Spatially Dependent Fluorescent Probe for Detecting Different

Subscriber access provided by University of Newcastle, Australia

Article

A spatial-dependent fluorescent probe for detecting different situations of mitochondrial membrane potential conveniently and efficiently Xuechen Li, Minggang Tian, Ge Zhang, Ruoyao Zhang, Ruiqing Feng, Lifang Guo, Xiaoqiang Yu, Ning Zhao, and Xiuquan He Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03842 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A spatial-dependent fluorescent probe for detecting different situations of mitochondrial membrane potential conveniently and efficiently Xuechen Li,† Minggang Tian, † Ge Zhang, † Ruoyao Zhang, † Ruiqing Feng, † Lifang Guo, † Xiaoqiang Yu,* † Ning Zhao,*‡ Xiuquan He*§ †

Center of Bio & Micro/Nano Functional Materials, State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, P. R. China, Fax: +86 0531 88364263; E-mail address: [email protected] ‡ Shandong Key Laboratory for Adhesive Materials, Advanced Materials Institute Shandong Academy of Sciences. Jinan 250014, Shandong, P. R. China. Fax: +86 0531 82959240; E-mail address: [email protected] § Department of Anatomy, Shandong University School of Medicine, Jinan 250012, P. R. China. Fax: +86 0531 88364263; Email adress: [email protected] ABSTRACT: The feedback from mitochondrial membrane potential (MMP) in different situations (normal, decreasing, and vanishing) can reflect different cellular status, which can be applied in biomedical research and diagnosis of the related diseases. Thus, the efficient and convenient detection for MMP in different situations is particularly important yet the operations of current fluorescent probes are complex. In order to address this concern, we presented herein a spatial-dependent fluorescent probe composed of organic cationic salt. The experimental results from normal and immortalized cells showed that it could accumulate in mitochondria selectively when MMP was normal. Also, it would move into nucleus from mitochondria gradually with the decreasing of MMP and finally targeted nucleus exclusively when MMP vanished. According to the cell morphology, there is a straightforward spatial boundary between the nucleus and cytoplasm where mitochondria locate, thus the three situations of MMP can be point-to-point indicated just by fluorescent images of the probe: that all probes accumulate in mitochondria corresponds to normal MMP; that probes locate both in mitochondria and nucleus corresponds to decreasing MMP; that probes only target nucleus corresponds to vanishing MMP. It's worth noting that counterstaining results with S-11348 indicated that the spatial-dependent probe could be applied to distinguishing dead from viable cells in same cell population. Compared with the commercial Cellstain-Double Staining Kit containing Calcein-AM and Propidium Iodide (PI), this probe can address this concern by itself and shorten the testing time, which brings enormous convenience for relevant researches.

Mitochondrial membrane potential (MMP) is a key parameter representing mitochondrial functions and its decrease can imply the disruption of mitochondrial electron transport chain, which results in cellular dysfunction even death. 1, 2 So, the feedback from different situations of MMP, such as normal, decreasing and vanishing, is practically useful in biomedical research and diagnosis of the related diseases.3-6 Generally, MMP of healthy cells is normal; MMP decreasing indicates occurrence of dysfunctions, for example, the decrease of sperm MMP indicates its activity reduce;7 MMP vanishing shows that cells are in disease status, for instance, MMP vanished in some parasites killed by artemisinin.8 Thus, developing a convenient method to efficiently reveal different situations of MMP is of great importance in biology and medicine. Recently, some fluorescent probes to detect the MMP have been studied extensively.9-11 To date, there are mainly two kinds of probes: 1) Fluorescent intensity probes, such as rhodamine 123, TMRE and TMRM with aggregation-caused quenching (ACQ) character.3, 8, 11 They can intensively accumulate in mitochondria with normal MMP and emit weak fluorescence due to the quenching effect from higher concen-

tration. When MMP decreases, parts of probes release to cytosol and the others still remain in mitochondria, while these probes at low concentrations emit stronger fluorescence. When MMP vanishes, all the probes release to cytosol and keep low concentration and fluorescent intensity is similar to one in decreasing situation. Therefore, it is difficult to distinguish MMP decreasing from vanishing just by fluorescent image. 2) Fluorescent dichromatic probes, such as JC-1.6, 7, 11 It can emit red fluorescence in aggregations or green fluorescence in monomer when MMP is high or low, respectively. So it can be used to judge the variation trend of MMP by comparing the changes of JC-1 fluorescent color. However, the staining concentration needs to be adjusted repeatedly before every experiment: it is difficult to form J-aggregates when using too low staining concentrations and high concentrations may produce block precipitates due to its very poor water solubility, which brings great inconvenience.7, 11 Thus, a fluorescent probe which can easily and efficiently show normal, decreasing and vanishing status of MMP is lack yet valuable. As mentioned above, rhodamine 123 releases to cytosol when MMP decreases. Because boundary between mitochon

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. The structures of CAI, LAD, LAD-1 and 9E-BMVC (a), synthetic routine of LAD and LAD-1 (b).

dria and cytosol is ambiguous, one cannot judge whether some probes have released to cytosol or not just by fluorescent images. Inspired by this phenomenon and considering there is the straightforward spatial boundary between the nucleus and cytosol, we conceive such a probe with characters as followed: it can accumulate in mitochondria selectively when MMP is normal; also, it will move into nucleus from mitochondria gradually with the decreasing of MMP and finally target nucleus exclusively when MMP vanishes. If so, the three situations of MMP will have three point-to-point fluorescent images: that all probes accumulate in mitochondria corresponds to normal MMP; that probes locate both in mitochondria and nucleus corresponds to decreasing MMP; that probes only target nucleus corresponds to vanishing MMP. In order to attain the objective, it should be rational to design such a probe based on organic cationic compounds (OCCs). Because some OCCs can accumulate in mitochondria with normal MMP. 12, 13, 14 On the other hand, some other OCCs just locate in nucleus due to its static interaction with DNA as well as inserted layer effect with DNA grooves.15,16 Thus, we believe that by balancing the interaction of OCC between MMP and DNA to finely control its targeting ability, a spatial-dependent probe can be obtained which meets the requirements above, but it is still challenging. Previously, our group has reported two OCCs probes: CAI 17 and 9E-BMVC 18 . More importantly, the two molecules shared same fluorophore, although they exclusively label mitochondria (CAI) and nucleus (9E-BMVC), respectively. From Figure 1, the nhexyl endows CAI with appropriate hydrophobic ionic radius (HIR) 13, so CAI can promptly penetrate mitochondrial inner

Page 2 of 11

membrane and accumulate in mitochondria preferentially. Compared with CAI, 9E-BMVC has an additional cationic pyridine salt to increase the static interaction with DNA and its enlarged conjugation plane enhances the insert effect with DNA grooves. In addition, methyl in 9E-BMVC reduces its HIR, which increases its difficulty in entering mitochondrial inner membrane. Most importantly, the calculation results of DNA titration experiment showed that the intrinsic binding constant (k) to DNA of 9E-BMVC was 6.46 × 106 M-1 while that of CAI was almost close to zero (Figure S1). Inspired by this, a new OCC probe LAD with spatialdependent character was designed and synthesized (Figure 1b) by balancing the structure of CAI and 9E-BMVC. LAD remained the appropriate HIR using butyl and added a planarizable pyridine group which increased inserting effect with DNA grooves. In addition, the experiment results showed that the k values of LAD was 8.30 × 105 M-1 which was appropriately less than 9E-BMVC but larger than CAI. A series of experimental results demonstrated that LAD accumulated in mitochondria when MMP was normal, immigrated to nucleus gradually with MMP decreasing, and located in nucleus completely when MMP vanished. Moreover, LAD has been successfully applied to distinguishing dead cells without MMP from viable ones with normal MMP in same cell population, which has great significance in biology and medicine. Also, the mechanism of LAD was discussed in detail and provided a theoretical guidance for the development of this type of probes. Recently, Moritomo and co-workers reported a probe (BP6) with similar properties. 19 However, the staining time is up to 12-16 hours due to very low membrane permeability of BP6, which greatly limits its application in convenient and efficient detection of MMP. Comparatively, the incubating time of LAD is only 15 min. Moreover, as a spatial-dependent fluorescent probe detecting different situations of MMP, the action mechanism of LAD has been explained in detail. Thus, LAD1, a homologous compound of LAD has been synthesized, which is also a spatial-dependent fluorescent probe for detecting MMP. LAD-1 needs longer time to enter cells in comparison with LAD, mainly due to the weaker lipotropy of methyl in LAD-1 than butyl in LAD. Very importantly, according to our experimental results in living HeLa cells, the sensitivity of LAD based on spatial-dependent mechanism to MMP changes is higher than that of JC-1 on fluorescence colors, indicating that a spatial-dependent fluorescent probe has huge potentials in detecting MMP. Yet, relative researches have just started.

EXPERIMENTAL SECTION Synthesis. The details of the synthesis of LAD have been given in the Supplementary Information. LAD was synthesized by the Knoevenagel condensation reaction between 1butyl-4-methylpyridin-1-ium iodide and (E)-9-(2ethoxymethyl)-6-(2-(pyridine-4-yl) vinyl)-9H-carbazole-3carbaldehyde using piperidine as the catalyst. Apparatus and general methods.The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a HITACH U-2910 spectrophotometer using a quartz cuvette


Page 3 of 11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


having 1 cm path length. Single-photon excited fluorescent (SPEF) spectra were obtained on a HITACH F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. The ideal deadcell stain SYTOX Blue nucleic acid stain (S-11348) and MitoTracker Red FM (MTR) were purchased from Molecular Probes. Viability of the cells was assayed using cell proliferation Kit I with the absorbance of 492 nm being detected using a Perkin-Elmer Victor plate reader. Measurement of binding parameters of carbazole derivatives to DNA. The intrinsic binding constants (k) were obtained by the one-photon fluorescence titration method. The excitation wavelengths of 458 nm for LAD and the fluorescence intensity monitored at 550 nm were analyzed with the Scatchard equation according to equation (1) r / Cf = kn–kr (1) 20 Where k is binding constant, n is the number of dye sites of perphosphate, r is the ratio of the concentration of the binding dye to the concentration of DNA (in phosphate) and Cf is the concentration of free dye. The concentration of the binding compound was calculated using equation (2). Cb = Ct [(F - F0) / Fmax- F0)] (2) 21 Ct is the total compound concentration, F is the observed fluorescence intensity at given DNA concentration, F0 is the fluorescence intensity in absence of DNA, and Fmax is the fluorescence intensity of the totally binding compound. Cell culture and staining. All cells were grown in a 5% CO2 incubator at 37 oC. SiHa and HeLa cells were grown in H-DMEM supplemented with 10% fetalbovine serum (FBS) and 1% penicillin and streptomycin. Human umbilical vein endothelial (HUVEC) cells were grown in M199 medium supplemented with 10% FBS and 2 ng/mL FGF-2. For living cell staining experiments, cultured cells were stained with 2 µM LAD in culture medium for 15 min at 37 °C and then imaged with fluorescence microscopy. For fixed cell staining experiments, cultured cells were pretreated according to the following procedure: cells were first fixed by 4% paraformaldehyde for 30 min and then with 2 µM LAD in culture medium for 15 min at 37 °C. For the investigating of LAD staining effect, living cells were incubated with 2 µM LAD firstly for 15 min, then these cells were treated by H2O2 (3‰,10 µL) for different time. Every time the cells were washed to remove the unbound probes before adding H2O2. After rinsing with PBS twice, cells were imaged immediately. Secondly, the living SiHa cells were pre-loaded with 2 µM LAD for 15 min and then 0.2 µM Carbonylcyanide-m-chlorophenylhydrazone (CCCP ） was added to the culture medium. Every time the cells were washed to remove the unbound probes before adding CCCP. After rinsing with PBS twice, cells were imaged immediately. After treated 20 min by CCCP, culture medium was sucked out to remove CCCP and the cells were imaged continuously until MMP recovering. To co-staining experiments, living cells were incubated with LAD firstly for 15 min, then stained with 5 µM S-11348 for 30 min, or stained with 0.2 µM MTR for 30 min. Every time the cells were washed to remove the unbound probes before staining with another probe. After rinsing with PBS twice, cells were imaged immediately. For cell-viability assay, the study of the effect of LAD on viability of cells was carried out using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. SiHa cells growing

in log phase were seeded into 96-well plates (ca. 1 × 104 cells/well) and allowed to adhere for 24 h. LAD (200 µL/well) diluted in DMEM at concentration of 2 µM was added into the wells of the treatment group, and DMSO (200 µL/well) diluted in DMEM at final concentration of 0.2% to the negative control group, respectively. The cells in one of the 96-well plates were incubated with 2 µM LAD for 2, 8, and 24 h at 37oC under 5% CO2, and the cells in another 96-well plateswere incubated with 2 µM, 5 µM, 10 µM LAD for 2 h at 37 o C under 5% CO2, then MTT (5 mg/mL in PBS) was added into each well. After 4 h incubation, the culture medium in each well was removed and DMSO (200 µL) was added to dissolve the purple crystals. After 20 min incubation, the optical density readings at 492 nm were taken using a plate reader. Each individual cytotoxic experiment was repeated for three times. In addition, for the study of the cytotoxicity of LAD on viability of cells in mitochondria level, the living SiHa cells were firstly incubated with LAD (2 µM, 15 min) and then were placed for a period of time (2 h, 5 h, and 10 h). Thereafter, these cells were stained with 0.2 µM MTR for 30 min, respectively. Then, these cells were imaged immediately, after rinsing with PBS twice. For the sensitivity assay, the living HeLa cells were incubated with 20 µg/ml of JC-1 firstly for 30 min and then the cells were washed to remove the unbound probes. After rinsing with PBS twice, cells were imaged immediately and the fluorescent signal from green channel (500-550) and red channel (580-630 nm) were collected, respectively. Consequently, these cells were treated by H2O2 (3‰, 10 µL) and the fluorescent signal from two channels above at different incubation time of H2O2 (1 min, 5 min, 7 min, 9 min, 10 min) were collected again. Each experiment was repeated for three times. And then, the fluorescent intensity ratio between green channel and red one at different incubation time of H2O2 was calculated as a parameter of the sensitivity of JC-1 to MMP. Fluorescent imaging. Confocal fluorescent images were obtained with an LSM 780 confocal laser scanning microscope, an Olympus FV 300 Confocal Microscope or Olympus FV 1200 Confocal Microscope. The overlap coefficients and fluorescence intensity of the images were determined by the software with the LSM 780 confocal microscope.

RESULTS AND DISCUSSION Photophysical properties. The absorption (Figure S2a) and fluorescence spectra (Figure S2b) of LAD have shown in the Supporting Information. The structure of LAD was characterized by 1H NMR, 13C NMR, MS, and IR, as showed in Supporting Information. Its maximum absorption and fluorescence wavelengths in various polarity solvents are in the range of 428-452 nm and 525-566 nm, respectively. Moreover, from Figure S1, the fluorescent intensity of LAD decreases with the increasing of solvent polarity, while maximum emission wavelength red shifts, indicating intramolecular charge transfer (ICT) process of the probe. These properties are similar to that of previous reported two probes CAI and 9E-BMVC.17, 18 The ICT process can be reasonably attributed to the electron donor of the carbazole moiety and the electron acceptor of the pyridinium cationic. In addition, the maxima wavelengths of absorption (λab) and emission (λem), the molar absorption coefficients (ε) and fluorescence quantum yields (Φ) in all solvents were obtained (Table S1). As shown in Table S1, the Φ of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 11

LAD is higher in 1, 4-dioxdane with lower polarity. Also, the absorption and fluorescence spectra of LAD-1 have been shown in Figure S3. From Figure S3, its maximum absorption and fluorescence wavelengths in EtOH are at 450 nm and 560 nm, respectively, which are similar to LAD. In addition, the fluorescent stability of LAD against different physicochemical conditions (ionic changes, temperature, pH and viscosity) was tested respectively (Figure S4). Because LAD was in a lipophilic environment when it accumulated mitochondria or the groove of DNA, the mixed solvent of 1, 4dioxane and H2O (V:V= 1:1) was used when investigating the influence of ionic changes, temperature, and pH values on fluorescence of LAD, while glycerol (Gly) was used to study the viscosity effect. As shown in Figure S4a, various ions expressed in cytoplasm and intracellular environment (Na+, K+, Ca2+, Mg2+, Mn2+, Ba2+, Br-, Cl-) were added to the solution of LAD, and the fluorescent intensity of LAD did not change obviously. At the same time, the influence of temperature (060 oC) was detected. From Figure S4b, the fluorescent intensity of LAD fluctuated within a small scope in a temperature range from 0 oC-60 oC. Importantly, the fluorescent intensity of LAD was always stable at the physiological temperature range (30-40 oC). In addition, the influence of pH values in the physiological range (4.0-9.0) on fluorescent intensity of LAD was also investigated. As shown in Figure S4c, the fluorescent intensity of LAD in lipophilic environment (1, 4-dioxane) was much higher than that in the mixed solvents with pH values of 4.0-9.0. Thus, the fluorescent intensity of LAD to detect MMP was not affected by the change of pH. Meanwhile, LAD showed enhanced emission in high-viscosity conditions (Figure S4d). This should be attributed to the limitation of intramolecular motions in such conditions. We think this property of LAD would benefit its application, because mitochondria and nucleus can supply high-viscosity environments and enhance its fluorescent signals. Exclusively staining mitochondria with normal MMP. Probe LAD was firstly used to stain vigorous cells to check its selectivity to mitochondria with normal MMP. As shown in Figure 2, when staining SiHa cells and HUVEC cells, LAD obviously displayed strong fluorescence in cytosol. The emission in cytosol sketched filament morphologies, which indicated that LAD may concentrate in mitochondria with normal MMP. Furthermore, due to the ICT properties of LAD, it displayed rather weak fluorescence in high-polarity water and thus showed no background signals when staining the cells and no fluorescence was observed in extracellular regions from the merged images. More importantly, according to the results in Figure 2, no fluorescence emitted in nucleus regions, which indicated LAD does not stain nucleus in cells with normal MMP. In addition, as shown in Figure S5, LAD-1 obviously displayed strong fluorescence with filament morphologies in cytosol, indicating that LAD-1 may concentrate in mitochondria with normal MMP. To strictly and accurately confirm the selectivity of LAD to mitochondria, conventional co-localization experiments have been performed with MTR 17, a commercially available mitochondria probe. As shown in Figure S6a, the absorption spectra of the mixed solution of LAD and MTR was similar to the

Figure 2. Fluorescent images, differential interference contrast (DIC) photos, and their merged pictures of SiHa cells (a) and HUVEC cells (b) stained by LAD (2 µM, 15 min). λex = 488 nm, λem > 565 nm. Bar = 20 µm. Confocal Microscope: Olympus FV 300.

linear superposition (LAD + MTR) of their absorption spectra, which indicated no chemical interaction between them. Very importantly, when illuminated by 405 nm (Figure S6b), only LAD could fluoresce in the mixed solution; while when excited by 561 nm (Figure S6c), only MTR fluoresced. These results demonstrated that LAD and MTR do not disturb with each other. Therefore, the green fluorescence in Figure 3 excited by 405 nm was solely from LAD, and the red signals excited with 561 nm came exclusively from MTR. According to the co-localization results, the co-localization coefficient of LAD and MTR was up to 0.87, and as shown in Figure3d, the fluorescence from LAD and MTR showed similar distribution in cells. Therefore, LAD actually stained mitochondria with normal MMP in vigorous cells. Also, from Figure S7, there would not be optical interferences between the two dyes. Therefore, the green fluorescence in Figure S8 excited by 405 nm was solely from LAD-1, and the red signals excited with 543 nm came exclusively from MTR. According to colocalization results, the co-localization coefficient of LAD and MTR was up to 0.94. Thus, LAD-1could also actually stain mitochondria with normal MMP in vigorous cells. High selectively targeting nucleus when MMP vanishes. To verify the ability of LAD in high selectively staining nucleus in cells whose MMP have vanished, the SiHa cells have been fixed by paraformaldehyde and the strictly staining experiments of LAD were performed. According to Figure 4a, all nucleic had been stained in fixed cells, and the position and shape stained by LAD were the same as that of nucleus in the DIC image, implying that LAD accurately labelled the nucleus in MMP vanishing cells. Most importantly, no fluorescence was observed in cytosol regions from the merged image of MMP vanishing cells. In addition, as we know, long period incubation of CCCP could also eliminate the MMP. So, the staining experiments of LAD in SiHa and HUVEC cells incubated by CCCP were performed. From Figure 4b and c, similarly with staining results in fixed cells, LAD also exclusively labelled the nucleus, which also indicated the high selectivityto nucleus of LAD when MMP vanished. Also, from the Figure S9, LAD-1could exclusively target nucleus in the cells incubated 30 min


Page 5 of 11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3.Confocal fluorescent images of SiHa cells stained with LAD (2 µM, 15 min) and MTR (0.2 µM, 20 min): (a) Images of LAD, λex = 405 nm, λem = 450–700 nm; (b) Images of MTR, λex = 561 nm, λem = 570–700 nm; (c) Overlay images of a and b; (d): The relative emission intensity distributions of MTR and LAD in various pixel points along the red arrow in c. Bar = 10 µm. Confocal Microscope: Zeiss LSM 780.

by CCCP, which showed the same targeting ability with LAD.

Figure 4. Fluorescent images, DIC photos, and their merged pictures of SiHa cells (a, b) and HUVEC cells (c) stained by LAD (2 µM, 15 min) after fixed by paraformaldehyde (30 min) (a) or incubated with CCCP (30 min, b, c). λex = 488 nm, λem > 565 nm. Bar = 20 µm. Confocal Microscope: Olympus FV 300.

To explain the mechanism that LAD and LAD-1 target nuclei in MMP vanished cells, the fluorescence DNA titration experiment 18 was performed, and the calf thymus DNA (ctDNA) was used. As shown in Figure 5 and Figure S10, both LAD and LAD-1 have showed weak fluorescence in buffer solutions, and the fluorescence gradually enhanced with the addition of ctDNA solution. This turn-on behavior of the probe could also be interpreted by the ICT properties, because binding to DNA also resulted in the isolation of probes from water. The k values have been obtained as 8.30 × 105 M-1 and 2.47 × 106 M-1 respectively by analyzing the fluorescence titration data with the Scatchard equation 20, 22. As shown in Figure S1, the k value of DNA probe 9E-BMVC was 6.46 × 106 M-1 and the fluorescence intensity of mitochondria probe CAI barely increase with the addition of DNA, indicating no interaction between the CAI and DNA. In order to provide good insights, the targeting abilities of LAD, LAD-1, CAI and 9EBMVC were compared in detail in Table S2. Therefore, it is very necessary to possess appropriate affinity to DNA for a spatial-dependent probe for detecting MMP. According to our experiments, the k values to DNA at the magnitude of 105 M-1 or 106 M-1 may be suitable. Locating both in mitochondria and nucleus when MMP

Figure 5. Fluorescence titration of LAD with DNA (a), the fitted curve of the graph according to Scatchard equation (b). λex = 458 nm and λem = 550 nm for LAD. [LAD] = 5 µM, [phosphate in DNA] = 0–20 mM for LAD. For LAD, the binding constant is 8.30 × 105 M1 , the corresponding site is 1.72 phosphates/dye, goodness-of-fit: R2= 0.979.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 11

Figure 6. Confocal fluorescent images of SiHa cells stained by LAD (2 µM, 15 min) before (a, Control) and after incubated by H2O2 (3‰, 10 µL) at 1, 2, 5, 8, 10, 12 min (b, c, d, e, f, g). h: the merged image of a and g whose color was replaced by green. λex = 488 nm, λem > 565 nm, Bar = 50 µm. i: the ratios of fluorescent intensity in nucleus to that in whole cells with different incubation time of H2O2. Data were expressed as mean ± SD, control, experiment times n = 3. Confocal Microscope: Olympus FV 300.

decreased. According to the results above, LAD located in mitochondria with normal MMP, and it targeted nucleus when MMP vanished. From this, we may deduce that LAD should locate both in mitochondria and nucleus when MMP decreased. In order to detect this inference, we investigated the staining effect of LAD in cells treated by two MMP reducing agents CCCP and H2O2. In the first experiment, the living SiHa cells were loaded with LAD and the staining effect was recorded. Consequently, these cells were treated by H2O2 and influence of the treating time on the targeting site of LAD was investigated in detail (Figure 6 and Video S1). As shown in Figure 6, LAD was initially accumulated in mitochondria before adding H2O2. After this, with the extension of H2O2 treating time, fluorescence gradually appeared in nucleus, indicating that LAD gradually moved to nucleus. After 12 min, almost all fluorescence emitted from the nucleus in the cells. Generally, the longer time treated by H2O2 and the more MMP decreased, which would result in that the more probes target nucleus. Accordingly, LAD located both in mitochondria and nucleus in Figure 6b-f when MMP decreased. At the same time, an important fact that the fluorescent intensity

ratios between nucleus and whole cells increased from 0.1 to 0.8 with extension of H2O2 treating time supported this deduction above. (The calculation method about the ratio was instructed in detail at Supporting Information, Figure S11.) Also, in order to more visually show the influence of H2O2 treating on targeting, the Figure 6h was given by merging Figure 6a and Figure 6g whose fluorescent color was replaced by green. From Figure 6h, the position emerged by green color nicely padded the nucleus regions in Figure 6a, which indicated again that LAD can stain both in mitochondria and nucleus when MMP decreased. The same results have been also obtained in HeLa cells (Figure 7 and Video S2). From Figure 7a, LAD exclusively stained mitochondria with normal MMP. When the H2O2 treating time was 1 min to 10 min, LAD labelled the mitochondria and nucleus simultaneously (Figure 7b-f), and the corresponding fluorescent intensity ratios between nucleus and whole cells increased from 0.1 to 0.8. This fact proved again that LAD located both in mitochondria and nucleus when MMP decreased.

Figure 7. Confocal fluorescent images of HeLa cells stained by LAD (2 µM, 15 min) before (a) and after incubated (b-f; 1, 3, 6, 8, 10 min) with H2O2 (3‰, 10 µL). λex = 488 nm, λem> 565 nm, Bar = 50 µm. g: the ratios of fluorescent intensity in nucleus to that in whole cells with different incubation time of H2O2. Data were expressed as mean ± SD, control, experiment times n = 3. Confocal Microscope: Olympus FV 300.


Page 7 of 11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Confocal fluorescent images of SiHa cells stained with LAD (2 µM) for 15 min (a) then incubated with CCCP (20µM) for different time (b-f, 5-28 min), and after removal of CCCP (g-j, 8-20 min). k: The merged image of a and f whose color was replaced by green; l: The merged image of f and its DIC. λex = 473 nm, λem > 560 nm, Bar = 50 µm. m: The ratios of fluorescent intensity in nucleus to that in whole cells when incubating and removing CCCP at different time. Data were expressed as mean ± SD, control, experiment times n = 3. Confocal Microscope: Olympus FV 300.

Affected by various physiological and pathological conditions, the MMP is often fluctuant and its change is reversible sometimes. Thus, the reversibility of staining sites of LAD was also studied in detail in the process of MMP reversible change. For this purpose, CCCP was added to reduce MMP and then MMP could be recovered by washing out CCCP (Figure 8 and Video S3). As shown in Figure 8a, LAD was initially accumulated in mitochondria before adding CCCP. From Figure 8b-f, with the extension of CCCP treating time, fluorescence gradually appeared in nucleus, indicating that LAD gradually moved to nucleus. After treating the cells by CCCP for 20 min, almost all fluorescence emitted from thenucleus. Importantly, as shown in Figure 8g-j, after washing out CCCP, the MMP recovered and the fluorescence reappeared gradually in mitochondria. Moreover, after 18 min, almost all probes moved back to mitochondria. Therefore, LAD can detect the process of not only MMP decreasing but also MMP recovering, which could be used to long-time observe MMP fluctuation. Also, in order to intuitively show the influence of CCCP treating on targeting site of LAD, the Figure 8k was given by merging Figure 8a and Figure 8f whose pseudocolor was replaced by green. From Figure 8k, the position emerged by green color nicely padded the nucleus regions in Figure 8a. Also, from Figure 8l, the position and shape stained by LAD were the same as that of nucleus in its DIC image, implying that LAD accurately labelled the nucleus in cells incubated for 28 min by CCCP. Further, the ratio values of fluorescent intensity between nucleus and the whole cells were also obtained. As shown in Figure 8m, the ratio gradually increased from 0.1 to 0.9 during 28 min after addition of CCCP and decreased from 0.9 to 0.1 during 20 min after washing out CCCP. This fact proved that LAD could be used to monitor the process of MMP reversible change. Most importantly, it demonstrated again that LAD could locate both in mitochondria and nucleus when MMP decreased. The comparison of the sensitivity to MMP between LAD and JC-1. The fluorescent images of HeLa cells stained by

Figure 9.(a) Confocal fluorescent images of HeLa cells stained by JC-1 (20 µg/ml, 30 min) before (a, Control) and after incubated by H2O2 (3‰, 10 µL) at 1, 5, 7, 9, 10 min. Green, λex = 473 nm, λem = 500-550 nm; Red, λex = 543 nm, λem = 580-630 nm, Bar = 50 µm. (b) the fluorescent intensity ratios in nucleus to that in whole cells from LAD (black line, coming from Figure 7g) and the fluorescent intensity ratios between green channel to red one from JC-1 at different incubation time of H2O2. Data were expressed as mean ± SD, control, experiment times n = 3. Confocal Microscope: Olympus FV 1200.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 11

Figure 10. Confocal fluorescent images in SiHa cells stained with 2 µM LAD and 5 µM S-11348 in sequence. Each incubation 15 min. (a) LAD fluorescence image, λex = 473 nm, λem = 500-600 nm; (b) S-11348 fluorescence image λex = 405 nm, λem = 410-460 nm; (c) DIC; (d) Overlay images of a, b and c. Bar = 20 µm. Confocal Microscope: Olympus FV 1200.

JC-1 were firstly obtained (Figure 9a). Previously, the sensitivity of LAD to the change of MMP has been detected in the study of the MMP decreasing process. Also, the fluorescent intensity ratio in nucleus to that in whole cells from LAD was given again (Figure 9b, black line), which could serve as a parameter of the sensitivity of LAD to MMP. Comparatively, the sensitivity of JC-1 to MMP was also investigated in cells treated by H2O2 with the same experimental conditions to LAD in Figure 7. The fluorescent images from green and red channels as well as their merge images have been shown in Figure 9a. According to Figure 9a, the red fluorescent signal was bright while the green fluorescence was very weak before the H2O2 treating, indicating that the MMP was normal. With the extension of H2O2 treating time, the green fluorescence enhanced and red fluorescence waned gradually, which illustrated that the MMP decreased and JC-1 transformed from J-aggregates into monomers. Also, the fluorescent intensity ratio between green channel and red one at different incubation time of H2O2 was calculated and plotted in Figure 9b (red line). As shown in Figure 9b, compared to JC-1, LAD can sense the change of MMP more sensitively, indicating that a spatial-dependent fluorescent probe has huge potentials in detecting MMP. Application in distinguishing dead cells from viable ones in same cell population. According to the sensing MMP character of LAD, we speculate that its fluorescent images can be used to distinguish dead cells from viable ones in same cell population, which has great significance in biology and medicine. For examples, it can help to screen anticancer drug 23 and evaluate cell viability 24. Also, it is useful in estimating whether certain environment condition, such as in bioreactors, is suitable for cell culture or not. 25 In order to verify the speculation, counterstain experiments of LAD with S-11348, a dye only staining dead cells, have been performed (Figure 10). As shown in Figure S12, upon binding DNA, S-11348 has an absorption band in the region of 380-470 nm with emission at 450-550 nm. From Figure S12, the absorption of LAD is about 400-500 nm and the emission is about 480-650nm. Consequently, S-11348 displays rather low absorption at 473 nm, while LAD shows little fluorescencein the emission range 410-460 nm. In consequence, red fluorescence excited by 473 nm should only come from LAD, while blue fluorescence selected from 410 to 460 nm should be attributed to S-11348 excited by 405 nm. Consequently, the

relevant confocal microscope photos of a group of SiHa cells incubated with 2 µM LAD and 5 µM S-11348 in sequence are shown in Figure 10. From Figure 10a, LAD stained the almost cellular mitochondria except for two cells whose nucleus were stained. Importantly, the two exceptional cells in Figure 10a were just stained by S-11348 (Figure 10b). In addition, from the DIC picture (Figure 10c) the two exceptional cells have broken. More importantly, the two exceptional cells in Figure10a and two cells stained by S-11348 in Figure 10b as well as the two broken cells in Figure 10c are fully overlapped in their merged image (Figure 10d), which indicated that the two exceptional cells in Figure 10a were dead and other cells should be intact and viable. The results confirmed that a single probe LAD can distinguish dead cells from viable ones in same cell population conveniently and efficiently. The distinguishing principle is based on that LAD located in mitochondria with normal MMP and it targeted nucleus when MMP vanished. To date, the commercial fluorescent dye commonly used to distinguish dead and viable cells is Cellstain-Double Staining Kit. In this Kit, Calcein-AM and Propidium Iodide (PI) can stain viable and dead cells, respectively26. Thus, this method needs to incubate the two dyes in sequence. When imaging viable cells stained by Calcein-AM, the 470 nm excitation and 535 nm emission wavelengths are used, and 540 nm excitation and 660 nm emission wavelengths can be used to image dead cells. Comparatively, a single probe LAD can accomplish the task of Cellstain-Double Staining Kit using single excitation wavelength and single detection range. In fluorescent photos, fluorescence in viable cells comes from only mitochondria in cytosol but fluorescence in dead cells only from nucleus. More importantly, using a single probe can greatly shorten the testing time and improve efficiency, which will bring enormous convenience in biology and pharmacology research. In practical application, another key consideration is toxicity of LAD itself to living cells. Thus, cytotoxicity of LAD was evaluated in living SiHa cells by MTT assays. The influence of LAD on SiHa and HeLa cells at different incubation time and concentrations were studied, and the cell viability assay data were quantified (Figure 11 and Figure S13). The results showed that both SiHa and HeLa cells showed > 85% viability after 24 h incubation with 2 µM LAD and 2 h incubation with 10µM LAD. Thus, the LAD of concentrations at a micromolar


Page 9 of 11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11.(a) MTT results of SiHa cell viabilities after incubation with LAD (2 µM) for 2 h, 8 h, and 24 h; (b) MTT results of SiHa cell viabilities after incubation with LAD for 2 h at different incubation concentration (2 µM, 5 µM,10 µM).Data are expressed as mean ± SD (experiment times n = 3).

order of magnitude and short incubation time (such as < 30 min) of LAD can be regarded as non-cytotoxicity to living cells and the death of the cells is not induced by LAD. In addition, the cytotoxicity of LAD to mitochondria was studied in mitochondria level. The SiHa cells were firstly stained by LAD and then placed for 2h, 5h and 10h, respectively, and then MTR was used to stain these cells (Figure S14). As shown in Figure S14a, after stained by LAD for 10 h, in the green channel, the SiHa cells still exhibited filaments shaped fluorescence in cytoplasm and none fluorescence in nucleus, which indicated that LAD may still target mitochondria and the MMP was normal. Moreover, from the red channel, MTR could still accumulate to mitochondria after 10h incubation of LAD, which further confirmed that the MMP was still normal. Also, from the Figure S14b, the colocalization coefficients of LAD and MTR was up to 0.90 after 10h incubation of LAD, which demonstrated LAD still target mitochondria very well and the MMP is still normal. In a word, the probe LAD of concentrations at a micromolar order of magnitude (such as 2 µM) in short incubation time (such as < 30 min) should not bring damage to MMP.

CONCLUSIONS We have presented a spatial-dependent fluorescent probe (LAD) to conveniently and efficiently detect different situations of MMP. The three MMP situations can be indicated clearly by three point-to-point fluorescence images: when MMP is normal, LAD could exclusively accumulate in mitochondria; once MMP decreases, probes locate both in mitochondria and nucleus; LAD will only target nucleus when MMP vanishes. In addition, the fluorescent intensity ratios between nucleus and whole cells may be used to detected MMP semi-quantitively. The success of LAD should be ascribed to the elaborately adjusting of chemical structure of OCCs, such as the conjugate planes and hydrophobic properties. Most importantly, counterstaining results with S-11348 indicates that a single probe LAD can be applied for the distinguishing dead cells from viable cells in same cell population. In addition, to find such a spatial-dependent fluorescent probe, some principles should be followed: firstly, it can accumulate in mitochondria with normal MMP; meanwhile, the probe should have appropriate affinity to DNA. The fluores-

cent organic cations with k values to DNA at the magnitude around 105 and 106 may achieve this task.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Experiment details of synthesis and structural characterization of all compounds, photophysical properties of LAD and LAD-1, additional fluorescent images of LAD and LAD-1 cells, MTT assay for LAD, the comparisons of LAD, LAD-1, CAI and 9EBMVC, instruction of the calculation method about the fluorescent intensity ratio between nucleus and whole cell. (PDF) Videos of cells stained by LAD before and after incubated by different reagents (H2O2 or CCCP). (AVI)

AUTHOR INFORMATION Corresponding Author Xiaoqiang Yu Fax: +86 0531 88364263; E-mail address: [email protected] Ning Zhao

Fax: +86 0531 82959240 E-mail address: [email protected] Xiuquan He Fax: +86 0531 88364263 E-mail address:[email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Our Laser confocal scanning imaging of living cells was performed at The Microscopy Characterization Facility, Shandong



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

University. For financial support, we thank the National Natural Science Foundation of China (51273107 and 51303097), Natural Science Foundation of Shandong Province, China (ZR2012EMZ001), Open Project of State Key Laboratory for Supramolecular Structure and Materials (SKLSSM201524).

Page 10 of 11

21. B.-d. Wang, Z.-Y. Yang and T.-r. Li, Bioorgan. Med. Chem., 2006, 14, 6012-6021. 22. Y. Guan, R. Shi, X. Li, M. Zhao and Y. Li, J. Phys. Chem. B, 2007, 111, 7336-7344. 23. A. Y. Oral, B. Cevatemre, M. Sarimahmut, C. Icsel, V. T. Yilmaz and E. Ulukaya, Bioorgan. Med. Chem., 2015, 23, 4303-4310.

REFERENCES 1. I. G. Shabalina and J. Nedergaard, Biochem. Soc. Trans., 2011, 39, 1305-1309. 2. Y. Pan, Exp. Gerontol., 2011, 46, 847-852. 3. L. V. Johnson, M. L. Walsh and L. B. Chen, P. Natl. Acad. Sci.USA, 1980, 77, 990-994. 4. E. Gottlieb, S. M. Armour, M. H. Harris and C. B. Thompson, Cell Death Differ, 2003, 10, 709-717.

24. Y. Hong, Y. Yao, S. Wong, L. Bian and A. F. Mak, J. Biomech., 2016, 49, 1305-1310. 25. R. Vecchiatini, L. Penolazzi, E. Lambertini, M. Angelozzi, C. Morganti, S. Mazzitelli, L. Trombelli, C. Nastruzzi and R. Piva, J. Periodontal. Res., 2015, 50, 544-553. 26. D. Kuksin, C. A. Kuksin, J. Qiu and L. L. Chan, Anal. Biochem., 2016, 503, 1-7.

5. L. V. Johnson, M. L. Walsh, B. J. Bockus and L. B. Chen, J. CellBiol., 1981, 88,526-535. 6. M. E. Widlansky, J. Wang, S. M. Shenouda, T. M. Hagen, A. R. Smith, T. J. Kizhakekuttu, M. A. Kluge, D. Weihrauch, D. D. Gutterman and J. A. Vita, Transl. Res., 2010, 156, 15-25. 7. M. Binet, C. Doyle, J. Williamson and P. Schlegel, J. Exp. Mar. Biol. Ecol., 2014, 452, 91-100. 8. C. L. Peatey, M. Chavchich, N. Chen, K. J. Gresty, K.-A. Gray, M. L. Gatton, N. C. Waters and Q. Cheng, J. Infect. Dis., 2015, 212, 426–34. 9. N. Zhao, S. Chen, Y. Hong and B. Z. Tang, Chem. Commun., 2015, 51, 13599-13602. 10. L. Zhang, W. Liu, X. Huang, G. Zhang, X. Wang, Z. Wang, D. Zhang and X. Jiang, Analyst, 2015, 140, 5849-5854. 11. S. W. Perry, J. P. Norman, J. Barbieri, E. B. Brown and H. A. Gelbard, Biotechniques, 2011, 50, 98-115. 12. S. Huang, R. Han, Q. Zhuang, L. Du, H. Jia, Y. Liu and Y. Liu, Biosens. Bioelectron., 2015, 71, 313-321. 13. M. F. Ross, G. F. Kelso, F. H. Blaikie, A. M. James, H. M. Cocheme, A. Filipovska, T. Da Ros, T. R. Hurd, R. A. J. Smith and M. P. Murphy, Biochemistry -Moscow, 2005, 70, 222-230. 14. H. Xiao, P. Li, X. Hu, X. Shi, W. Zhang and B. Tang, Chem. Sci., 2016, 7, 6153-6159. 15. M. Q. Wang, W. X. Zhu, Z. Z. Song, S. Li and Y. Z. Zhang, Bioorg. Med. Chem. Lett., 2015, 25, 5672-5676. 16. G. Licari, P.-F. Brevet and E. Vauthey, Phys. Chem. Chem. Phys, 2016, 18, 2981-2992. 17. F. Miao, W. Zhang, Y. Sun, R. Zhang, Y. Liu, F. Guo, G. Song, M. Tian and X. Yu, Biosens. Bioelectron., 2014, 55, 423-429. 18. Y. Zhang, J. Wang, P. Jia, X. Yu, H. Liu, X. Liu, N. Zhao and B. Huang, Org. Biomol. Chem., 2010, 8, 4582-4588. 19. H. Moritomo, K. Yamada, Y. Kojima, Y. Suzuki, S. Tani, H. Kinoshita, A. Sasaki, S. Mikuni, M. Kinjo, J. Kawamata. Cell Struct. Funct., 2014, 39, 125-133. 20. G. Scatchard, Ann. N.Y. Acad. Sci., 1949, 51, 660-672.


10

Page 11 of 11

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of Contents (TOC):


11

Spatially Dependent Fluorescent Probe for Detecting Different

Recommend Documents