Excimer Emissions for Dual

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Biological and Medical Applications of Materials and Interfaces

Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-color Live Cell Imaging and Cell Damage Diagnosis Xijuan Chao, Kangnan Wang, Lili Sun, Qian Cao, Zhuofeng Ke, Duxia Cao, and Zongwan Mao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12521 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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ACS Applied Materials & Interfaces

Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-color Live Cell Imaging and Cell Damage Diagnosis

Xi-Juan Chao a, †, Kang-Nan Wang

a, †

, Li-Li Sun a, Qian Cao a, Zhuo-Feng Ke a,

Du-Xia Cao b, *, Zong-Wan Maoa, *

a

MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of

Chemistry,

Sun

Yat-Sen

University,

Guangzhou

510275,

China.

E-mail:

[email protected];

b

School of Material Science and Engineering, University of Jinan, Jinan 250022,

Shandong, China. E-mail: [email protected];

†

These authors contributed equally to this work

KEYWORDS:

monomer/excimer-like

emission,

nucleolus,

lysosome

and

mitochondrion, dual color imaging, cell damage diagnosis

1

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ABSTRACT: Studies on the development of fluorescent organic molecules with different emission colors for organelles imaging and their biomedical application are gaining lots of focus recently. Here we report two cationic organochalcogens 1 and 2, both of which exhibit very weak green emission (Ф1=0.12%; Ф2=0.09%) in dilute solution as monomers, but remarkably enhanced green emission upon interaction with nucleic acids and large red-shifted emission in aggregate state by the formation of excimers at high concentration. More interestingly, the monomer emission and excimer-like emission can be used for dual color imaging of different organelles. Upon passively diffusing into cells, both probes selectively stain nucleoli with strong green emission upon 488 nm excitation; whereas upon 405 nm excitation, a completely different stain pattern by staining lysosomes (for 1) or mitochondria (for 2) with distinct red emission is observed, due to the highly concentrated accumulation in these organelles. Studies on the mechanism of the accumulation in lysosomes (for 1) or mitochondria (for 2) are found that the accumulations of the probes are dependent on the membrane permeabilization, which make the probes have great potential in diagnosing cell damage by sensing lysosomal or mitochondrial membrane permeabilization. The study is demonstrative, for the first time, of two cationic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell based on monomer and excimer-like emission, respectively, and more importantly, for diagnosing cell damage. 1. INTRODUCTION The fluorescence imaging of cells and biological tissues based on fluorescent molecules has attracted considerable attention in biological and medical fields. Specific localized imaging of sub-cellular organelles such as nucleoli and mitochondria (or lysosomes) is of great significance for in-depth understanding of biological processes and pathways for early disease diagnosis and drug development in biomedicine.1,2 For example, nucleolus is the key site in the nucleus that synthesizes, processes and assembles the ribosomal RNAs (rRNA).3 Its function is tightly related to cell growth and proliferation.4 Enlarged or aberrant nucleolus and 2


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the associated number are often indicative of particular types of cancer and other human disorders.5,6 As for mitochondrion and lysosome, their membrane permeabilization constitutes one of the major checkpoints in inducing apoptotic and necrotic cell death. Both of them play pivotal roles in cell life and death.7,8 Nowadays, a lot of mitochondrial (or lysosomal) probes and nucleolar probes have been reported widely for different purposes.7-24 However, to the best of our knowledge, probes with different color emissions for imaging nucleoli and mitochondria (or lysosomes) simultaneously have rarely been reported so far.25,26 Even though fluorophores with different color emissions for cell imaging have been widely reported,13,22,23,27-36 they either simply show two different colors from the same target or have no specific targets in cell at all. It would be attractive if a single fluorophore can image two or more organelles with distinct emissions at the same time. Such a probe can not only simplify the cell operation but also save cost and time. Therefore, probes with distinct colors to co-stain different organelles simultaneously are needed. Pyrene is a classical example of fluorophore capable of monomer–excimer transition. In dilute solution, fluorescence emission at 375 nm from the pyrene monomer is detected upon UV irradiation; at increased concentrations, the aggregation of pyrene monomer molecules could produce a face-to-face dimer and accordingly a red-shifted broad emission at 450–530 nm can be observed.37 Besides, the monomer emission could be recovered through the de-aggregation process. Based on this dual luminescence signal, pyrene moieties have been included in the design of numerous optical sensors as reporters.30,38-41 In spite of pyrene's unique characteristics, its poor aqueous solubility, excitation in the bio-damaging UV region and the blue emission greatly limit its applications. Recently, Kim's and Bouffard's groups reported a family of organic chromophores that, like pyrene, formed red emissive excimers with large Stokes shifts.42 Inspired by the report, we introduced a positive charge into the benzothiazole group in the similar molecules (Figure 1A). We expect that the fluorophore would still exhibit excimer emission upon aggregate; meanwhile, the positively-charged benzothiazole would ensure electrostatic interactions with negatively-charged nucleic acid, which may allow the monomer emission to be 3



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recovered by competitive binding of nucleic acid. Based on the different emissions from monomer and excimer of the molecules, we aim to achieve distinctly different color imaging of different organelles in live cells. 2. EXPERIMENTAL SECTIONS 2.1 General Materials and Methods All starting materials were used as received from commercial sources unless otherwise indicated.

Solvents

were

purified

and

degassed

by

standard

procedures.

The

2-Methyl-1-methylbenzothiazolium iodide and 1-Ethyl-Indole-3-carbaldehyde were synthesized according

to

the

procedures.43-45

literature

MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Aldrich), PI (propidium iodide, Sigma Aldrich), SYTO59 (a cell-permeant nucleic acid stain displaying stronger fluorescence upon binding to RNA than to DNA, with excitation at 633 nm and emission at 645 nm),

acridine

orange

(AO),

RNA-select

dyes,

JC-1

(5,5',

6,6'-tetrachloro-1,1',

3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide, a mitochondrial selective aggregate dye,60 used as positive comparison), LTDR (Lyso Tracker Deep Red) and MTDR (Mito Tracker deep Red) were all purchased from Life Technologies, USA. Plasmid DNA and RNA (from baker's yeast) were purchased from Sigma Aldrich. NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer at room temperature. Microanalysis (C, H, and N) was carried out using an Elemental Vario EL CHNS analyzer (Germany). Fluorescence spectra were recorded on a Shimadzu RF5301 spectrofluorophotometer, and fluorescence lifetime was recorded on a combined fluorescence lifetime and steady state spectrometer FLS 920 (Edinburgh). UV–vis spectra were recorded on a Varian Cary 300 spectrophotometer. ESI-MS were recorded on a Thermo Finnigan LCQ DECA XP spectrometer (USA). The quoted m/z values represent the major peaks in the isotopic distribution. Fluorescence microscopy of cells was performed in Carl Zeiss LSM 710. 2.2 X-ray Crystallographic Single crystals suitable for X-ray diffraction analysis were gained by slow evaporation and diffusion. The data were collected at 150 K on a Rigaku Pilatus diffractometer equipped with Mo Kα radiation (λ = 0.71073 Å). Structure solution and refinement were performed using SHELX-97 suite program. In the final stage of least-squares refinement, non-hydrogen atoms were refined anisotropically. Crystallographic data and details of the data collection and structure refinements are listed in Table S1. 2.3 Absorption and Emission The spectroscopic investigations were carried out in PBS (pH=7.4) and organic solvents used without further purification. The absorption spectra were recorded with a Varian Cary 300 spectrophotometer at 298K. The emission spectra and solid-stated absorption and emission were 4


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recorded on an Edinburgh FLS 920 Spectrometer at 298K. Decay curves of compounds were recorded by an Edinburgh FLS 920 Spectrometer at 298K. The analysis of the fluorescence decay profiles was accomplished with decay-analysis software provided by the manufacturer, and the quality of the fit was assessed with the χ2 value close to unity and with the residuals regularly distributed along the time axis. 2.4 Determination of Quantum Yield The relative fluorescence quantum yields were determined by using Rhodamine 6G in methanol (0.94) as standard46 and were calculated through the following equation: ɸx=ɸs*(Fx/Fs)*(As/Ax)*(nx/ns)2 where ɸ represents quantum yield; F is integrated area under the corrected emission spectrum; A absorbance at the excitation wavelength; n is the refractive index of the solution; and the subscripts x and s refer to the unknown and the standard, respectively. 2.5 Dynamic Light Scattering (DLS) DLS measurements were performed on Nanophox (EliteSizer). No aggregates by DLS were detected at the concentration of 50 µM for probes 1 and 2, while relatively large sized aggregates (size: ~ 260 nm for 1 and ~ 350 nm for 2) were formed at 100 µM and too large ones (> 1 µm) were formed at concentration over 400 µM 2.6 Cell Culture A549, HepG2 cells were cultured in RPMI 1640 medium with 10% fetal bovine serum (FBS), HeLa cells were cultured in DMEM medium with 10% FBS, all with 1% penicillin-streptomycin, at 37 °C under a 5% CO2 atmosphere. 2.7 Flow Cytometry A549 cells were cultured in 6-well tissue culture plates for 24 h and then treated with complex 1 or 2(10 µM) for 1 h. Data were collected by a flow cytometer (FACS Calibur TM, Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with FlowJo 7.6 software (Tree Star, OR, USA). 2.8 Confocal Microscopy For living cell staining experiments, cultures cells grown on confocal petri dish were stained with 1 or 2 in complete medium for 1 h at 37°C and then imaged with both one- and two-photon 5



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Microscopy (ex: 810 nm).Temperature dependence studies used cells that had been cooled at 4 °C and incubated with 1 or 2 at 4 °C for 1 h. For inhibitors, cells were previously treated with NH4Cl, chlorpromazine at the stated concentrations for 30 minutes, and then incubated with 1 or 2 for 1 hours before imaging. For cell counterstain experiment: A549 cells were stained with 20 µM 1 or 2 for 1 h. After rinsing with PBS twice, the same sample was stained with 2.5 µM SYTO59 for 10 min or (100 nm LTDR or 100 nm MTDR for 30 min) and then imaged by 633 nm excitation by confocal microscopy (LSM 710, Carl Zeiss, Göttingen, Germany) immediately. Different concentration (0, 125, 250, 1000 µM) of H2O2 for inducing cell apoptosis and death were pretreated for 1 h before treatment with probes 1 or 2. And 20 µM CCCP were pretreated for 1 h for inducing mitochondrial membrane potential decrease. 2.9 Digest Test For DNase and RNase digest test, A total of 1 mL clean PBS (as control experiment), 100 µg/mL DNase-Free RNase (GE) or 100 U DNase was added into three sets of prefixed A549 cells for 30 min, and then cells were stained with 20 µM 1 or 2 for 1 h. Cells were rinsed by clean PBS twice before imaging. 2.10 MTT Assay for the Cell Cytotoxicity This involves the reduction of MTT tetrazolium to MTT formazan pigment by the metabolic activity of living cells. Cells were seeded at a density of 1 *105 cells/mL in a 96-well plate. After 24 h of cell attachment, cells were treated with 1 or 2 for 12 h. Six replicate wells were used for each control and tested concentrations. After incubation for 12 h, the medium was removed and cells were washed with PBS twice. MTT tetrazolium solution (100 mL of 0.5 mg/mL in PBS) was added to each well, and the cells were further incubated at 37°C for 4 h in a 5% CO2 humidified atmosphere. Excess MTT tetrazolium solution was then carefully removed and the colored formazan was dissolved in 100 mL dimethyl sulfoxide (DMSO). The plate was shaken for 10 min and the absorbance was measured at 570 nm using a microplate reader.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization Compounds

1

and

2

were

synthesized

by

condensation

of 6


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2-Methyl-N-methylbenzothiazolium

iodide

with

indole-3-carboxaldehyde

or

1-Ethyl-Indole-3-carbaldehyde in ethanol according to references (Scheme S1).43-45 The synthesis processes and structural characterization using ESI-MS, 1H NMR, 13C NMR and elemental analysis are provided in the supporting information (Figure S1-6). The X-ray crystallography structures of 1 and 2 were grown by slow diffusion of diethyl ether into a methanol solution of those compounds. The crystal data and structural refinements are shown in Figure 1A and Table S1.

Figure 1. Optical spectra of compounds 1 and 2. A, Chemical structures and crystal structures of compounds 1 and 2. Thermal ellipsoids set 30% probability. All hydrogen atoms, counterions and solvent are omitted for clarity. B, Absorption (compound 1: λ=447 nm; compound 2: λ2=462 nm) and emission (compound 1: λ=520 nm; compound 2: λ=530 nm) spectra of compounds 1 and 2 (10 µM) in PBS buffer. C, Emission spectra of solid 1 and 2 with excitation of 450 nm.

3.2. Photophysical Properties of the Compounds The UV-visible absorption spectra of 1 and 2 under physiological conditions (PBS buffer, pH=7.4) exhibit bands with major peaks at 447 and 462 nm, respectively (Figure 1B). These bands are characteristic of ICT (intramolecular charge transfer) π→π* transitions occurring from the donor (indole group) to the acceptor (thiazole group).47 In dilute solution, both 1 and 2 exhibit relatively weak fluorescence with 7



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visible emission centered around 520 nm and 530 nm, respectively (Figure 1B and Table S2). It is quite obvious based on their D-π-A (Donor-π-Acceptor) chemical structures that the intramolecular rotation of the probes relaxes the excitation energy and further results in significant quenching of fluorescence in PBS buffer.48,49 Next, the emission spectra of the solid powders were also measured. As Figure 1C shows, strong red fluorescence is observed with 1 showing two emission peaks at 580 nm and 640 nm and 2 showing emission peak at 660 nm. The solid-state photophysical properties of probes 1 and 2 made us further investigate their potential for excimer emission. By gradually increasing the concentration of the probes in aqueous solution, the fluorescence intensity centered around 520 nm (1) or 530 nm (2) (450 nm excitation) decreased gradually, but weak emission around 600-700 nm became observed and increased (Figure 2A). The excitation wavelength upon increased concentration measured was found to blue shift to 250-400 nm (Figure S7). Therefore, by switching to excite with UV light (250-400 nm), at low concentration, the main emission peak was much lower than that with excitation of 450 nm, however with increasing concentration, progressively red-shifted and enhanced fluorescence emission was observed, and emission intensity at around 600-700 nm finally became much stronger compared with that by 450 nm excitation (Figure 2B).50 The changes of emission spectra upon increased concentration probably indicate that the intermolecular interaction induces aggregation, along with the formation of excimers.30,42,50 The new arising spectroscopic features upon increased concentration become similar to those detected in the solid state (Figure 1C and Figure S8). This further proves aggregation is triggered by the increase of concentration. Besides, the aggregate formation of probes 1 and 2 was further confirmed by dynamic light scattering (DLS) measurements and crystal structures. In DLS experiments, different sizes of aggregates have been recorded; with increased concentration, the size of the aggregate becomes larger (Figure S9). The details of the molecular conformation and packing structure obtained by single-crystal X-ray diffraction study show that compounds 1 and 2 are nearly planar in the crystal with close intermolecular stacking distances (3.40 Å for 1 and 3.39 Å for 2; Figure S10). Finally, fluorescence lifetimes 8


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of molecule 1 at low and high concentrations under excitations with both 472 nm and 354 nm lasers were recorded. In dilute solution, where emission (520 nm) occurs mainly from monomeric molecules, very fast fluorescence decay (0.1 ns) is observed with excitations of both 472 nm and 354 nm lasers. In contrast, in solution with high concentration, lifetime recorded with 472 nm becomes a little longer (0.16 ns), but much longer (0.64 ns) by excitation of 354 nm laser (emission at 650 nm) (Table S3), which is consistent with the new red-shifted and enhanced emission with UV excitation. Time-dependent density functional theory (TD-DFT) calculations were also carried out on the monomer and dimer of probe 1 (Figure S12-13, Table S4, See SI for detailed description). Hence, all the results above demonstrate that intermolecular interaction induces aggregation with increased concentration, thus causing red-shifted and excimer-like emission.

Figure 2. Spectral emissions measured upon increased concentration. Emission detected with increased concentration by 450 nm and 350 nm excitations, respectively.

Spectral emission in viscous solution and solvents with different polarities were 9



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also measured. In viscous glycerol solution, enhancement in green emission intensity of the probes 1 and 2 is observed (Figure S14). The enhancement is attributed to viscous microenvironment surrounding the probes which leads to the freezing of intramolecular rotation of the probes and facilitates the intramolecular charge transfer.47,51-53 Even though the position of the emission peak is slightly red-shifted by excitation with either 450 nm or 350 nm (UV light), the emission band observed in the solid state is not detected. For fluorescence emission measured in different solvents with excitations by both 450 nm and 350 nm (UV light), no obviously red-shifted emission is observed, either (Figure S15). The results further demonstrate that red-shifted and excimer-like emission upon increased concentration results from intermolecular interaction induced aggregation. Next, fluorescence emission in the presence of negatively-charged nucleic acid was investigated. As expected, in dilute solution, the green emission intensity increased markedly in the presence of nucleic acid (Figure 3 and Figure S16). It should be noted that no obvious enhancement was observed in the presence of Bovine Serum Albumin (BSA) (Figure S17). Upon binding with nucleic acid, the intramolecular rotation process becomes weak largely due to the interactions of cationic unit of probes with the negatively charged phosphate backbone of nucleic acids, 47 thereby reducing the probability of non-radiating pathways and thus restoring fluorescence. What's more, we found that the progressively red shift upon increased concentration was hindered largely in the RNA-containing solution (Figure S18). The result suggests the RNA competitive binding of the probes can hinder the intermolecular self-assembly of the molecules, 14 which is exactly what we expected. Moreover, the fluorescence lifetime measurements were also made in the presence of nucleic acid. As Table.S3 shows, very fast fluorescence decay (0.1 ns) is observed in their dilute solutions due to the intramolecular relaxation in the excited state. However, the lifetimes of the probes enhance dramatically in the presence of DNA or RNA (Figure S19A and Table S5), and even longer than that recorded in the glycerol environment (Figure S19B), which demonstrates interactions between the dye molecules and nucleic acids. 10


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Figure 3. Fluorescence emission of compounds 1 and 2 in the presence of nucleic acid. Fluorescence emission of compounds 1 and 2 (10 µM) in the presence of varied concentration of DNA and RNA (0-340 µg/mL) in PBS buffer. Excitation with 450 nm.

3.3. Dual Color Emissions for Imaging of Nucleolus and Lysosome (for 1)/Mitochondrion (for 2) in Live Cell The enhanced green emission with nucleic acid in dilute solution and red excimer-like emission in aggregate state intrigued us to further investigate their potential for different color imaging of different organelles in cells. Given that low cytotoxicity is one of the key criteria for live cell imaging, and the fluorescent probes used for long-term cell tracing and imaging need to have minimal cell toxicity, MTT assays were then carried out to evaluate cell cytotoxicity in A549 cell and other two cell lines (HeLa and HepG2). The derived IC50 values show that 2 has no high toxicity over a 12 h incubation period even when the concentration as high as 100 µM, while 1 displaying a more toxic behavior with derived IC50 of 18 µM over a 12 h incubation period probably due to its faster cellular uptake behavior (Table S6 and Figure S20). Based on their 12-hour IC50 values, we chose concentration of 10 µM to treat cells for 11



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1 h. First we detected the cellular uptake ability by flow cytometry in A549 cells. As Figure 4A shows, the fluorescence intensity of cells treated with probe 1 or 2 enhances dramatically by FITC (Fluorescein isothiocyanate) channel, which indicates these probes are quite cell permeable. Then confocal microscopy imaging was carried out to examine the distribution of the probes in cells with excitations of 488 nm and 405 nm, respectively. After probes 1 and 2 entering a live cell, the green fluorescence shows distinguishable nucleolar staining accompanied with faint cytoplasm that contains ribosomal RNA upon excitation with 488 nm (Figure 4B). And notably, by switching the excitation light from 488 nm to 405 nm, distinct red signals in certain organelle in the cytoplasm were detected in a pattern completely different from that of the green ones in the cytoplasm (Figure 4B). The imaging results prove that the probes 1 and 2 are indeed capable of dual color imaging of different organelles simultaneously.

12


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Figure 4. Flow cytometry analysis and confocal dual-color imaging of compounds 1 and 2 in live A549 cells. A, Flow cytometry analysis of cells incubated with 1 or 2 (10 µM) for 1 h, by FITC channel. B, Dual color emission effects of cells incubated with 1 and 2 (20 µM) for 1 h, images were obtained at the emission ranges of 500-560 nm (green) and 600-670 nm (red) corresponding to excitations at 488 nm and 405 nm, respectively. (C, D) Localization in cell proved by co-staining with SYTO 59 (2.5 µM) and digest test with RNase (100 µg/mL). The excitation wavelength for 1 and 2, 488 nm. And emission range at 500-560 nm. The excitation for SYTO 59 is 633 nm with emission range at 650-700 nm. (E, F) A549 cells were treated with 1 or 2 (20 µM) for 1 h, then MTDR and LTDR were co-stained, respectively. The excitation wavelength for 1 and 2 is 405 nm with emission range at 600-670 nm. MTDR and LTDR were both excited at 633 nm and emission range at 650-700 nm. Pseudo color (green) were used here for clarification.

To confirm whether the bright green spots observed in the nucleus were related to 13



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the fluorescence of the probes localizing at the nucleoli, co-localization experiments were undertaken by co-staining with the commercially available nuclear stain SYTO 59. As shown in Figure 4C, the co-staining results demonstrate that the green fluorescence of probes 1 or 2 from nucleoli colocalizes well with the red fluorescence of SYTO 59 in the nucleoli. The colocalization coefficients of probes 1 and 2 with SYTO 59 in nucleoli are 0.92 and 0.96, respectively. This strongly suggests that the green emissions are from nucleoli. Besides, both deoxyribonuclease (DNase) and ribonuclease (RNase) digestion tests were performed to further confirm the distribution of 1 and 2 in nucleoli. Only DNA in cell would be hydrolyzed in the presence of DNase while only RNA would be hydrolyzed in the RNase digest by contrast. After DNase digestion, the fluorescence intensity in nucleoli (containing RNA) largely remains as that without treatment (Figure S21). On the contrast, after RNase digestion, the fluorescence diminishes dramatically in nucleoli (Figure 4D). All these results above confirm that the strong green emission comes from nucleoli. Even though molecule 1 has been reported as a DNA imaging probe in nucleus,47 in our study we found both molecules 1 and 2 were more favorable for imaging nucleoli by showing much brighter green emission in nucleoli than in nucleus. However, according to the titration experiment with nucleic acid in the solution above (Figure 3), the probes have not shown much specificity to RNA than to DNA. As Table S5 shows there is also no much difference of the lifetimes between DNAand RNA-bound for both 1 and 2, which indicates lifetime is not a factor in determining the stronger signal in nucleoli than that in nucleus. By searching literatures, we found the same kind of phenomenon have also been observed by other groups,13,54 the explanations they suggested were possibly due to the difference between structure of nucleic acids in solution and their real state in cells and specific interaction between different probes and nucleic acids. Another possible reason we think might be that the viscosity of DNA is higher than that of RNA with the same concentration in solution in the experiment, thus leading to high fluorescence in DNA containing solution for the probes. As for the red signals observed in the cytoplasm with excitation at 405 nm, based 14


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on what we have found above (Figure 2), the accumulation of the dye molecules is probably highly concentrated in certain organelles and causes aggregation caused red-shifted emission. The aggregates in these organelles are found to increase with incubation time (Figure S22). To find out the exact localization of the red emission, close inspection of the subcellular distribution of the compounds by co-localization with both MTDR and LTDR was performed (Figure 4E and F). Confocal microscopy analysis reveals that the fluorescence of probe 1 mainly overlays with that of LTDR in lysosomes (colocalization coefficient: 0.80); while probe 2 mainly stains mitochondria (colocalization coefficient: 0.85) with no distribution in lysosomes at all. The distinctly different cytoplasmic distribution of probes 1 and 2 may be explained by their slightly different structures of substitute groups at atom N in the indole groups.55 In addition to one-photon excitation, we found the probes can image in live cells also upon 810 nm excitation by two-photon fluorescence microscopy, which indicates 1 and 2 can also be used as two-photon imaging agents for nucleoli or organelles in cytoplasm (Figure S23). Besides, similar phenomenon of dual color imaging is observed in other two cell lines (HeLa and HepG2) as well (Figure S24). Their dual color emissions of both probes 1 and 2 make them attractive probes for different color imaging of RNA-rich nucleoli and mitochondria (or lysosomes) simultaneously, in most kinds of cell lines by both one- and two- photon excitations. 3.5. Mechanism Discussion in Lysosomal(1) or Mitochondrial(2) Accumulation The probes, especially 1, even at a concentration as low as 2.5 µM, target the nucleoli in minutes (Figure S20). Studies on the mechanism of cellular uptake demonstrate a passive pathway mechanism for 1 and a non-endocytotic, but temperature-dependent passive pathway for 2 (Figure S25, See Experimental section for details). For further discussing the mechanism by which they selectively image mitochondria (for 2) or lysosomes (for 1), it is essential to understand the structural character of the targets. For mitochondrion, it has a very large membrane potential up to −180 mV.56 Thus, cationic species tend to accumulate in the mitochondria rather 15



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than other organelles.57 For lysosome, it is a spherical vesicle containing hydrolytic enzymes with acid environment (pH=4.7).7 First for probe 1, H- substitute at atom N in the indole part probably makes it preferentially distribute within the acid vacuolar cellular compartment like lysosomes or exhibit high fluorescence in acid environment.55,56,58 The emission intensities of probe 1 at both low and high concentration under varied pH solutions were then detected. In dilute solution, the green fluorescence (450 nm excitation) is decreasing with decreased pH values; whereas in solution with high concentration, the red fluorescence intensity is increasing with decreased pH values under UV (350 nm) excitation (Figure 5). These results are well consistent with the aggregation caused red-shifted and intense emission in lysosomes. The gradually decreasing green fluorescence (450 nm excitation) for diluted probe 1, along with the weak and negligibly varied red fluorescence (350 nm excitation) with decreasing pH values further confirms red-shifted emission is caused by aggregation but not by its low pH in lysosomes. Then for probe 2, to see whether the targeting to mitochondria is driven by the huge membrane potential, cells were pretreated with CCCP, a type of protonophore that could decrease of mitochondrial membrane potential (MMP).59 Control cells and cells treated with CCCP were stained with probe 2 and JC-1, respectively. As expected, the red signal from aggregated state of JC-1 shifted to green signal of isolated state in mitochondria for cells pre-treated with CCCP (Figure 6A). Similar phenomenon for cells stained with probe 2 was also observed with the red fluorescence intensity of aggregated probe 2 molecules within mitochondria becoming weaker while the green fluorescence of probe 2 molecules becoming stronger in nucleolus and cytoplasm (Figure 6B). The similar phenomenon observed between JC-1 and probe 2 suggests that probe 2 is also a membrane potential dependent probe.

16


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Figure 5. Contrast pH response of probe 1 in condensed state or dilute solution. The red fluorescence intensity is increased with decreasing pH values in condensed state. While in contrast, the green fluorescence intensity is decreased with decreasing pH values in dilution solution, and the red fluorescence intensity is very weak with negligible changes in dilute solution at different pH solution. Emission under 550 nm was filtered for emission spectra excited by 350 nm.

Figure 6. Probe 2 as a mitochondrion membrane potential dependent probe. Cells were pretreated without or with 20 µM CCCP for 1 h, then stained with (A) JC-1 and (B) probe 2, respectively.

Acridine orange (AO) is a lysosomotropic metachromatic fluorochrome that emits red fluorescence at high concentrations (in lysosomes) and green fluorescence at low concentrations (in the cytosol and the nucleus).7,58 The similar dual color imaging behavior between probe 1 and AO inspired us to speculate whether probe 1 would function as AO in detecting lysosomal health, as manifested by reduced red fluorescence

and increased

green fluorescence after lysosomal membrane

permeabilization (LMP). Meanwhile, the similar dual color imaging behavior between probe 2 and JC-1 also prompted us to see whether probe 2 could function as JC-1 in detecting mitochondrial health by sensing membrane potential change. So, 17



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experiments were performed to verify our speculation. Here, H2O2, widely reported in causing cell apoptosis and death by inducing decrease of their membrane permeabilization,56,57 was used to induce decrease of mitochondrial and lysosomal membrane permeabilization. First we excluded the possibility of H2O2 itself in reducing the emission intensity of probes. As shown in Figure S26, no change of emission intensity is observed in solution system containing different concentration of H2O2. Then cells were pretreated with different concentration of H2O2 to induce cell apoptosis or death, and stained by probe 1 or 2. As shown in Figure 7 and Figure S27, similar phenomena with commercial dyes (AO or JC-1)58, 60 were observed for probes 1 and 2, respectively, as manifested by an increase in cytoplasmic diffuse green fluorescence, or a decrease in granular red fluorescence. These results confirm that probes 1 and 2 have similar function as AO and JC-1, respectively, in detecting lysosomal or mitochondrial membrane permeabilization. Mitochondrial or lysosomal membrane permeabilization are reported to be one of the major checkpoints of apoptotic and necrotic cell death,7 which further indicates probes 1 and 2 have the potential in diagnosing cell damage.

Figure 7. Aggregation caused red-shifted emission in organelles used for detecting cell apoptosis and death. Cells were pretreated with different concentrations of H2O2 for 1 h in serum-free medium, then incubated with 1 or 2 (20 µM) for 1 h. Obvious decreasing aggregation caused red-shifted emissions in lysosomes (for 1) or in mitochondria (for 2) were observed. Images were obtained at the emission ranges of 500-560 nm (green) and 600-670 nm (red) corresponding to excitations at 488 nm and 405 nm, respectively. 18


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3.6. Unique Features of Probes and Their Potential Biological Implication Conventional fluorescent dyes for nucleoli or mitochondria (lysosomes) staining have been widely developed.7-24 However, poor permeability, poor selectivity and tedious synthetic steps of most probes have limited their broad applicability in live cell imaging. Hence the development of efficient probes for live cell imaging that are easy to synthesize, nontoxic, and provide high sensitivity and selectivity is receiving attention, especially the probes that are capable of multicolor imaging for different organelles in live cells. To the best of our knowledge, the kind of probe has rarely been reported so far.25,26, 29 Most reported fluorophores with different color emissions can't realize multicolor imaging for different organelles,13,22,23,27-36 The probes we present here are not only capable of dual-color imaging nucleoli and mitochondria (or lysosomes), but also may function as JC-1 (or AO), in detecting mitochondrial (or lysosomal) health. Moreover, as nucleolar probes, compared with the only commercial RNA dye SYTO RNA-select, probes 1 and 2 possess good features including simple synthesis, faster cell permeability, better sensitivity and selectivity and higher photostability (Figure S28). All these make 1 and 2 multifunctional probes with great potential application in a biological context. 4. CONCLUSIONS In conclusion, we report, for the first time, two cationic small organic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell and for diagnosing cell damage based on monomer/excimer emission. Both probes exhibit very weak green emission in dilute solution, but enhanced green fluorescence upon interaction with nucleic acids by restriction of intramolecular rotation mechanism and large red-shifted emission due to self-assembly induced aggregation at high concentration. Upon diffusing into cells, the probes diffuse across the cell membrane to the nucleoli very fast and allow clear visualization of nucleoli with green emission; Meanwhile, they highly accumulate in lysosomes (1) or mitochondria (2) and display distinct red emission in these organelles. The great features of simple synthesis, fast cell permeability, low cytotoxicity (especially 2), 19



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high selectivity, two-photon effect, and potential utility in detecting lysosomal or mitochondrial health, making them excellent dual color probes with great interest in biological and medical fields. From the fundamental point of view, this study may also provide a platform for the rational design of probes with efficient different emission colors for different organelles imaging.

Associated Content The Supporting information is available free of charge on the ACS Publications website. Synthetic scheme; 1H NMR;

13

C NMR; ESI-MS; Optical spectra of probes in the

presence of nucleic acid; Optical property of probes in solution and solid state; MTT assay; The digest experiment; Lifetime measurement; DLS measurement; Cellular uptake; The aggregation in organelles increased with time; Fluorescence intensity in the presence of H2O2; Commercial dye JC-1 and AO in detecting mitochondrial and lysosomal

membrane

integrity;

Photostability

assay;

Photophysical

data;

Physicochemical parameters; crystallographic data (PDF) Crystallographic data for Probe 1 (CIF) Crystallographic data for Probe 2 (CIF) AUTHOR INFORMATION Corresponding Authors *

E-mail: [email protected]; [email protected].

†

These authors contributed equally to this work.

ORCID iD Zong-Wan Mao: 0000-0001-7131-1154 Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This study was supported by the National Science Foundation of China (Nos. 21231007

and

21572282),

the

973

program

(Nos.

2014CB845604

and 20


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1036-1048.

Table of Contents (TOC): Dual-color emission behavior for organelle-specific imaging in live cells. Title: Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-color Live Cell Imaging and Cell Damage Diagnosis

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Excimer Emissions for Dual

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