Specific Fluorescence Probes for Lipid Droplets Based on Simple

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Specific Fluorescence Probes for Lipid Droplets Based on Simple AIEgens Zhiming Wang, Chen Gui, Engui Zhao, Jing Wang, Xiaodong Li, Anjun Qin, Zujin Zhao, Zhen-Qiang Yu, and Ben Zhong Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01282 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 10, 2016

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ACS Applied Materials & Interfaces

Specific Fluorescence Probes for Lipid Droplets Based on Simple AIEgens Zhiming Wang, †,‡,∇, # Chen Gui, †,∇,§ Engui Zhao,∇,§Jing Wang, # Xiaodong Li,∇ Anjun Qin, ‡ Zujin Zhao,*,‡ Zhenqiang Yu,∇ Ben Zhong Tang*,‡,∇,§,^

State Key Laboratory of Luminescent Materials and Devices, South China University of Technology (SCUT), Guangzhou 510640, China; HKUST-Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park, Nanshan, Shenzhen 518057, China; School of Petrochemical Engineering, Shenyang University of Technology (SUT), Liaoyang 111003, China; Department of Chemistry, The Hong Kong University of Science & Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, China; Hong Kong Branch of Chinese National Engineering Research Center (CNERC) for Tissue Restoration and Reconstruction, Hong Kong, China. ‡

SCUT

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HKUST-Shenzhen Research Institute

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HKUST

ACS Paragon Plus Environment

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^CNERC

ABSTRACT. Lipid droplets (LDs), as dynamic complex organelles, are involved in various physiological processes, and their numbers and activity are related to many diseases, even cancer. Hence, locating and concentration monitoring of LDs are very important to scientific bioresearch and health care. In this work, we prepared two simple luminogens (FAS and DPAS) via very facile synthetic procedures and purification. They feature aggregation-induced emission (AIE) and excited state intramolecular proton transfer (ESIPT) characteristics. They exhibit large Stokes shifts and bright orange and yellow emissions in the aggregated state, and the emissions can be reversibly turned “off” and “on” for many cycles by controlling buffer’s pH values. Both FAS and DPAS are cytocompatible, and can selectively accumulate in and light up the LDs in living cells with superior resolution and high contrast. They also outperform the commercial LD probes in terms of photostability. Combining the advantages of high LD-specificity, good biocompatibility, surperb photostability and low preparation cost, FAS and DPAS may become powerful tools to the study on LDs-related intracellular activities, such as LDs-based pathology and pharmacology.

KEYWORDS: aggregation-induced emission, excited-state intramolecular proton transfer, lipid droplets, fluorescence probe, photostability.

INTRODUCTION


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Lipid droplets (LDs) are subcellular organelles surrounded by a phospholipid monolayer, and contain diverse neutral lipids such as triacylglycerol and cholesteryl esters. 1-3 LDs are generally considered to be inert and static aggregates of neutral lipids since its discovery, but this viewpoint is challenged in recent years.4 Some reports demonstrate that LD is a dynamic complex organelle involving various physiological processes, and its metabolic balance and stability play a key role in living organisms. The abnormity of LD may even cause certain cancers according to modern medicine study.5-10 Hence, locating and concentration monitoring of LDs are highly important to scientific bioresearch and health care. In addition to the biomedical significance, LDs in plants are potential sources for biofuels to alleviate global energy crisis, which makes it important to increase LD production by screening plants (especially algae) rich in LDs.11

Owing to the high sensitivity, visibility and tunability, fluorescence imaging technique is emerging as one of the most powerful and popular approaches in biophysical studies on membrane structure, phase separation and domain dynamics, etc.12,13 Many fluorescent probes targeting LDs have been developed and commercialized for fluorescence imaging, such as Nile Red and BODIPY dyes (Scheme 1).14-16 However, most of these probes can only been used in dilute solutions because their fluorescence is severely quenched in high-concentration solutions or in the aggregated states, that is aggregation-caused quenching (ACQ).17 Although ACQ problem can be alleviated by reducing the amount of fluorescent probe in some degree, a new


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problem of photobleaching arises.18,19 The fluorescence signals from dilutely dispersed probe molecules in cells or tissues become faint or disappear after several scans because of selfdecomposition of the probe in confocal microscope measurements. Additionally, the commercial fluorescence probes for LDs also have some drawbacks. For example, Nile red can respond to the polarity in different cytoplasmic environments, exhibiting yellow fluorescence in nonpolar conditions and red fluorescence in polar conditions, which enables it to discriminate nonpolar LDs from other organelles. However, the sensitivity of Nile red is relatively low because of fluorescence signal overlap between the yellow and red channels.14 The BODIPY dyes usually have small Stokes shifts, which cause severe self-absorption phenomenon, and thus weaken the fluorescence.15,16 These technical problems undermines the performance of current fluorescence probes in dynamic detection and quantitative analysis. On the other hand, most traditional fluorophores require complicated synthetic procedures and troublesome purification, making the cost prohibitive when apply them to ordinary therapy or large-scale research work. It is therefore highly favorable to develop fluorescence probes with ACQ-free emissions, large Stokes shifts and high photostability in an easy and low-cost way.


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Scheme 1. Chemical structure of LD bioprobes: Nile red, BODIPY dyes, and TPE-AmAl (AIEgen).

Recently, aggregation-induced emission (AIE), an opposite photophysical phenomenon to ACQ, is attracting intense interest because of the fundamental importance and promising practical applications.20-22 The fluorogens with AIE characteristics (AIEgen) are generally weakly fluorescent or nonfluorescent in solutions, but can fluoresce intensely in nanoparticles or solid films. In view of the utility in fluorescence bioimaging techniques, the AIEgens are of intrinsic merits. They are free of ACQ problem, and thus can be used as nanoaggregates (AIE dots) in aqueous environment with high fluorescence, which decreases the interference of the autofluorescence from background and enhances the contrast of the images.23 The formation of AIE dots can protect the inner probe molecules from photobleaching and photooxidation, consequently resulting in high photostability of the probes. Therefore, it is envisioned that employing AIEgens to probe LDs could be a promising approach with high sensitivity and stability.

In our previous work, we developed an AIEgen, TPE-AmAl, for LD imaging in living HeLa cell, liver LO2 cells and green algae.24 Thanks to the AIE characteristic, TPE-AmAl showed higher selectivity and better photostability compared with those of commercial Nile Red dye.24 However, the fluorescence of TPE-AmAl was responsive to the environmental polarity, and varied from orange in aqueous media to greenish blue when internalized in cells, within the self-


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fluorescence range of many living organisms. To address this issue, in this contribution, we prepared two novel AIEgens (FAS and DPAS) with a salicylaldehyde schiff-base structure. They are readily accessible from cheap raw materials (salicylaldehyde or hydrazine) via very simple synthetic procedures and facile purification of crystallization. They exhibit excited-state intramolecular proton transfer (ESIPT) process, and gives large Stokes shift and enhanced orange and yellow fluorescence associated with their keto forms (K) emissions in the aggregated state.25-27 They are permeable to cell membrane and can specifically locate and accumulate in LDs with lower cytotoxicity and better photostability than commercial LD probes. All of these merits enable them to be perfect fluorescence probes for detecting LDs with great potential for commercial use in pathology and pharmacology.28

RESULTS AND DISCUSSION Synthesis and Characterization The moelcular sturctures and synthetic routes of FAS and DPAS are shown in Scheme 2. The synthesis was started from cheap commercial product of fluorenone (or benzophenone for M2), which refluxed with excess hydrazine hydrate in ethanol for 2h to afford light yellow (or white for M2) needle crystals of M1 in 98% yield (or 95% for M2) after cooling to room tempreture (RT). After filtration, the obtained crystals without further purification were treated with salicylaldehyde in refluxed ethanol for 2h, and yellow micro crystals of the target FAS were generated in 95% yield (or light yellow crystals for DPAS in 90% yield) when the mixture was


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cooled down to RT. The obtained FAS and DPAS were of high purity, and did not need further purification by recrystallization or chromatography. After fully characterizing by NMR spectra, high resolution mass spectra (HRMS) and X-ray crystallography, the fine structures of FAS and DBAS were confirmed (Experimental section).

O

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Scheme 2. The synthetic routes to FAS and DPAS.

Single crystals of FAS and DPAS were grown from the mixture of petroleum ether and dichloromethane, and analyzed by X-ray crystallography. As shown in Figure 1F and 1G, FAS crystals emit orange-red light, while DPAS crystals show yellow fluorescence under illumination of a UV lamp. The ORTEP drawings of the crystal structures of FAS and DPAS were displayed in Figure 1A and 1B. FAS shows a relatively planar conformation and the whole moelcule becomes rigid due to the intramolecular hydrogen bonding (1.898 Å) as indicated in Figure 1A. The FAS molecules are stacked in a face-to-face and offset pattern (J-aggregate), and the distance between the neighbouring planes are 3.314 Å, indicative of strong intermoleular π-π stacking interactions.29 DPAS, however, adopts a distorted conformation, where one of the phenyl ring is located perpendicularly to the conjugated plane with a dihedral angle of ~90o,


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which prevents the close π-π stacking between the neighbouring molecules.30 Multiple C-H…π and C-H…O interactions are observed in these crystals, indicating the molecules are rigidified and the intramolecular rotation are restricted greatly. According to the crystal structures, FAS should possess a larger conjugation degree and stronger intermolecular interactions in the aggregation state than DPAS, which is in good agreement with the fluorescence colors of their crystals.

Figure 1. The ORTEP drawings of single crystal structures of (A) FAS (CCDC 1432238) and (B) DPAS (CCDC 1432239). The molecular packing patterns of FAS (C: side view, D: top view) and DPAS (E: side view) in the crystalline state. The fluorescence images of crystals (F) FAS and (G) DPAS.

Photophysical Properties

Both FAS and DPAS exhibit typical ESIPT characters, of which the photophysical process is illustrated in Figure S1. The absorption maxima of FAS and DPAS are located at 322 and 301


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nm, respectively, which are associated with the transition of enol form from ground state to excited state (E→E*), and no obvious changes are observed in the absorption spectra in different solvents. FAS shows two emission bands in solutions, where the short-wavelength emission at 430 nm is originated from E form emission (E*→E), and the long-wavelength emission at 600 nm is assigned to the emission of K form (K*→K). 25-27 Similar emission bands at 425 and 550 nm are also observed for DPAS. Although FAS and DPAS have a similar framework, the subtle structural alteration has led to apparent difference in emission intensity of K and E forms. The emission from K form is more conspicuous in DPAS than in FAS in different solvents (Figure S2), suggesting DPAS is easier to undergo ESIPT process. This is also evidenced by the shorter hydrogen bond between phenolic hydroxyl group and nitrogen atom (O-H...N) in DPAS as indicated in the crystal structure (Figure 1A), which promotes the proton transfer.

The AIE features of both fluorescence probes are investigated in THF/water mixtures, and the measured PL spectra are displayed in (Figure 2).23 Whereas both FAS and DPAS show weak emissions in THF solutions, their emissions are enhanced greatly as the addition of a large amount of water into THF. Since the probes are insoluble in water, they should have aggregated into nanoparticles in aqueous media, and the enhanced emission is thus caused by the aggregate formation, demonstrating their AIE attributes. The aggregates of FAS and DPAS show orange (595 nm) and yellow (565 nm) emissions, respectively, which stem from K forms of the molecules. The absolute fluorescence quantum yield (ФF) of DPAS is 3.0%, which is higher than


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that of FAS (2.1%) in the solid state, probably due to the easier ESIPT process in DPAS. The ESIPT effect gives rise to large Stokes shifts of ~ 200 nm in aggregated state (Figure S3) (Nanoparticles in fw = 90% THF/water mixture: λabs-max-FAS = 380 nm, λPL-max-FAS = 595 nm; λabsmax-DPAS

= 360 nm, λPL-max-DPAS = 565 nm), which are very important to enhance fluorescence

imaging resolution and contrast.

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Figure 2. The fluorescence spectra of (A) FAS and (B) DPAS in THF-water mixtures with different water fractions (fw) ; Plots of I/I0 versus water fractions of (C) FAS and (D) DPAS, where I0 is the emission intensity in THF (Inset: fluorescence images at fw = 0 % and 90 %).

As schiff base derivatives, the structural stability and optical property of FAS and DPAS in aqueous media, especially in acidic and basic conditions, should be concerned.31 The two probes were dispersed in buffers with pH values in the range of 1‒13, and the absorption and emission spectra were measured (Figure S4). The absorption and emission spectra of both probes change little in buffers with pH values ranging from 1 to 11, implying they are structurally stable even in the extreme conditions. When the pH values of buffers reach 12 or above, their absorption profiles are varied, with obviously red-shifted absorption maxima, and drastically weakened emission intensity (Figure 3A and 3B), suggesting that the ESIPT process is forbidden. The NMR data of the two probes measured in CD3OD with excess KOH reveal that their molecular frameworks are kept stable, but the probes exist in a salt form in highly basic condition due to the acidity of the phenolic hydroxyl group (Figure S5 and S6).32 Along with the disappearance of active proton in phenolic hydroxyl group, the ESIPT process cannot occur and thus the emissions from K forms vanish. After the addition of excess acid, the salified probes revert to the original form, and the ESIPT process is activated, which turns on the emissions. These processes are highly reversible, and the fluorescence turn-off and turn-on can be switched efficiently for many times by controlling buffer’s pH values (Figure 3C and 3D).


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Figure 3. The pH plots of I/I0 versus pH value for (A) FAS and (B) DPAS, where I0 is the emission intensity in pH = 7.0 (Inset: the PL spectra in different buffers (where fw = 99.5 %). The reversible switching in acidic medium (turn-on) and basic medium (turn-off) of (C) FAS and (D) DPAS, where I0 is the emission intensity in pH = 7.0.

Fluorescent Imaging Properties


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The in vivo fluorescence bioimaging potential of FAS and DPAS was evaluated by costaining experiments using BODIPY as a reference. As shown in Figure 4, FAS and DPAS selectively accumulate in LDs and show bright orange and yellow fluorescence, respectively, which are similar to the emissions from their aggregates in aqueous media.24 Unlike environment-sensitive fluorescence of TPE-AmAl, FAS and DPAS show stable fluorescence color in LDs. Both FAS and DPAS can specifically target and light up the LDs in lung cancer cells A549 with good resolution and high contrast (the clear appearances of LDs are shown in merged images in Figure 4E and 4J), with high overlap ratios of 97% for FAS and 98% for DPAS referred to commercial LDs probe BODIPY (green emission) (Figure 4B and 4G). At the same time, the two newly prepared probes had high overlap ratios of ~99% in co-strained process (Figure 4O). These results suggest that it is feasible to replace BODIPY with FAS and DPAS in LDs imaging, and the cost can be much lower because of the very easy preparation method.


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Figure 4. Images of A549 cells incubated with FAS, DPAS or BODIPY (FAS and DPAS: 7.5 µM; BODIPY: 50 ng/mL) for 15 mins (Bright-field images: A, F and K; Fluorescence images: B, C, G, H, L and M; Merged images: D, I and N) and the enlarged regions (E, J and O). BODIPY is excited at 460-490 nm, FAS at 400-440 nm, and DPAS at 330-385 nm. The overlap ratios: 97.70% for BODIPY and FAS, 98.53% for BODIPY and DPAS, and 98.84% for FAS and DPAS. All the images share the same scale bar of 30 µm (except E, J and O).

For most traditional fluorescence probes with ACQ effect, working concentration is very important and sensitive. High concentrations will weaken the fluorescence signals, while too low concentrations will cause photobleaching after several laser scans.18,19 On the contrary, FAS and DBAS are not influenced by concentrations as much as traditional fluorescence probes, and can work at high concentrations with brighter images owing to the AIE attribute. To test the concentration impact, HeLa cells were pretreated by oleic acid (50 µM, 6h) for stimulating cells to produce more LDs in cytoplasm, and then they were incubated with different concentrations of FAS or DPAS (2.5, 5.0, 7.5 and 10.0 µM) (Figure 5). After incubating for 30 min, the dotshaped LDs with bright orange or yellow emissions were observed, and their strong fluorescence made them clearly distinguishable from the background. These results demonstrate that the two probes can be used at a much wide concentration range. The more clear fluorescence images of DPAS indicates its better bioimaging performance than FAS, which may result from higher sensitivity to yellow light of human eyes as well as its higher ФF values.


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Figure 5. Bright-field (A-D, I-L) and fluorescent (E-H, M-P) images of HeLa cells stained with FAS (A-H) and DPAS (I-P) at different concentrations for 30 min after incubation in the presence of 50 µM oleic acid for 6h. Probes concentration: (A, E, I, M) 2.5 µM; (B, F, J, N) 5.0 µM; (C, G, K, O) 7.5 µM; (D, H, L, P) 10.0 µM. All the images share the same scale bar of 30 µm.

Since fast incubation will increase screening efficiency in real experiments and applications, the impact of staining time of FAS and DPAS towards LDs was further investigated. The incubation time was set at 15, 30 and 60 min for the probes at a concentration of 7.5 µM. It can be seen from the fluorescence images (Figure 6) that DPAS enters cells and accumulates in LDs more quickly than FAS, and fluorescence images with good resolution and high contrast can be


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obtained within 15 min. This is probably due to a more flexible molecular structure of DPAS, which enables DPAS to easily adjust its conformation according to the structures of cell membrane or transport receptors, and thus rapidly get to LDs.33

Figure 6. Bright-field (A-C, G-I) and fluorescence (D-F, J-L) images of HeLa cells stained with FAS (A-F) and DPAS (G-L) at 7.5 µM for different time after incubation in the presence of 50µM oleic acid for 6h. Stained time: (A, D, G, J) 15 min; (B, E, H, K) 30 min; (C, F, I, L) 60 min. All the images share the same scale bar of 30 µm.

Most of the hydrophobic probes prefer to accumulate in LDs, in the absence of strong interactions with other organelles, because the LDs are the most hydrophobic organelles in cells,


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which is consistent to the theory of similarity and intermiscibility. FAS and DPAS are small molecules free of specific groups that can strongly target, interact or bond with intercellular organelles. Once they are internalized in cells via endocytosis, they prefer to aggregate in LDs because of their hydrophobic nature. In this way, the free intramolecular motions are restricted and the ESIPT process is activated, which turns on their fluorescence. This is probably the sensing mechanism of these simple hydrophobic LD probes.

Cytotoxicity and Photostability

The cytotoxicity of FAS and DPAS were evaluated by the method of 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure 7A).22,24 No distinct inhibitory effect was observed in HeLa cell growth in culture medium with high concentrations of up to 10 µM of FAS and DPAS. The excellent biocompatibility of FAS and DPAS inspired us to investigate their further applications in cellular imaging, such as dynamic monitor of LD formation, metabolism and death in living cells, and so on in our further work.


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Figure 7. (A) Cytotoxicity of FAS and DPAS on HeLa cells determined by MTT assay. (B) The signal loss of fluorescence intensity of FAS, DPAS and BODIPY under different light irradiation time.

For real-time tracking of dynamic systems, the photostability of the probes is a major concern. Since the fluorescence imaging process is achieved in the dark field under the excitation light source, the prolonged excitation time usually leads to decrease of fluorescence intensity and finally fluorescent signal loss of ACQ probes at low working concentrations. Commercial LDs probe of BODIPY (1µg/mL), for example, would lost 90% signal of fluorescence intensity after 5 min irradiation with 2% laser power energy as displayed in Figure 7B. However, the fluorescence intensity of FAS and DPAS in cellular LDs had almost no decrease after 60 min irradiation (continuous scanning) with 2% laser power energy, which was not only much better than conventional ACQ probes but also AIE probe TPE-AmAl (the loss is about 20% after 10


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min irradiation with 2% laser power energy).24 At the same time, the dynamic processes were recorded as videos and shown in Figure S7, and Viedo S1 and S2 (the whole record time is about 60 min, which is speeded up to 11 s). The movement and behavior of LDs in HeLa cells can be clearly observed, implying the cells are alive, and the two probes have low cytotoxicity even under long-time continuous excitation. The fluorescence intensity is steady, which should result from the stable chemical structures of the probes. LDs-specific probes with such high photostability and low cytotoxicity, to our knowledge, have been rarely reported before. These results make it possible to study the function of lipid droplets in vivo, relationships between the LDs contents and diseases as well as the drug treatment effect. The FAS and DPAS may become promising candidates for LD-related researches.

CONCLUSION In this work, two AIE-active LDs-specific probes with ESIPT characteristics, namely FAS and DPAS, were designed and synthesized. They can be facilely and efficiently prepared from commercial raw materials via very simple synthetic procedures and purification. FAS and DPAS can selectively target and accumulate in cellular LDs, and give bright orange and yellow fluorescence, respectively. They can be used to image the LDs in HeLa cells and A549 cells with good resolution and high contrast. They also have merits of large Stokes shifts, alterable incubation concentration and time, strong photostability as well as high specificity towards LDs. These results enable the two probes localizing LDs accurately, which can provide more insights


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into LDs-related intracellular activities such as diagnosis of the related diseases and screening for algae with high content of lipid.

EXPERIMENTAL SECTION Synthesis FAS: Into a 500 mL three necked round-bottom flask with a thermometer and a reflux condenser were placed 10 g (55.6 mmol) of 9H-fluoren-9-one and 400 mL ethanol, and stirred until completely dissolved under 40 °C. The excess hydrazine hydrate (85%, 20 mL) was added, and heated to reflux for 2h. The mixture was cooled to room temperature, and light yellow needle crystals were obtained (M1, yield 98 %). After filtrating, the crystals (5g, 25.7 mmol) were placed to 250 ml round-bottom flask with 150 mL ethanol, and the slightly excess salicylaldehyde (1.02 eq) was added into the mixture. The mixture was refluxed for 2h, and then cooled to room temperature. Yellow crystals were of FAS were obtained in yield = 95 %, which did not need further purification. 1H NMR (400 MHz, d-DMSO, ppm): 10.68 (s, 1H, -OH), 8.91 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 7.9 Hz, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.87-7.84 (m, 3H), 7.577.50 (m, 2H), 7.45-7.37 (m, 3H), 7.04-7.00 (m, 2H). 1H NMR (400 MHz, CD3OD, ppm): 8.83 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.74-7.62 (m, 3H), 7.52-7.38 (m, 3H), 7.35-7.29 (m, 2H), 7.04-6.97 (m, 2H) (No signal from -OH was detected.). HRMS (C20H14N2O): m/z 298.1118 (M+, calcd 298.1106).

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C NMR (100 MHz, d-DMSO, ppm), 159.22, 158.86,


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158.31, 141.82, 140.82, 136.08, 133.42, 132.03, 131.58, 130.57, 129.45, 128.61, 128.57, 128.42, 122.49, 120.72, 120.55, 119.81, 119.56, 116.60.

Crystal data for FAS (CCDC 1432238): C20H14N2O; MW = 298.33; monoclinic; P2(1)/c; a = 20.379(8), b = 4.7351(9), c =31.360(9) Å; β = 106.25(4)o; V = 2905.4(15) Å3; Z = 8; ρcalcd = 1.364 g cm-3; µ = 0.677 mm-1 (MoKα, λ = 1.5418 Å); F(000) = 1248; T = 99.9(5) K; 2θmax = 66.5 (98.58)o; 13060 measured reflections; 5145 independent reflections (Rint = 0.1302); GOF on F2 = 1.000; R1 = 0.0835; wR2 = 0.1805 (all data); ∆e 0.374 and -0.378 e Å3.

DPAS: The procedure was analogous to that described for FAS. White needle crystals of M2 were obtained in 95% yield, and light yellow ones of DPAS were obtained in 90% yield. 1H NMR (400 MHz, d-DMSO, ppm): 11.28 (s, 1H, -OH), 8.75 (s, 1H), 7.64-7.62 (m, 2H), 7.54-7.41 (m, 7H), 7.28-7.24 (m, 3H), 6.29-6.26 (m, 1H), 5.95 (d, 1H). 1H NMR (400 MHz, CD3OD, ppm): 8.79 (s, 1H), 7.75-7.68 (m, 2H), 7.52-7.44 (m, 4H), 7.42-7.37 (m, 3H), 7.31-7.23 (m, 3H), 6.90 (t, J = 7.9 and 8.1 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H) (No signal from –OH was detected.). HRMS (C20H16N2O): m/z 300.1278 (M+, calcd 300.1263). 13C NMR (100 MHz, d-DMSO, ppm), 167.64, 163.42, 158.81, 136.57, 135.29, 133.01, 132.11, 131.08, 129.05, 128.57, 128.54, 128.51, 128.22, 119.34, 117.95, 116.32.

Crystal data for DPAS (CCDC 1432239): C20H16N2O; MW = 300.35; monoclinic; P21; a = 11.1098(5), b = 5.7159(2), c =13.0553(5) Å; β = 109.388(5)o; V = 782.03(6) Å3; Z = 2; ρcalcd = 1.275 g cm-3; µ = 0.629 mm-1 (MoKα, λ = 1.5418 Å); F(000) = 316.0; T = 99.94(16) K; 2θmax =


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66.5 (98.8%)o; 3385 measured reflections; 2198 independent reflections (Rint = 0.0280); GOF on F2 = 1.003; R1 = 0.0398; wR2 = 0.0950 (all data); ∆e 0.14 and -0.23 e Å3.

Preparation of deprotonated salt FAS (or DPAS, 10 mg, 0.035 mol, 1 eq) was dissolved in 0.3 mL CD3OD, and the solution of 1.5 mg (KOH)/0.2 mL CD3OD (5eq) was added to complete the deprotonated process. The NMR was detected to analyze the structure change in basic condition, where the salt production after neutralization reaction from FAS or DPAS with KOH and were named as FAS-K or DPAS-K FAS-K: 1H NMR (400 MHz, CD3OD, ppm): 9.20 (s, 1H), 8.64 (d, 1H), 7.97 (d, 1H), 7.88 (d, 1H), 7.75-7.66 (m, 2H), 7.45-7.38 (m, 2H), 7.35-7.27 (m, 2H), 6.70 (d, J = 8.2 Hz, 1H), 6.516.45 (m, 1H). DPAS-K: 1H NMR (400 MHz, CD3OD, ppm): 9.07 (s, 1H), 7.62-7.58 (m, 2H), 7.45-7.27 (m, 9H), 7.03-6.96 (m, 1H), 6.62 (d, J = 7.9 Hz, 1H), 6.30-7.25 (m, 1H).

ASSOCIATED CONTENT

Supporting

Information. The experimental details, the UV-visible absorption and

photoluminescence spectra in different solvent (different kind, THF-water mixtures and pH buffers), the ESIPT forms and their values of K/E, the NMR spectra and their deprotonated


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form, and the stained fluorescent images with different irradiation time of FAS and DPAS also given in Supporting Information. The crystal (*.cif) files, check-files (checkcifs.PDF) and photostability videos for FAS and DPAS are uploaded as separated files. These material are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Prof. Z. Zhao (e-mail: [email protected]). Prof. B. Z. Tang (e-mail: [email protected]). Author Contributions †

Both authors contributed equally to this work.

ACKNOWLEDGEMENT This work was partially supported by National Science Foundation of China (51203091, 51273053), National Basic Research Program of China (973 Program, 2013CB834701), the Guangdong Natural Science Funds for Distinguished Young Scholar (2014A030306035), Guangdong Innovative Research Team Program of China (201101C0105067115), ITCCNERC14S01, China Postdoctoral Science Foundation Grant (2014M562213, 2015T80919), Science and Technology Plan of Shenzhen (JCYJ20140425170011516), Natural Science Fund of


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Guangdong Province (2014A030313659) and Fundamental Research Funds for the Central Universities (2015PT020, 2015ZY013).

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FIGURE CAPTION Scheme 1. Chemical structure of LD bioprobes: Nile red, BODIPY dyes, and TPE-AmAl (AIEgen). Scheme 2. The synthetic routes to FAS and DPAS. Figure 1. The ORTEP drawings of single crystal structures of (A) FAS (CCDC 1432238) and (B) DPAS (CCDC 1432239). The molecular packing patterns of FAS (C: side view, D: top view) and DPAS (E: side view) in the crystalline state. The fluorescence images of crystals (F) FAS and (G) DPAS. Figure 2. The fluorescence spectra of (A) FAS and (B) DPAS in THF-water mixtures with different water fractions (fw) ; Plots of I/I0 versus water fractions of (C) FAS and (D) DPAS, where I0 is the PL intensity in pure THF solution (Inset: fluorescence images at fw = 0 % and 90 %). Figure 3. The pH plots of I/I0 versus pH value for (A) FAS and (B) DPAS, where I0 is the PL intensity in pH = 7.0 (Inset: the PL spectra in different buffers (where fw = 99.5 %). The reversible switching in acidic condition (turn-off) and basic one (turn-on) of (C) FAS and (D) DPAS, where I0 is the emission intensity in pH = 7.0. Figure 4. Images of A549 cells incubated with FAS, DPAS or BODIPY (FAS and DPAS: 7.5 µM; BODIPY: 50 ng/mL) for 15 mins (Bright-field images: A, F and K; Fluorescence images: B, C, G, H, L and M; Merged images: D, I and N) and the enlarged regions (E, J and O). BODIPY is excited at 460-490 nm, FAS at 400-440 nm, and DPAS at 330-385 nm. The overlap


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ratios: 97.70% for BODIPY and FAS, 98.53% for BODIPY and DPAS, and 98.84% for FAS and DPAS. All the images share the same scale bar of 30 µm (except E,J and O). Figure 5. Bright-field (A-D, I-L) and fluorescent (E-H, M-P) images of HeLa cells stained with FAS (A-H) and DPAS (I-P) at different concentrations for 30 min after incubation in the presence of 50 µM oleic acid for 6h. Probes concentration: (A, E, I, M) 2.5 µM; (B, F, J, N) 5.0 µM; (C, G, K, O) 7.5 µM; (D, H, L, P) 10.0 µM. All the images share the same scale bar of 30 µm. Figure 6. Bright-field (A-C, G-I) and fluorescence (D-F, J-L) images of HeLa cells stained with FAS (A-F) and DPAS (G-L) at 7.5 µM for different time after incubation in the presence of 50µM oleic acid for 6h. Stained time: (A, D, G, J) 15 min; (B, E, H, K) 30 min; (C, F, I, L) 60 min. All the images share the same scale bar of 30 µm. Figure 7. (A) Cytotoxicity of FAS and DPAS on HeLa cells determined by MTT assay. (B) The signal loss of fluorescence intensity of FAS, DPAS and BODIPY under different light irradiation time.


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Table of Contents Graphic and Synopsis


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Specific Fluorescence Probes for Lipid Droplets Based on Simple

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