Zincke's Salt-Substituted Tetraphenylethylenes for Fluorometric Turn

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Zincke’s Salt-Substituted Tetraphenylethylenes for Fluorometric Turn-On Detection of Glutathione and Fluorescence Imaging of Cancer Cells Chi Zhan,†,‡ Guanxin Zhang,*,† and Deqing Zhang*,†,‡ †

CAS Key Laboratory of Organic Solids, CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: In this paper, we report Zincke’s saltsubstituted tetraphenylethylenes 1a and 1b with Cl− and PF6− as counteranions, respectively. The crystal structure of 1b was determined. Both 1a and 1b are almost nonemissive even in the aggregated states. This is attributed to the photoinduced electron transfer from 2,2-bis(4-methoxyphenyl)-1-phenylvinyl-phenyl unit to 1-(2,4-dinitrophenyl) pyridinium unit within 1a and 1b. The results demonstrate that the emissions of 1a and 1b in aqueous solution can be switched on upon either reaction with GSH or light irradiation. On the basis of the reaction between 1a and GSH, 1a can be utilized for the fluorescence turn-on detection of GSH selectively, and GSH with concentration as low as 36.9 nM can be detected. The transformation of 1b into 2 under light irradiation results in the fluorescence imaging of Hela and U2OS cells and phototoxicity toward Hela and U2OS cells after the protonation of pyridine unit in 2 because of the acidic environment of tumor cells. Aggregates of 1b can be up-taken by Hela and U2OS cells and fluorescence imaging has been successfully recorded with CLSM. Moreover, the protonated form of 2 can function as photosensitizer and 1b shows phototoxicity toward tumor cells such as Hela and U2OS cells. KEYWORDS: aggregation-induced emission, tetraphenylethylene, Zincke’s salt, glutathione, photodynamic therapy mechanisms are synergistically operated remain limited.25,26 In this paper, we report a Zincke’s salt-substituted tetraphenylethylene (compound 1a with chloride as the counteranion in Scheme 1), which is capable of selective and sensitive detecting glutathione (GSH) based on the synergistic operation of AIE and PET mechanisms. The results reveal that compound 1a is nonemissive (Φ < 0.01) even in the aggregated state because of intramolecular photoinduced electron transfer (PET) from TPE to N-(2,4-dinitrophenyl) pyridinium. However, the N(2,4-dinitrophenyl) group can be selectively removed after incubation with GSH under mild condition to inhibit the PET process, and simultaneously generate a hydrophobic compound 2 (Scheme 1) which afterward aggregates in aqueous solution, leading to remarkable fluorescence enhancement. In this way, a selective and sensitive detection of GSH can be established with 1a. It is well-acknowledged that glutathione (GSH) as well as cysteine (Cys) and homocysteine (Hcy) act as critical roles in

1. INTRODUCTION Fluorogens with aggregation-induced emission (AIE) characteristics have received increasing attentions in recent years.1−20 A number of AIE fluorogens have been reported. Among them, tetraphenylethylene (TPE) and its derivatives have been extensively investigated because TPE compounds can be easily synthesized and functionalized.1,3,4 TPE molecules with either charged or reactive moieties have been successfully utilized for constructing chemo-/biosensors.2−13The sensing mechanism is based on the manipulation of aggregation and deaggregation of AIE molecules, which is achieved with the specific chemical and enzyme-catalyzed reactions. Moreover, TPE molecules with targeting groups have been used for cell imaging, which possesses a low signal-to-noise ratio and can be performed without washing steps.14−20 It is known that conventional fluorescent sensors are designed based on photoinduced electron transfer (PET) and intramolecular charge transfer (ICT) mechanisms.21−24 The combination of AIE and PET mechanisms is expected to offer new design rationale for highly selective and sensitive sensors, which may have higher signal outputs and lower background signals compared to those based on AIE or PET mechanism, respectively. In fact, chemo-/biosensors in which AIE and PET © XXXX American Chemical Society

Special Issue: AIE Materials Received: September 23, 2017 Accepted: November 1, 2017

A

DOI: 10.1021/acsami.7b14446 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Chemical Structures of Compounds 1a and 1b, and Possible Rationales for GSH Detecting As Well As PhotoTriggered Cancer Cell Imaging and Photodynamic Therapy

with dichloromethane. The organic phase was washed with brine and dried over anhydrous Na2SO4. After removal of solvents, the residue was purified by silica chromatography with CH2Cl2/CH3OH (v/v, 10/ 1). 1b (0.22 g, 0.31 mmol) was obtained as a dark red solid in 93.3% yield. 1H NMR (400 MHz, CD2Cl2): δ 9.15 (s, 1H), 8.74 (d, J = 8.4 Hz, 1H), 8.62 (d, J = 8.0 Hz, 2H), 8.27 (d, J = 6.0 Hz, 2H), 8.05 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 7.17−6.95 (m, 9H), 6.69−6.65 (m, 4H), 3.71 (s, 3H), 3.73 (s, 3H). 13 C NMR (100 MHz, CD2Cl2): δ 159.38, 159.11, 158.88, 151.57, 149.77, 144.37, 143.60, 143.56, 143.20, 138.44, 137.68, 135.69, 135.62, 133.34, 132.84, 132.71, 131.74, 131.54, 130.67, 129.95, 128.20, 128.10, 126.85, 124.37, 122.69, 113.56, 113.18, 55.29, 55.24. HRMS: m/z calcd for C39H30N3O6 [M-PF6]+: 636.2129; found: 636.2128. Anal. Calcd for C39H30ClN3O6·0.5H2O: C, 59.25; H, 3.95; N, 5.31. Found: C, 59.08; H, 4.26; N, 5.65. 2.2. Detection of GSH in PBS Buffer and in Human Serum. Stock solutions (40 mM) of Hcy, Cys, GSH, L-serine (L-Ser), Lglycine (L-Gly), L-histidine (L-His), Na2S2O4, Na2S2O3, Na2SO3, Na2S, and NaClO were prepared in distilled water. The stock solution of 1a (2 mM) was prepared in DMSO. The solutions for sensing studies were prepared by diluting the respective stock solutions with PBS buffer (10 mM, pH 7.4), and each solution contains 0.5% DMSO (v/v). The serum sample was deproteinized for the detection of GSH with the following procedure: 1500 μL of serum was added into ultrafiltration tube (15 mL, Millipore MWCO 3000), and the sample was centrifuged at 7,000 g for 20 min at 4 °C. After centrifugation, the filtrate was collected and it was further diluted with PBS buffer (10 mM, pH 7.4) to form 10% (v/v) serum solution. Then, 1a (10 μM in PBS buffer) was added into the 10% serum solution, and the mixture solution was incubated for 20 min at 37 °C before recording the fluorescence spectrum. Alternatively, different concentrations of GSH (10, 13, and 18 μM) were added into the serum solutions, and the respective fluorescence spectra were measured after incubation with 1a in the same manner. 2.3. Cell Imaging. Cells (∼10000 mL−1) were seeded in 35 mm glass-bottomed dishes and incubated overnight for adhesion. The old medium was discarded carefully, and the cells were treated with 1 mL of newly prepared culture medium solution of 1b (20 μM). After incubation for 30 min, the cells were washed for three times with culture medium. Fluorescence imaging experiments were performed on a FV 1000-IX81 CLSM (Olympus, Japan). The objective used for imaging was a UPLSAPO 100× oil-immersion objective (Olympus). Image processing and analysis was performed on Olympus software (FV10-ASW).

maintaining biological redox homeostasis, which is crucial for a plenty of physiological and pathological processes.27−29 Sudden or abnormal changes of the GSH level is suggested to associating with cancers, Alzheimer’s disease, leucocyte loss, liver damage, as well as HIV infection.30−32 It is thus essential to monitor the concentration of GSH, which can be the signal of diseases, and help to diagnose and treat ailments. In fact, a number of molecular probes for GSH have been reported.33−43 Furthermore, the analogue of 1a (1b in Scheme 1) with PF6− as the counteranion can be also prepared via anion exchange. In particular, the aggregates of 1b can be selectively up-taken by cancer cells and the N-(2,4-dinitrophenyl) group in 1b can be also removed under light irradiation to generate 2 to achieve the fluorescence imaging of cancer cells. Additionally, the results manifest that 2 can be protonated under acidic condition and functionalized as photosensitizer under visible-light irradiation. Accordingly, the light irradiation of 1b triggers the formation of 2, which can be utilized in fluorescence imaging of tumor cells and also displays phototoxicity to tumor cells such as Hela simultaneously.

2. EXPERIMENTAL SECTION 2.1. Synthesis of 1a and 1b. Synthesis of 1a. A mixture of 2,4dinitrochlorobenzene (0.61 g, 3.0 mmol) and compound 2 (0.47 g, 1.0 mmol) in 50 mL of ethanol was refluxed at 95 °C for 12 h. After evaporation of solvent, the residue was subjected to chromatography column with CH2Cl2/CH3OH (v/v, 7/1). 1a was obtained as dark red solid (356 mg, 0.53 mmol) in 53% yield. 1H NMR (300 MHz, CD3OD): δ 9.27 (d, J = 2.4 Hz, 1H), 9.12 (d, J = 7.2 Hz, 2H), 8.93− 8.89 (m, 1H), 8.60 (d, J = 7.2 Hz, 2H), 8.28 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 7.19−6.93 (m, 9H), 6.73−6.66 (m, 4H), 3.73 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 158.75, 158.55, 156.91, 149.86, 149.50, 146.11, 143.72, 143.66, 142.6, 139.00, 137.95, 135.63, 135.61, 132.69, 132.57, 131.34, 130.68, 130.62, 128.73, 128.61, 127.15, 123.91, 121.91, 113.97, 113.72, 55.49. HRMS: m/z calcd for C39H30N3O6+ [M-Cl]+ 636.2129, found: 636.2127. Anal. Calcd for C39H30ClN3O6·0.5CH2Cl2: C, 66.39; H, 4.37; N, 5.88. Found: C, 65.90; H, 4.75; N 5.95. Synthesis of 1b. Silver hexafluorophosphate (0.15 g, 0.39 mmol) in 0.5 mL of deionized water was added to a solution of 1a (0.2 g, 0.3 mmol) in 5 mL of methanol, and the mixture was stirred for 4 h at room temperature. The solution was poured into water and extracted B

DOI: 10.1021/acsami.7b14446 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 2. Synthetic Approach for 1a and 1b

2.4. Detection of 1O2 with ABDA. The generation of 1O2 was detected chemically according to literature using 9,10anthracenediylbi(methylene) dimalonic acid (ABDA) as the indicator for the formation of 1O2.44,45 ABDA was bleached by 1O2 to its corresponding endoperoxide. The reaction was monitored spectrophotometrically by recording the decrease of absorbance at 378 nm for ABDA. 2.5. Photocytotoxicity Assays. The photocytotoxicity of 1b to cancer cell lines was determined by enhanced cell counting kit-8 (CCK-8).46 First, cells were seeded into 96-well plates with cell density of 5000−6000 each well, and incubated overnight. The cells were then cultivated with fresh culture medium containing different concentrations of 1b (ranged from 5 to 35 μM). After incubation in the dark for further 1.5 h, cells stained with 1b were irradiated by a LED light source (450 nm, 13 mW/cm2) for 15 min. After exposure to light irradiations, the cells were put back into incubator. After incubation for 48 h, 1 μL of CCK-8 solution was added to each well, and the cells were incubated for another 1 h. Finally, the absorption of each well of the plate was measured with the microplate reader at 450 nm. The cell viability was calculated with the following equation: cell viability = A/ A0100%, where A was the absorption value of the experiment group and A0 was that of cells cultured in high-glucose DMEM medium without 1b treatment. The cytotoxicity assays were repeated for three independent experiments. The control cells were treated similarly except for light irradiation.

Figure 1. Absorption spectra of 1a (10 μM, black line) and 1b (10 μM, red line) in PBS buffer (10 mM, pH 7.4) .

photoinduced electron-transfer (PET) reaction from 2,2-bis(4methoxyphenyl)-1-phenylvinyl-phenyl unit to 1-(2,4-dinitrophenyl) pyridinium unit within 1a and 1b. The oxidation and reduction potentials of compounds 4 and 5 (see Scheme S1) as the reference electron donor and acceptor units of 1a and 1b were estimated with the respective cyclic voltammograms (see Figure S3). Accordingly, the ΔG value for PET reaction from 2,2-bis(4-methoxyphenyl)-1-phenylvinyl-phenyl unit to 1-(2,4dinitrophenyl) pyridinium unit within 1a and 1b was estimated to be −1.86 eV (see the Supporting Information). Accordingly, the PET reaction is expected to occur and the emission will be quenched for 1a and 1b. 3.2. Selective and Sensitive Fluorescence Detection of GSH. While Zincke’s salts were usually utilized to prepare Nsubstituted pyridinium salts after reactions with the respective amines, they could also be transformed into neutral pyridine derivatives via a single-electron transfer (SET) process upon reaction with aminothiols.48 It is expected that this reaction of Zincke’s salts can provide a new platform for designing fluorescent sensors for GSH with 1a or 1b according to the following fact that GSH, Cys and Hcy are all bioactive reductant and GSH shows the strongest reduction ability among them.49 Figure 2a shows the fluorescent spectra of 1a after incubation with different amounts of GSH in PBS buffer (10 mM, pH 7.4) at 37 °C for 20 min. Before incubating with GSH, the ensemble was almost nonemissive. However, the emission around 502 nm emerged after incubation with GSH. Moreover, the fluorescence intensity at 502 nm increased by enhancing the concentration of GSH. For instance, the fluorescence intensity at 502 nm increased by more than 600 times when the concentration of GSH reached 80 μM in the ensemble. In fact, such fluorescence enhancement can be distinguished with naked-eye as shown in the inset of Figure 2a, where photos of solutions of 1a before and after incubation with GSH under UV light illumination are displayed. Figure 2b shows the variation of the fluorescence intensity at 502 nm after incubation with

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization. As shown in Scheme 2, the synthesis of compound 1a started from compound 2, which was synthesized by following the reported procedures.47 The reaction of 2 with 2,4-dinitrochlorobenzene in ethanol under refluxing for 12 h yielded 1a as dark red powder in 53% yield. The analogues of 1a with other counteranions were accessible by anion-exchange reactions. For instance, compound 1b was obtained after reaction of 1a with silver hexafluorophosphate. The crystal structure of 1b was determined (see the Supporting Information). Figure S1a shows the structures of the cation and anion of 1b. The TPE framework within the cation is twisting, and the pyridinium forms a dihedral angle of 72.07° with the connected benzene ring. Short interatomic contacts (2.49−2.66 Å) such as F···H are observed within the crystal structure of 1b (see Figure S1b). Figure 1 shows the absorption spectra of 1a and 1b in PBS buffer solutions. Broad absorptions around 439 and 443 nm were observed for 1a and 1b, respectively. On the basis of our previous studies on pyridinium substituted TPE,47 these absorptions around 439 and 443 nm for 1a and 1b can be attributed to the intramolecular charge transfer between TPE unit and Zincke’s salt. Both 1a (20 μM) and 1b (20 μM) were found to form aggregates with sizes of ca. 55.1 and 93.7 nm in aqueous solutions based on the DLS data shown in Figure S2a. It is noted that both 1a and 1b were almost nonemissive in aqueous solutions and solid states. This is attributed to the C

DOI: 10.1021/acsami.7b14446 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. (a) GSH concentration-dependent fluorescence spectra of compound 1a (10 μM) in PBS buffer (10 mM, pH 7.4) at 37 °C after 20 min incubation, λex = 379 nm; the inset shows photographs of PBS solution of compound 1a under UV light (365 nm) illumination (A) before and (B) after incubation with GSH (60 μM). (b) Plot of fluorescence intensity at 502 nm vs the concentration of GSH; the inset illustrates the linear relation between the fluorescence intensity and the concentration of GSH in the range of 0−26 μM.

Figure 3. Fluorescence microscope images of 1a (10.0 μM) (a) before and (b) after the reaction with GSH (40 μM); the scale bar was 10 μm; the reaction was carried out under 37 °C in PBS buffer (10 mM, pH 7.4) for 20 min before recording the images; λex = 379 nm.

after incubation with GSH (40 μM) based on the fluorescence microscopic images (see Figure 3b). On the basis of DLS data (see Figure S2b), aggregates of ca. 262 nm were formed for 1a (10.0 μM) in PBS buffer (10 mM, pH 7.4) before incubation with GSH, but larger aggregates of ca. 1000 nm were generated for the solution of 1a (10.0 μM) after incubation with GSH (40.0 μM). These DLS data also support the transformation of 1a into 2 after reaction with GSH. It is noted that large aggregates of ca. 93.7 nm were formed for 1b (20 μM) in aqueous solution on the basis of the DLS data (see Figure S2a). Moreover, fluorescent aggregates were not observed for 1b after incubation with GSH under the same condition. In fact, the ensemble of 1b and GSH kept weakly emissive after incubation at 37 °C for 60 min as shown in Figure S9. The more hydrophobic nature of PF6− aids the formation of larger aggregates of 1b in aqueous solution. For example, as shown in Figure S2c, aggregates about 80 nm were observed for 1a in PBS buffer at 37 °C, while aggregates with sizes around 400 nm were found for 1b in PBS buffer at 37 °C. Such larger aggregates of 1b may retard the reaction between the Zincke’s salt-substituted TPE and GSH. Next, we investigated the variation of the fluorescence spectra of 1a after incubation with other biological relevant analytes such as Cys, Hcy and other amino acids under the same condition as for GSH. Figure 4 shows the variation of fluorescence intensity at 502 nm for 1a after incubation with each possible interference species with a concentration of 400 μM and those after incubation with GSH (40 μM) together

different concentrations of GSH. Interestingly, the fluorescence intensity at 502 nm increases almost linearly with the concentration of GSH in the range of 0−26 μM. Therefore, the detection limit of GSH with 1a was estimated to be 36.9 nM (n = 11 and S/N = 3). Hence, this fluorometric assay with 1a is proved effective for the detection of GSH with good sensitivity. The fluorescence enhancement observed for the ensemble of 1a after incubation with GSH is attributed to the transformation of 1a into 2 as shown in Scheme 1. In comparison with 1a, compound 2 is more hydrophobic and thus aggregation occurs easily in aqueous solution, leading to fluorescence enhancement. This assumption is supported by the following results: (i) the fluorescence spectrum of the ensemble of 1a after incubation with GSH overlaps well with that of the authentic sample of 2 in the same PBS buffer solution; (ii) the mass signal at m/z = 470.2115, corresponding to the molecular weight of [2+H]+, was found after the reaction solution of 1a and GSH (Figure S7). According to Scheme 1, the L-γ-glutamyl-S-(2,4-dinitrophenyl)-L-cysteinylglycine (compound 3) was formed simultaneously. In fact, a signal at m/z = 472.0781, corresponding to the molecular weight of [3H]−, was observed (Figure S8). The formation of fluorescent aggregates was confirmed by both fluorescence microscopic and dynamic light scattering (DLS) studies. As displayed in Figure 3a, there were no fluorescent aggregates for the solution of 1a (10.0 μM) before incubation with GSH; however, fluorescent aggregates emerged D

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less than 6%, indicating that 1a can quantitatively detect GSH in practical biological samples with good performance. 3.3. Fluorescence Imaging of Cancer Cells and Phototoxicity. Interactions of 1a and 1b with tumor and normal cells were investigated with confocal laser scanning microscope (CLSM). Hela (a kind of adenocarcinoma cell line)/U2OS (osteosarcoma cell line) and HEK 293T cells were selected as the cancer and normal cells, respectively. Hela and HEK 293T cells were separately incubated with aggregates of 1a (20 μM) and 1b (20 μM) separately in high-glucose DMEM cell culture medium at 37 °C for 30 min. The samples were carefully washed and investigated with CLSM. Fluorescence images were detected for Hela cells after incubation with 1a (see Figure S10) and 1b (see Figure 5a, b). But, no fluorescence was observed for HEK 293T cells under the same conditions (see Figures 5d, e), even after incubation for 6 h (see Figure S11). As discussed above, 1a can be transformed into 2, which aggregates in aqueous solution and becomes emissive, after reaction with intracellular GSH. Thus, the fluorescence imaging of Hela cells may be owing to the reaction of 1a with GSH. However, the aggregates of 1b keep nonemissive after incubation with GSH. Further studies reveal that the 2,4dinitrophenyl group in the cations of 1a and 1b can be removed to generate 2 upon exposure to light (405−450 nm) irradiation for less than 15 min as illustrated in Scheme 1. The formation of 2 after exposure of the aqueous solution of 1b to light irradiation was confirmed by the following results: (i) HPLC analysis showed the presence of a new compound in the reaction mixture with the same retention time as for compound 2 under the same condition (see Figure S13); (ii) mass spectral signal at m/z = 470.36 was detected for the reaction mixture (see Figure S14), corresponding well to m/z of [2+H]+; (iii) the fluorescence spectrum of the aqueous solution of 1b after light irradiation overlapped well with that of 2 in aqueous solution (see Figure S15). As discussed above, fluorescence images were observed for Hela cells after incubation with 1b and further light irradiation, but no fluorescence images were detected for HEK 293T cells under the same conditions (see Figure 5). In order to understand this observation, the aqueous solution of 1b was subjected to DLS and zeta potential measurements. On the basis of DLS data (see Figure S2a), molecules of 1b were aggregated to form particles. Zeta potential of particles of 1b was measured to be 55.08 mV. According to previous studies,51 particles with positive zeta potentials are easily up-taken by tumor cells such as Hela cells. This is indeed consistent with previous report that aggregates of pyridinium-substituted TPE salts with certain counteranions can selectively stain tumor cells.51 Interestingly, both green-fluorescence images from the channel of 450−550 nm and red-fluorescence image from the channel of 570−670 nm were recorded with CLSM for Hela and U2OS cells after incubation with 1b as shown in Figure 5 and Figure S12. Compound 2 showed emission around 502 nm in aqueous solution. Thus, the green-fluorescence image can be attributed the emission from 2 which was generated from 1b under light irradiation. Considering the fact that the intracellular environment of tumor cells such as Hela is acidic, the red-fluorescence image is probably owing to the emission from the protonated form of 2 (see Scheme 1). This assumption is supported by the fact that the emission of 2 becomes broad ranging from 470 to 720 nm, covering both green and red

Figure 4. Selectivity profiles of 1a (10 μM) in the presence of various species in PBS (10 mM, pH 7.4): fluorescence intensity at 502 nm (λex = 379 nm) in the presence of competing species [10 equiv of Cys, Hcy, L-Ser, L-Gly, L-His, Na2S2O4, Na2S2O3, Na2SO3, Na2S, and NaClO (black bars) and those after further addition of 40 μM GSH (red bars)]. The concentration of each competing species was 400 μM.

with each of the species. The results manifest that only minor fluorescence enhancement was observed after 1a (10 μM) incubated with Cys (400 μM) and no obvious fluorescence increment was detected for 1a after incubation with the following species: Hcy, L-Ser, L-Gly, L-His, Na2S, Na2S2O3, Na 2 SO 3 , H 2 O 2 , Na 2 S 2 O 4 , and NaClO. The negligible interferences from Cys and Hcy can be ascribed to the fact that GSH shows higher reduction ability than Cys and Hcy.49 Further incubation of the ensemble of 1a with each of the species and GSH led to remarkable fluorescence enhancement as depicted in Figure 4. These competition experiments indicate that the fluorescence detection of GSH with 1a can be carried out without noticeable interferences from these species, and thus 1a shows good selectivity toward GSH. By considering the good selectivity and sensitivity of 1a toward GSH, we employed 1a for selective and sensitive detection of GSH in human serum. The detection of GSH was performed with 10% deproteinated human serum which was prepared by diluting serum with PBS buffer solution (10 mM, pH 7.4). When the diluted serum solution was incubated with 1a (10 μM) in PBS buffer (10 mM, pH 7.4) at 37 °C for 20 min, the emission around 502 nm was detected. On the basis of the linear relation between the fluorescence intensity at 502 nm vs GSH (see the inset of Figure 2b), the concentration of GSH in 10% the diluted human serum sample was determined to be 1.301 ± 0.033 μM. Hence, the GSH concentration in the human serum was about 13.01 μM, which agrees well with the fact that the concentration of GSH in human serum is in the range of 5−50 μM.50 Moreover, different amounts of GSH were spiked into the diluted serum, and the recovery was determined with probe 1a. As listed in Table 1, the recoveries of GSH ranged from 101.64 to 104.72% with a relative error of Table 1. Determination of GSH in 10% Deproteinized Human Serum Samples with 1a sample 10% serum 10% serum + GSH 10% serum + GSH 10% serum + GSH

added GSH (μM)

found (μM)

recovery (n = 3 RSD (n = 3, , %) %)

0 10

1.30 11.49

101.64

2.57 5.29

13

14.98

104.72

1.31

18

19.67

101.90

1.49

E

DOI: 10.1021/acsami.7b14446 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 5. CLSM images of (a−c) Hela cells and (d−f) HEK 293T after incubation with 1b (20 μM) at 37 °C for 30 min: (a, d) images from channel of 450−550 nm; (b, e) images from channel of 570−670 nm; (c) merged image of a, b, and the bright-field image; (d) merged image of d, e, and the bright-field image. Scale bar: 20 μm.

(50 μM) in the presence of 2 (20 μM) in D2O (pH 5) after light (λ = 450 nm) irradiation for different times. The absorptions in the range of 320−410 nm of ABDA gradually decreased by prolonging the irradiation times in the presence of 2 in D2O (pH 5). Figure S17b clearly shows the decrease of the absorption at 378 nm after light irradiation, but the absorption intensity of ABDA remains almost unaltered in the absence of 2 under the same condition. Such absorption spectral changes indicate that 2 in the protonated form is able to function as photosensitizer to generate singlet oxygen. Therefore, 1b is potentially useful for photodynamic dynamic therapy (PDT) as it can be transformed into 2 under light irradiation. To demonstrate the PDT potency of 1b toward tumor cells, we exposed Hela and U2OS cells after incubation with 1b to light irradiation (450 nm, 13 mW/cm2) for 15 min. The cell viabilities were then monitored via enhanced CCK-8. For comparison, the cells, which were treated with 1b without exposure to light irradiation, and those exposed to light irradiation without incubation with 1b, were also monitored in the same way. HEK 293T cells were equally treated and monitored. As depicted in Figure 7, HEK 293T cells remained viable after incubation with 1b and further light irradiation. This clearly testified that HEK 293T cannot be photoabated with 1b. In fact, this agrees with the observation that aggregates of 1b cannot be up-taken by HEK 293T as discussed above; however, viabilities decreased to 31% and 42% for Hela and U2OS cells, respectively, after incubation with 1b and further exposure to light irradiation. Such phototoxicity is induced by the transformation of 1b into 2, which leads to the generation of 1O2 in the acidic environment under light irradiation as illustrated in Scheme 1. In short, 1b can be selectively up-taken by tumor cells such as Hela and U2OS cells, and further light irradiation results in the transformation 1b into 2 and its protonated form, achieving both fluorescence imaging and

emission regions by lowering the pH of the solution from 7.4 to 4 as shown in Figure 6. Simultaneously, the absorption of 2 is

Figure 6. Absorption (dashed line) and emission (solid line) spectra of 2 (30 μM) in SDS (Sodium dodecyl sulfate, 6.8 mM) aqueous solutions with different pH values (red curve, pH 4.0; black curve, pH 7.4).

red-shifted by 40 nm and it is extended to the visible light region. The red-shifts of both absorption and fluorescence spectra are ascribed to the enhancement of intramolecular charge-transfer characters after the protonation of pyridine in 2.52 Also, 1b shows good photostability in living cells. For example, as shown in Figure S16, the fluorescence signal loss for the CLSM images was only 13.6% after 60 scans. Furthermore, the protonation of 2 can not only red-shift the absorption and emission spectra, but also enhance the photosensitization capability under visible light irradiation. Figure S17a shows the variation of absorption spectra of ABDA F

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treatment with GSH, cyclic voltammograms of 4 and 5, optimization of GSH detection condition, time-dependent absorption spectra of THF and CH3OH solution of 1a in the presence of GSH, mass spectrum of 1a after treated with GSH, fluorescence spectra of 1b after treatment with GSH for different time, photostability of 1b for fluorescence imaging of HeLa cells, the transformation of 1b into 2 after light irradiation, detection of 1 O2 with ABDA for 1b after light irradiation, and characterization of compounds 1a and 1b (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.Z.). *E-mail: [email protected] (G.Z.). Figure 7. Cell viability of HEK 293T cells, U2OS cells, and Hela cells after the following treatments: light irradiation only (black bars); incubation with 1b (20 μM) only (red bars); incubation with 1b (20 μM) and further light irradiation (blue bars).

ORCID

Deqing Zhang: 0000-0002-5709-6088 Notes

The authors declare no competing financial interest.

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phototoxicity for Hela cells and U2OS cells. Further investigations include the design and synthesis of new analogues of 1b with more conjugated structures to further red-shift the absorption and emission spectra. This will enable to achieve red-fluorescence imaging of tumor cells and PDT with visible light irradiation and even with two-photon excitation.

ACKNOWLEDGMENTS Financial supports from National Natural Science Foundation of China (21672220, 21422207), the Ministry of Science and Technology of China (2013CB733700, 2013CB834700), and Beijing Natural Science Foundation (2162048) are gratefully acknowledged.

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4. CONCLUSION Zincke’s salt-substituted tetraphenylethylenes 1a and 1b, with Cl− and PF6− as the respective counteranions, were synthesized and investigated. Both 1a and 1b are almost nonemissive even in the aggregated states owing to the photoinduced electron transfer from 2,2-bis(4-methoxyphenyl)-1-phenylvinyl-phenyl unit to 1-(2,4-dinitrophenyl) pyridinium unit within 1a and 1b. However, the emissions of 1a and 1b in aqueous solution can be switched on upon either reaction with GSH or light irradiation. Accordingly, a new selective fluorescence turn-on detection of GSH can be constructed with 1a, and the limit of detection (LOD) can reach 36.9 nM. In comparison, the incubation of the aqueous solution of 1b with GSH cannot turn on the emission. However, 1b can be transformed into 2 by removal of 2,4-dinitrophenyl group under light irradiation in aqueous solution, and the subsequent aggregation of 2 turns on the emission. Moreover, fluorescence imaging studies reveal that aggregates of 1b can be up-taken by tumor cells (Hela and U2OS cells). The protonation of pyridine unit in 2 due to the acidic environment of tumor cells red-shifts the emission, and as a result both green and red fluorescence images have been observed. Moreover, the protonated form of 2 can function as photosensitizer and 1b shows phototoxicity toward tumor cells such as Hela and U2OS cells as a consequence of sequential transformation of 1b in to 2 and its protonated form within tumor cells after exposure to light irradiation.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b14446. Materials and instruments, single-crystal structure and crystallographic data for 1b, DLS profiles of 1a and 1b in water/PBS buffer and those of 1a before and after G

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