Visualizing Fluoride Ion in Mitochondria and Lysosome of Living Cells

Aug 4, 2015 - Two ratiometric probes fluoride ion, Mito-F and Lyso-F, were rationally designed and synthesized with positive charges at physiological ...
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Visualizing Fluoride ion in Mitochondria and Lysosome of Living Cells and in Living Mice with Positively Charged Ratiometric Probes Zhisheng Wu and Xinjing Tang * State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. Tel/Fax: 86-10-82805635; Email: [email protected] ABSTRACT: Two ratiometric probes fluoride ion, Mito-F and Lyso-F, were rationally designed and synthesized with positive charges at physiological conditions. The positive charges functioned as target moieties for subcellular mitochondria and lysosome of living cells, and effective sequesters of fluoride ion for fast and efficient fluorescent detection. In addition, in vivo imaging of fluoride ion in living mice was successfully achieved for the first time using probe Mito-F.

Fluoride ion (F−) has been considered as one of important anions that play crucial roles in health science. For example, fluoride ion is widely used as an additive in toothpaste and essential ingredient in pharmaceutical agents. It has also been added to drinking water to prevent dental caries and osteofluorosis1-4. However, excess fluoride ingestion affects various cell signaling processes5,6 and induce apoptosis at a higher concentration in mammalian cell7,8, results in dental and skeletal fluorosis9, and even leads to nephrotoxic changes10 and urolithiasis in humans11. As a result, there is a need to develop efficient methods for sensitive and accurate detection of fluoride ion in biological systems, even in living animals. Although analytic methods, such as ion-selective electrode and ion chromatography12-14 have been developed for assaying fluoride ion, they always require expensive equipment and facilities, which limits the rapid and convenient detection and is not suitable for cell and animal imaging. By contrast, fluorescence sensing is a powerful technology to study fluoride ion in biological systems, even in living animals. In the past decade, several fluorescent probes for detection of fluoride ion were developed in solution or cell imaging15-17. They are mainly based on fluoridehydrogen bonding18-20, boron-fluoride or stibium-fluoride complexation21-24, and desilylation25-39. However, most fluorescent probes for fluoride ion still have some limitations in detection and bioimaging. First, some probes could only detect tetrabutylammonium fluoride (TBAF) salt in organic or organo-aqueous solvents rather than sodium fluoride (NaF) in aqueous solutions, which limits their further applications in biological systems. If sodium fluoride was used, the detection kinetics was very low, and the addition of CTAB was necessary to increase the rate of desilylation in aqueous solution. Imaging fluoride distribution in subcellular structures (such as mitochondria and lysosome) or in vivo has not been investigated.

Recently, several water soluble fluorescence probes for fluoride detection have been developed by attachment of sugar moiety28,31. We also reported fluorescent fluoride chemosensors with attachment of quaternary ammonium which greatly increase the solubility of probes in water, and quaternary ammonium itself could sequester fluoride anion due to charge attraction and promote the desilylation of probes40. Based on above considerations and our previous study40, we continued to develop a series fluorescent fluoride ratiometric probes with the attachment of positively charged moieties such as triphenylphosphonium and dimethylamino under physiological pH value (Scheme 1). We envisioned that these novel probes are more water soluble and efficiently detect fluoride ion due to the sequestration effect of positive charges in physiological conditions. In addition, probes with the attachment of triphenylphosphonium and dimethylamino were localized in specific organelles such as mitochondria and lysosome, respectively and successfully used to image the fluoride ion in subcellular structures and in living mice. 4-amino-1,8-naphthalimide with intramolecular charge transfer (ICT) was chosen as fluorophore due to its advan-

Scheme 1 Molecular structures of three probes and the sensing mechanism of probes for detection of fluoride ion.

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The sensing ability of three probes for NaF was investigated in 20 mM HEPES buffer (pH 7.4) containing 50% DMSO (for the comparison with Con-F that is not soluble in HEPES buffer). Under these conditions, three probes emitted blue fluorescence with an emission peak at 475 nm. Upon the addition of 10 mM NaF, the emission intensity at 475 nm gradually decreased and a new emission peak at 540 nm appeared, exhibiting a ratiometric change with a large red-shift over 65 nm for all three probes (Figure S1). After incubation for 16 min, the ratios of F540/F475 for Mito-F and Lyso-F respectively increased from 0.31, 0.33 to 16.43, and 16.76, which are 53-fold and 51-fold enhancement for Mito-F and Lyso-F. However, the probe Con-F showed much less sensitivity, and only 3-fold enhancement in fluorescence ratio (F540/F475) was observed (Figure 1). By fitting the data of time-dependent fluorescence intensity at 475 nm, fluorescence turn-on rates of all three chemosensors were obtained. Pseudo-first-order kinetic rate constants of Mito-F (0.40 min-1) and Lyso-F (0.34 min-1) are much larger (more than 10.5 and 8.9- fold) than that of Con-F (0.038 min-1). The above result confirmed that positive changes of probes under physiological pH could sequester F− and promote desilylation of probes. However, for Con-F with only alkyl moiety, much less sensitivity and slower response could be observed. In addition, the response of lysosomal probe Lyso-F towards NaF in acidic system (pH 5.0) was also carried out and similar observation was found (Figure S6), indicating this probe may be applied in imaging of F− in acidic organelle of living cells.

Figure 1 (A) Time-dependent increase in fluorescence ratio (F540/F475) change of 10 µM Mito-F, Lyso-F and Con-F in the presence of 10 mM NaF from 0 to 30 min; (B) Intensity ratios (F540/F475) for Mito-F, Lyso-F and Con-F in the absence and presence of NaF after incubation for 16 min. The experiment was performed in HEPES buffer (20 mM, pH 7.4, 50 % DMSO, v/v), λex = 405 nm, slit width: 5 nm/10 nm.

tageous optical properties, such as strong absorption and emission in the visible region, high photostability, and large Stokes shift41. A silyl group, tert-butyldimethylsilyl (TBDMS) was chosen as reaction site for fluoride ion which was first reported by Kim and Swager42, and fluoride ion probes based on desilylation have proven to be applied in detection of F− in aqueous solutions and cells. An electron-withdrawing carbamate group is introduced to convert the 4-amino donor to a weak donor, the weak ICT effect of the fluorophore results in a blue shift of fluorescence emission. Once the Si-O bond is broken by F−, the probes may undergo self-immolation to dissociate carbamate group and release the yellow-green fluorescent 4-amino-1,8-napthalmide, thereby inducing ratiometric changes of fluorescence emission. The synthetic procedure for positively charged probes Mito-F and Lyso-F was outlined in Scheme S1. In addition, probe Con-F with the attachment of no positively charged alkyl moiety was also synthesized for control experiment.

Inspired by strong fluorescence response of Mito-F and Lyso-F towards NaF, we then assessed their sensitivity toward F− (NaF). Different concentrations of F− (from 0 to 11.4 ppm) was added to probe solutions and fluorescence emission spectra was collected after incubation for 60 min. Figure 2 (A, C) show the fluorescence emission of Mito-F

Figure 2 Titration of NaF with Mito-F and Lyso-F. (A, C) The fluorescence emission spectra of 10 µM Mito-F (A) or 10 − µM Lyso-F (C) containing various levels of F (from NaF) (0～11.40 ppm); (B, D) titration curve of Mito-F (B) and Lyso-F (D) was plotted by fluorescence intensity ratio − (F540/F475) as a function of F concentrations. λex = 405 nm, scan from 410 to 700 nm, slit width: 5 nm/10 nm.

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and Lyso-F in the presence of various levels of F−. As F− concentration increased, the fluorescence intensity at 475 nm decreased while a new fluorescence emission peak at 540 nm increased. Linear regression curve was fitted according to ratios of fluorescence intensity (F540/F475) in the range of F− from 0 to 11.40 ppm. The regression equations were y=0.11x+0.26 (R2=0.987) for Mito-F, and y=0.12x+0.27 (R2=0.990) for Lyso-F, respectively. The detection limits were calculated to be 0.067 ppm for Mito-F and 0.073 ppm for Lyso-F based on 3s/k. Next, we evaluated the selectivity of both probes for F−, various anion competitors such as Cl−, Br−, I−, CO32−, HCO3−, N3−, NO2−, AcO−, H2PO4−, and SO42− were chosen. As shown in Figure 3, these competitors almost had no effect on fluorescence emission of both probes. The unique emission red-shift was observed only with F− (from TBAF or NaF), which indicated the excellent selectivity for F− among all other anions. In addition, no obvious difference was observed for detection F− from TBAF and NaF, which made these probes more realistic in detecting F− in biological samples and cells or even in living animals.

indicated that both two probes can serve as ratiometric fluorescent probes for detection of F− in living cells.

−

Figure 4 Ratiometric imaging of F in HeLa cells. (A) Cells was incubated with 10 µM probe Mito-F only; (B) Cells were − treated with 1 mM F after incubation with probe Mito-F; (C) Cells were incubated with 10 µM probe Lyso-F only; (D) Cells − were treated with 1 mM F after incubation with probe MitoF. Scale bars: 50 µm. Figure 3 Fluorescence response of (A) Mito-F (10 µM) and (B) − − − 2− Lyso-F (10 µM) towards various species (Cl , Br , I , CO3 , − − − − 2− − HCO3 , NO2 , N3 , AcO , H2PO4 , SO4 , NaF, and TBAF). λex = 405 nm, scan from 410 to 700 nm, slit width: 5 nm/10 nm.

Encouraged by their good sensing performance for F−, we next evaluated their applications for bioimaging in living cells. Firstly, standard cell viability protocols (SRB assays) for probes (0~15 µM) were conducted for 24 h and the results showed that both Mito-F and Lyso-F had low toxicity (Figure S10). Subsequently, HeLa cells were incubated with Mito-F or Lyso-F at 10 µM for 30 min under physiological conditions. After final washing, cells were treated with and without NaF at 1 mM for 2 h. Then cells were imaged on a confocal microscope via a dual excitation/dual emission (Dex/Dem) ratiometric mode. As shown in Figure 4, cells incubated with Mito-F alone display blue fluorescence emission collected from blue channel (425 nm ~ 475 nm) and almost no fluorescence was observed from green channel (500~550 nm) when excitation at 488 nm. After treatment with NaF, cells afforded strong fluorescence from green channel and the fluorescence from blue channel decreased. Similar phenomena were observed for cells incubated with Lyso-F upon the addition of NaF. The ratiometric images (Fgreen/Fblue) were then created for both probes, which clearly show obvious ratiometric changes for HeLa cells with or without the treatment of NaF. The above results

To further investigate mitochondrial and lysosomal localization of Mito-F and Lyso-F, commercially available mitochondrial dye (MitoTracker Deep Red) and lysosomal dye (LysoTracker Red) were employed for colocalization study. HeLa cells were co-stained with MitoF and MitoTracker, and imaged under confocal fluorescence microscopy. As shown in Figure 5 and Figure S13, the blue-channel images for Mito-F merged well with

Figure 5. Sensors colocalizes to mitochondria and lysosome in HeLa cells. (A) Mito-F (10 µM), λex = 405 nm; λem = 425−475 nm); (B) MitoTracker Deep Red (0.46 µM), λex = 640 nm; λem = 663−738 nm); (C) Overlay of A and B; (D) Lyso-F (10 µM), λex = 405 nm; λem = 425-475 nm); (E) LysoTracker Red (0.5 µM); λex = 561 nm; λem = 570−620 nm); (F) Overlay of D and E. scale bar= 20 µm.

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red-channel images for MitoTracker dye (Pearson’s correlation 0.90). Similarly, blue images for Lyso-F merged well with red images for LysoTracker dye (Pearson’s correlation 0.71). Since Mito-F and Lyso-F was pre-localized in mitochondria and lysosome, the above observations clearly showed that F− could enter into mitochondria and lysosome, and triggered the fluorescence emission of probes, which will possibly provide new probes to study the effect of F− on function of subcellular structures, such as mitochondria and lysosome. Finally, we examined the suitability of these probes for visualizing fluoride ion in vivo. Mice were intraperitoneally injected with NaF (0.5 mM in PBS solution) and PBS solutions (control experiment) every 24 h for four days, respectively. Both mice were then intraperitoneally or intravenously injected with Mito-F in 24 h after the last injection of NaF or PBS. The two mice were imaged using a Carestream Health FX PRO small animal in vivo imaging system with a 430 nm excitation laser and a 535 nm emission filter. Figure 6 shows fluorescence images of living mice pre-injected with or without NaF solutions, following by probe Mito-F injection. If Mito-F was intravenously injected to mice, similar results were observed (Figure S14). It is obvious that control mice showed very weak background fluorescence while the mice with NaF injection show much stronger fluorescence according to the pseudo-color, indicating that probe Mito-F can serve as a fluorescent agent for fluoride ion imaging in vivo. This result also demonstrated that excess uptaken fluoride ion could accumulate in living mice. This represents the first example of imaging fluoride ion in living animals.

Figure 6. Representative fluorescence images of mice with continuous injection of (A) PBS (10 mM, 200 µL) and (B) NaF (0.5 mM PBS solution, 200 µL) for four days. Both mice were injected with 50 µM Mito-F and imaged after anaesthesia.

In summary, we have developed two novel ratiometric fluorescent sensors for sensitive and selective detection of fluoride ion. Both probes displayed a large red-shift of fluorescence emission and were applied to the imaging of fluoride ion in living cells. With the help of attachment of triphenylphosphonium and dimethylamino moiety, two sensors display more sensitive responses than Con-F in

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physiological conditions due to sequestration effect of positive charges. In addition, the attachment of triphenylphosphonium and dimethylamino moieties helped transport probes to mitochondria or lysosome specifically and detected fluoride ion in situ. In vivo imaging of fluoride ion in living mice using probe Mito-F was further successfully achieved for the first time. We anticipated this work will provide an approach to design fluoride ion chemosensor that could be applied to study fluoride ion biology in subcellular structures (such as mitochondria and lysosome) and in vivo imaging of living animals.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org/

AUTHOR INFORMATION Corresponding Author * Email: [email protected]; Fax: 8610-82805635.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (‘973’ Program; grant no. 2013CB933800), the National Natural Science Foundation of China (grant Nos. 21422201 and 21372018).

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