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BODIPY based two-photon fluorescent probe for real time monitoring of lysosomal viscosity with fluorescence lifetime imaging microscopy Lingling Li, Kun Li, Meng-Yang Li, Lei Shi, Yan-Hong Liu, Hong Zhang, Sheng-Lin Pan, Nan Wang, Qian Zhou, and Xiaoqi Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00590 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on April 1, 2018
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Analytical Chemistry
BODIPY based two-photon fluorescent probe for real time monitoring of lysosomal viscosity with fluorescence lifetime imaging microscopy Ling-Ling Li, Kun Li, * Meng-Yang Li, Lei Shi, Yan-Hong Liu, Hong Zhang, Sheng-Lin Pan, Nan Wang, Qian Zhou and Xiao-Qi Yu* Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu, 610064, China Fax: (86)-28-85415886; Tel: (86)-28-85415886; E-mail:
[email protected];
[email protected]. ABSTRACT: The viscosity of lysosome is reported to be a key indicator of lysosomal functionality. However, the existing mechanical methods of viscosity measurement can hardly be applied at the cellular or sub-cellular level. Herein, a BODIPY-based two-photon fluorescent probe was presented for monitoring lysosomal viscosity with high spatial and temporal resolution. By installing two morpholine moieties to the fluorophore as target and rotational groups, the TICT effect between the two morpholine rings and the main fluorophore scaffold endowed the probe with excellent viscosity sensitivity. Moreover, Lyso-B succeeded in showing the impact of dexamethasone on lysosomal viscosity in real time.
As an essential micro-environmental parameter, cellular and subcellular viscosity is known to contribute to biological functions through affecting the interaction and transportation of biomolecules and chemical signals within live cells.1-2 The abnormal changes of cellular viscosity are closely related to many disorders and diseases, such as diabetes, infraction and hypertension.3 Cellular viscosity varies significantly as function of sub-cellular environment, monitoring viscosity at subcellular level is greatly beneficial for not only fundamental cell biology but also disease diagnosis and pathological studies.4 As an important subcellular in most eukaryotic cells, lysosome involves in various cell processes, including protein degradation, secretion, plasma membrane repair and autophagy.5 The viscosity of lysosome is reported to be a key indicator of lysosomal functionality.6 However, the existing mechanical methods of viscosity measurement can hardly be applied at the cellular or sub-cellular level.7 Therefore, development of applicable techniques for monitoring and measuring lysosomal viscosity is in urgent demand. Small molecule fluorescent probes stand out as viable tools for reporting on cellular and subcellular viscosity changes, owing to their advantage of high spatial and temporal resolution, non-invasive detection of targets in living systems, as well as technical simplicity.8 As a matter of fact, fluorescent rotors, especially 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivatives, are well-established viscometers that are used to measure the viscosity of many biological systems.914 Most of the fluorescent molecular rotors are based on a distorted intramolecular charge transfer (TICT) mechanism, which is proposed to be one of the major nonradiative deexcitation pathways in fluorophores.15 The change of TICT by the surrounding viscosity is known to alter the fluorescence intensity and lifetime.16 For example, Xiao and his colleagues synthesized Lyso-V (Scheme 1a) by attaching a morpholine
Scheme 1. a) Previously published structure of lysosomal viscosity probe (Lyso-V and BDP-1) and structure in this work (compound B); (b) Working principle of lysosomal viscosity probe Lyso-B
moiety to a typical BODIPY moiety, and for the first time the probe realized real-time quantification of lysosomal viscosity in live cells through fluorescence lifetime imaging microscopy (FLIM).17 Based on the design of Lyso-V, Kuang group installed ethylene glycols to the probe to form BDP-1 (Scheme 1a), whose fluorescence intensities increased with the elevation of viscosity.18 To the best of our knowledge, these two papers are the only reported work regarding the detection of lysosomal viscosity. Both of the studies realized their sensitivity towards viscosity through controlling the free rotation
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around the single bond connecting the BODIPY core and the phenyl group. Many other BODIPY-based viscosity probes are designed according to the same mechanism.9-14, 19 However, one shortage of this design strategy is that to maintain the sensitivity of the probe to viscosity, little modification at 1, 1’position or meso-phenyl group can be done since it will bring great hindrance to the free rotation. Whereas, more modifiable sites are desired to develop viscosity probes with improved properties, such as better biocompatibility, lower background noise, and two-photon (TP) imaging abilities. TP imaging offers important advantages such as low phototoxicity, minimum background interference, and deeper tissue penetration.20,21 To fulfil these needs, we took a different strategy to design our probe for lysosomal viscosity detection. In the molecular design of Lyso-B (Scheme 1c), we attached two highly hydrophilic polyethylene glycol (PEG) chains to the probe to reduce its cytotoxicity, improve its water solubility and biocompatibility.22, 23 However, the steric hindrance between the bulky PEG group at 7-position and the methyl group at 1-position was reported to prevent free rotation of the meso-phenyl group.12, 21 To detect viscosity, a new rotation center will be needed. We therefore connected two morpholine groups to the BODIPY fluorophore through the phenylethynyl moiety. The TICT effect between the two morpholine groups and the main fluorophore scaffold can be altered by viscosity, which will cause the change in fluorescence intensity and lifetime, thus endowing the probe with viscosity sensitivity. In addition, as the most widely adopted lysosome targeting group, morpholine has long been proved to be effective for the selective binding of lysosomes.17-18, 24 According to previously published literature, morpholine has a pKa of 5−6 and can only be protonated in lysosomes (pH 3.8−5.5). That is to say, the probe is lysosome active and may possess high signal to noise ratio in lysosomal tracing. That happens because the electron-rich morpholine moiety can induce the fluorescence quenching of the BODIPY fluorophore through photoinduced electron transfer (PET). Whereas when the probe diffuses into lysosomes and is protonated, the morpholine unit will be transformed from electron-rich group to electronwithdrawing group. Thus, the PET process will be blocked, leading to the turn-on of the fluorescent probe. This particular characteristic will make sure the selective imaging of lysosomes through confocal laser scanning microscopy (CLSM) and lysosomal viscosity imaging through FLIM. Noticeably, lifetime is independent of concentration, photobleaching, absorption, and excitation intensity, it therefore holds natural advantage in unambiguous assessment of microviscosity.25 Moreover, the phenylethynyl groups that connect the morpholine groups and the fluorescent core greatly extend the conjugate plane, thus increasing their two-photon absorption (2PA) cross section and making the probe an efficient TP excited fluorescent sensor. The similar strategy was adopted by Qian group back at 2009, who built a donor-π-donor type BODIPY fluorophore with great two-photon properties.20 To better illustrate the sensing mechanism, we also synthesized compound B as the contrastive compound. Lyso-B and compound B (Scheme 1) were synthesized according to Scheme S1, their chemical structure and the structure of the intermediate products were characterized by 1H-NMR, 13C-NMR and HRMS or MALDI-TOF-MS studies (Figure S20-S33).
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EXPERIMENTAL SECTION Materials. Unless otherwise stated, all reagents and solvents were obtained from commercial suppliers, and were used without further purification. Column chromatography was performed on silica gel (Qingdao Haiyang) 300-400 or 200-300 mesh. All solvents used in spectra test systems were chromatographically pure. Aqueous solutions were freshly prepared using ultrapure water from Thermo Scientific Smart 2 Pure 6 UV/UF. General methods, instrumentation, and measurements. 1 H NMR and 13C NMR spectra were recorded on a Bruker AMX-400 with chemical shifts expressed in parts per million (in deuteriochloroform or DMSO-d6, Me4Si as internal standard). The High-resolution mass spectra were obtained on a Finnigan LCQDECA and a Bruker Daltonics Bio TOF mass spectrometer. UV−vis absorption spectra were recorded on a Hitachi PharmaSpec UV-1900 UV−visible spectrophotometer. Fluorescence spectra were determined using a HITAI F-7000 Spectro fluorophotometer. The intracellular photostability and the live cell imaging experiments were performed on a ZEISS LSM 780 confocal laser scanning microscope (CLSM). Twophoton fluorescence imaging experiments were carried out on a Nikon A1R MP+ laser scanning confocal microscope. TLC analyses were performed on silica gel GF 254 using UV light as visualizing agent. The pH values were determined by a Leici pH3c (digital display) pH meter. The viscosity of water/ glycerol or water/ organic solvent/ glycerol mixtures were measured using an advanced Brook field Rheo3000 R/S plus Rheometer. The temperature was kept at (20±0.1) °C with a bath circulator. Toxicity studies. HeLa cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1‰ antibiotics (penicillin– streptomycin, 10,000 U mL-1) at 37 ºC in a humidified atmosphere containing 5% CO2. Toxicities of Lyso-B toward HeLa cells were determined by using MTS (3-(4, 5-dimethylthiazol2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt) reduction assay following literature procedures. About 1.0×104 cells/well was seeded into 96-well plates. After 24 h, various concentrations of Lyso-B (2.5, 5,10, 20, 40 µM) were added to the cells. After another 24 h, 20 µL MTS and 80 µL PBS were added to each well and the plates were incubated at 37ºC for another 1h. Then the absorbance of each sample was measured using an ELISA plate reader (model imark 680, BioRad) at a wavelength of 490 nm. The cell viability (%) was obtained according to the manufacturer’s instruction. One photon and TP Confocal laser scanning microscopy (CLSM) experiments. HeLa cells were seeded at a density of 2.5 × 105 cells per well in 35 mm confocal dish (Φ = 15 mm), 24 h before the addition of Lyso-B or dexamethasone. The cells were treated with Lyso-B (5 µM, 30min) only, or coincubated with Lyso-B and Lyso Tracker Green DND-26. In the Lyso Tracker Green group, the cells were cocultured with Lyso Tracker Green DND-26 (1 µM) and Lyso-B (5 µM) for 30 min. All the cells were washed 3 times with PBS before being taken pictures using one or two photon CLSM. In the intracellular photo stability experiment, Lyso Tracker Red and Lyso-B were tested under exact same conditions (λex = 543
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Analytical Chemistry nm, power of the light source = 1mW); the power of the light source for Lyso Tracker Green was 25 mW (λex=488 nm). Fluorescence lifetime imaging experiments were carried out on LSM upgrade kit from picoquant company. The emission was collected through a 590 ± 15nm band pass filter. The cells were cultured according to similar procedure mentioned above. The dexamethasone group was first treated with dexamethasone (5 µM) for half an hour, then Lyso-B (5 µM) was added. The cells were co-incubated with the probe and the drug for another 30 min and washed with PBS (3*1 mL). For the real-time monitoring of the effect of dexamethasone on lysosomal viscosity, 5 µM Lyso-B was first incubated with Hela cells for 30 min at 37 ºC in a humidified atmosphere containing 5% CO2. After being washed with PBS (3*1 mL), 2 mL PBS solution containing dexamethasone (5 µM) was added and images were collected continuously at different time points (Figure S19).
RESULTS AND DISCUSSION Photophysical properties. Following the synthesis and characterization, we systematically studied the photophysical properties of Lyso-B and compound B. The photophysical data were summarized in Table S1. The results revealed that the fluorescence intensity of Lyso-B was dependent on both pH and viscosity (Figure 1a, S3, S5 and S6). The ultra-violate (UV) absorption and fluorescent spectra of Lyso-B and compound B was first tested in a serious of Britton-Robinson buffer solution (from pH 1.89 to 11.9). As indicated in Figure 1a and Figure S3, Lyso-B can only be fluorescent in acidic conditions (pH
