION The electrical potential of the mitochondria membrane is an important physiological parameter employed to monitor health states of cells.1−5 Recently, classical fluorescent dyes, which contain TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethylrhodamine ethyl ester), Rhodamine 123 (Rh 123), and JC-1 cationic groups, have been developed as membrane potential indicators.6−8 However, these probes suffer from limitations including high cytotoxicity, low selectivity, and poor photostability. Therefore, new probes for monitoring changes in membrane potentials, which do not suffer from these limitations, are in great demand. Recent efforts have shown that aggregation-induced emission (AIE) dyes, discovered by Tang and his co-workers, possess many advantageous features that are not shared by counterparts that display aggregation-caused quenching (ACQ).9 For example, AIE fluorophores are nearly nonfluorescent in the solution state yet they display concentration dependent fluorescence in aggregate states.10−14 Recently, Peng and Yuan’s group summarized the development of targetable fluorescent probes that allow subcellular imaging and sensing in organelles including mitochondria.15,16 By introducing a mitochondrial targetable group on an AIE species, it is possible to create a highly photostable fluorescence luminogen that can be utilized in studies of the biological functions of mitochondria.17 Recently, Tang and his co-workers developed the AIE luminogen 1 (Figure 1), which bears two triphenylphosphonium (TPP) groups, that can be employed for © XXXX American Chemical Society

Figure 1. Structures of TPP-based probe 1 and 2.

specific mitochondrial imaging and tracking.9 Moreover, Tang showed that 1 undergoes highly specific binding to mitochondria, and that it has superior photostability and appreciable tolerance to environmental changes. Even when treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a chemical inhibitor of oxidative phosphorylation that induces a decrease in the mitochondrial membrane potential, cells do not undergo a significant change in their Special Issue: AIE Materials Received: September 25, 2017 Accepted: November 8, 2017

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DOI: 10.1021/acsami.7b14548 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Synthetic Route Utilized To Prepare Probe 2

fluorescence intensities. In an effort aimed at designing new types of fluorescent probes, groups headed by Tang and Jiang independently demonstrated that AIE Luminogens,18,19 which are conjugated with cationic indolium groups, not only target mitochondria but can also be utilized to monitor mitochondrial membrane potential changes (Δψm). In spite of the fact that the triphenylphosphonium (TPP) group has a high lipophilicity, electrophoretic force, and mitochondrial targeting ability,20−24 the new AIE luminogen does not serve as a mitochondrial membrane potential indicator. We proposed that the existence of strong electrostatic repulsive interactions between the two TPP groups in probe 1 is the likely source of this phenomenon. If this proposal is correct, connecting only one TPP group to AIEgen, a probe that would be a competent mitochondrial membrane potential indicator. Herein, we describe studies we carried out to design, prepare and test the new TPP containing AIE Luminogen probe 2 (Figure 1), which that can be utilized for precise monitoring of membrane potential changes (Δψm). The results of this effort show that in comparison to the first generation AIE luminogen developed for mitochondria tracking, the new probe, in which the TPP group is conjugated to the AIE through a double bond, has a longer emission wavelength enabling avoidance of autofluorescence in the cell (Figure 1). Furthermore, the probe is stable in the present of both biothiols and ROS, and it is sensitive to membrane potential change.

Figure 2. Fluorescence spectra of probe 2 (10 μ M) in DMSO and H2O (λex = 370 nm, slit width: 3/3).

is stable in the presence of biothiols and ROS such as glutathione (GSH), cysteine (Cys), homocysteine (Hcy), hydrogen peroxide (H2O2), hyperchloride (OCl−), and peroxynitrite (ONOO−). Specifically, no observable fluorescence changes take place when the above analytes are added to DMSO and H2O suspensions of probe 2 (Figure 3).

2. RESULTS AND DISCUSSION 2.1. Design and Synthesis of Probe 2. The synthetic route used to prepare the TPP containing AIE luminogen probe 2 (Figure 1) is outlined in Scheme 1. The three-step sequence is initiated by production of the arylaldehyde 2 through Pd(PPh3)4 promoted coupling of the corresponding vinyl bromide 1 with 4-formylphenylboronic. Condensation of the aldehyde group in 2 with 2-cyanoacetic acid in the presence of piperidine as a catalyst produces the acrylamide carboxylate intermediate 3, which is then transformed to probe 2 by amide bond forming reaction with aminoethyl-triphenylphosphium bromide. 2.2. Assessment of AIE Features and Fluorescence Response. The AIE features of the new TPP containing probe were assessed. The probe 2 has a strong absorbance at a peak of 370 nm which was further selected as an excitation wavelength (Figure S1). Inspection of the emission spectrum displayed in Figure 2 shows that probe 2 is nearly nonfluorescent in pure DMSO and displays concentration dependent fluorescence in aggregate states (Figure S2). These observations show that probe 2 forms emitting aggregates in water nonfluorescing species when dispersed in DMSO. The results of SEM (scanning electron microscope) analysis also show that probe 2 forms nanoaggregates in water and the nanopartical size was determined to be 129 nm in average by dynamic light scattering (DLS) analysis of particle sizes (Figure S3). Furthermore, the results of appropriate investigations demonstrate that the probe

Figure 3. Fluorescence spectra of probe 2 (10 μM) in the presence of biothiols (GSH: 10 mM, Cys and Hcy: 0.1 mM) and ROS (0.01 mM) in PBS (λex = 370 nm, slit width: 3/3).

2.3. Cell Imaging of Probe 2 in Mitochondria. The new probe can be used to stain mitochondria. For this purpose, cervical cancer HeLa cells were incubated with 5 μM of probe 2 for 2 h followed by washing with buffer solution and incubation with 50 nM MitoTracker and 200 nM LysoTracker at 37 °C for 30 min. The medium was removed and the resulting cells were rinsed with PBS and imaged immediately by using a confocal laser scanning microscope. As can be seen by inspecting the microscope images shown in Figure 4 a, the probe is cell B

DOI: 10.1021/acsami.7b14548 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

membrane potential falls in the range of 140 to −180 mV, which is about six times larger than those of nonexcitable plasma cell membranes.19 Mitotracker Deep red and Rhodamine 123 were utilized as commercial membrane indicators to evaluate the sensitivity of probe 2. For this purpose, HeLa cells were incubated with 20 μM of the oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazine (CCCP) for 20 min to induce a mitochondrial membrane potential decrease and then with 5 μM probe 2, or 50 nM Mitotracker Deep red or 500 nM Rhodamine 123 for 30 min. As can be seen by inspecting the images in Figure 6A, cells treated with probe 2 display a more highly sensitive response than do those treated with Rhodamine 123. Moreover, treatment with CCCP leads to near complete loss of fluorescence from the treated cells. In contrast, Rhodamine 123 and MitoTracker Deep Red bring about a less sensitive response. Furthermore, it has been reported that H2O2 treatment induces a decrease in the mitochondrial membrane potential and apoptosis.25,26 The results of FACS analysis show that, in comparison with a normal cell population that displays a high fluorescence, a H2O2-treated cell population displays low fluorescence (Figure 6B, C). Furthermore, the probe itself is very stable in the present of H2O2, no fluorescence change was observed after treatment with 0.5 mM H2O2 (Figures S6 and S7). These findings strongly demonstrate that probe 2 undergoes aggregation in mitochondria and that this phemomenon can be used to monitor the potential and assess apoptosis of the mitochondria membrane.

Figure 4. HeLa cell mitochondria imaging using probe 2 (5 μM). (a) Fluorescence image of HeLa cells stained with 2. (b) Fluorescence images of HeLa cells stained with mitochondria tracker. (c) Merged image of probe 2 and mitochondria tracker treated cells.

permeable and displays strong blue fluorescence. MitoTracker red FM (MT) is a commercially available mitochondria imaging agent which was used to costain with probe 2 (Figure 4b). The results of the costaining experiment (Figure 4c) show that the probe and MitoTracker (MT) specifically target mitochondria with a perfect Pearson’s correlation coefficient of r = 0.97 (Figure S4). 2.4. Photostabilities of Probe 2. Furthermore, the photostabilities of probe 2 and Mitochondria tracker stained cells in the range of 0−100 bleaching times (3.0 s/bleaching time) were determined by using continuous irradiation with a 25 μW laser. The newly developed probe was found to be stable after being irradiated over 100 bleaching cycles, while the fluorescence intensity of mitotracker decreases observably (Figure 5). The results demonstrate that the new AIE probe

3. EXPERIMENTAL SECTION 3.1. Intracellular Localization of Probe. Cells were incubated in 5% CO2 and 5 μM of probe 2 at 37 °C for 2 h. In colocalization experiments, the cells were first washed with PBS, and then incubated with 50 nM MitoTracker and 200 nM LysoTracker at 37 °C for 30 min. The medium was removed and the cells were rinsed with PBS. The cells were imaged immediately by using confocal laser scanning microscopy. 3.2. Photostability Test. The fluorescence intensities of probe 2 (10 μM) and mitotracker (50 nM) stained cells in the range of 0−300 bleaching times (3.0 s/bleaching time) determined using laser irradiation. A probe excitation wavelength λex = 405 nm and an emission filter of λ = 490−590 nm were used. For MitoTracker, an excitation wavelength λex= 635 nm and emission filter of λem = 655− 755 nm were used. The laser power of the confocal microscope is 25 μW. 3.3. Cell Culture. HeLa cells (human epithelial adenocarcinoma) lines were purchased from the Korean Cell Line Bank (Seoul, Korea) and then grown in MEM (Minimum essential media) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. All cells were maintained in an incubator at 37 °C and 5% CO2 air environment. 3.4. Mitochondrial Membrane Potential Change. Cells were seeded to the 35 mm glass bottomed dishes at a density of 3 × 105 cells per dish in culture medium. After 24 h, cells were treated with 20 μM CCCP for 20 min and incubated with 5 μM probe or 50 nM MitoTracker Deep Red or 500 nM Rhodamine 123 for 30 min and fluorescence images were acquired using a confocal laser scanning microscope (Fluoview 1200, Olympus, Japan). Fluorescence image acquisition conditions: probe λex 405 nm/λem 490−590, MitoTracker Deep Red λex 635 nm/λem 655−755 nm, Rhodamine 123 λex 470 nm/ λem 490−590 nm. 3.5. FACS Analysis. To induce mitochondrial membrane potential disruption and apoptosis, we treated cells 0.5 mM H2O2 for 16 h and then stained with 5 μM probe for 30 min. After treatment, cells were harvested with trypsin-EDTA and washed with DPBS twice and finally dissolved with DPBS. Fluorescence intensity analysis accomplished

Figure 5. Fluorescence Intensity with the increasing number of bleaching.

has a higher photostability than does mitotracker. The cytotoxicity of probe 2 was evaluated using a 3-(4,5-dimethyl2-thiazolyl)2,5-diphenyltetrazolium bromide (MTT)-based assay.9 The results (Figure S5) show that HeLa cells remain viable when up to 5 μM of probe 2 is employed for 2 hours. 2.5. Performance of Probe 2 in Mitochondrial Membrane Potential. It is known that the mitochondrial C

DOI: 10.1021/acsami.7b14548 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 6. Confocal fluorescence microscope images of (A) HeLa cells incubated with 20 μM CCCP for 20 min and incubated with 5 μM probe or 50 nM Mitotracker Deep red or 500 nM Rhodamine 123 for 30 min (probe, excitation 405 nm/emission 490−590; Mitotracker Deep red, excitation 635 nm/emission 655−755 nm; Rhodamine 123, excitation 470 nm/emission 490−590 nm). Scale bar: 20 μm. (B) Magnitude of loss of the fluorescence signal versus the mitochondrial membrane potential change. Each value was calculated from A and fluorescence intensity of no treatment was determined as 100%. (C) FACS analysis of H2O2 treated cells and normal cells. using the BD LSR Fortessa. 10 000 cells were counted for each experiment and Qdot 565 (excitation Laser: 405 nm, BP filter: 560/ 20) fluorochromes were used for probe detection. 3.6. Synthetic Method of Probe. Compound 2 was synthesized according to the reported reference.16 Synthesis of Compound 3. A mixture of compound 2 (1 mmol, 360 mg) and 2-cyanoacetic acid (2 mmol, 170 mg) in anhydrous EtOH containing 2 drops piperidine was stirred at reflux overnight. The solvent was removed under reduced pressure, and the residue was subjected to silica column chromatography to form 100 mg of product 3 as a yellow solid. 1H NMR (300 MHz, DMSO-d6): 7.97 (s, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.15−6.97 (m, 16H). FAB-MS: calcd for [M + ]: 427.1572, found: 427.1575. Synthesis of probe 2. To a mixture of compound 3 (0.42 mmol, 180 mg), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (0.5 mmol, 112 mg) and 1-hydroxybenzotriazole hydrate (0.5 mmol, 73 mg) in 10 mL anhydrous DMF was added (2-aminoethyl)triphenylphosphonium bromide (0.42 mmol, 163 mg). The mixture was stirred at room temperature for 5 h and concentrated under reduced pressure.The residue was subjected to silica column chromatography to form the probe 2 as a yellow solid. 1H NMR (300 MHz, DMSO-d6): 8.96 (s, 1H), 8.04 (s, 1H), 7.89−7.70 (m, 17H), 7.20−7.14 (m, 11H), 7.04−6.97 (m 6H), 3.90−3.85(m, 2H), 3.60−3.51 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 161.68, 150.96, 148.35, 143.13, 142.82, 139.99, 135.43, 134.19, 134.05, 131.04, 130.27, 128.34, 127.36, 119.19, 118.05, 116.57, 104.92, 69.95, 34.17, 10.79. FAB-MS: calcd for [M +]: 715.2873, found: 715.2881.

for use as a biomarker for studies of the function of mitochondria.

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ASSOCIATED CONTENT

S Supporting Information *

The The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b14548. 1 HNMR, 13CNMR, HRMS, Fluorescence, and UV data. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 82-2-3277-2385. ORCID

Juyoung Yoon: 0000-0002-1728-3970 Author Contributions

J.L. and N.K. contributed equally to this work. The manuscript was written with contributions from all authors. All authors have given approval for publication of the final version of the manuscript. Notes

The authors declare no competing financial interest.

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4. CONCLUSION The investigation described above led to development of the new fluorescent probe 2 for monitoring changes in mitochondria membrane potentials and cell apoptosis. Compared with previously described fluorescence membrane potential indicators, the new probe contains a triphenylphosphonium (TPP) group that enables strong electrostatic interactions to occur with the mitochondrial membrane that not only lead to selective targeting but also to a higher sensitivity than those displayed by the commercial indicator Rhodamine 123. Therefore, probe 2 appears to be ideally suited

ACKNOWLEDGMENTS This study was supported by grants from the National Creative Research Initiative programs of the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (2012R1A3A2048814). Mass spectral data were obtained from the Korea Basic Science Institute (Daegu) using a Jeol JMS 700 high-resolution mass spectrometer.

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DOI: 10.1021/acsami.7b14548 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX