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Mitochondria-targeted Ratiometric Fluorescent Probe Based on Diketopyrrolopyrrole for Detecting and Imaging of Endogenous Superoxide Anion in Vitro and in Vivo Jian Wang, Lingyan Liu, Weibo Xu, Zhicheng Yang, Yongchao Yan, Xiaoxu Xie, Yu Wang, Tao Yi, Chengyun Wang, and Jianli Hua Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00014 • Publication Date (Web): 02 Apr 2019 Downloaded from http://pubs.acs.org on April 2, 2019
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Analytical Chemistry
Mitochondria-targeted Ratiometric Fluorescent Probe Based on Diketopyrrolopyrrole for Detecting and Imaging of Endogenous Superoxide Anion in Vitro and in Vivo Jian Wang,a Lingyan Liu,b Weibo Xu,c,d Zhicheng Yang,a Yongchao Yan,a Xiaoxu Xie,a Yu Wang,c,d Tao Yi,*,b Chengyun Wang,a and Jianli Hua*,a aKey
Laboratory for Advanced Materials and School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China bDepartment of Chemistry, Fudan University, 2005 Songhu Road, Shanghai 200438, PR China cDepartment dDepartment
of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China. of Head and Neck Surgery, Fudan University Shanghai Cancer Center, Shanghai 200032, China.
ABSTRACT: Intracellular reactive oxygen species involved in a wide variety of physiological and pathological processes. In this work, we have developed a new mitochondria-targeting probe (DPP-S) for superoxide anion detection with ratiometric fluorescence response. DPP-S exhibited obvious colour change from violet to orange along with a distinct fluorescence change with maximum emission peak from 652 nm to 545 nm in response to superoxide anion. The limit of detection of DPP-S for superoxide anion was calculated to be 20.5 nM. Imaging studies taken in MCF-7 and RAW264.7 cells showed that DPP-S could be employed as a ratiometric fluorescent probe for endogenous superoxide anion detection and imaging in living cells with large emission shift. Furthermore, the co-localization study indicated that DPP-S can localize in mitochondria specifically. Finally, the fluorescent probe was successfully applied for superoxide anion imaging in mouse.
As a reactive oxygen species (ROS), superoxide anion was produced by single electron reduction of oxygen.1,2 Many proteins and enzymes are involved in the above reactions, including mitochondrial respiratory chain complexes I-IV,3,4 NADPH oxidase, 5 cyclooxygenase 6 and xanthine oxidase 7,8 etc. The produced superoxide anion will reside temporarily or transmit in the cells, then react with various intracellular reducing substances or biological macromolecules, regulating the related redox signals.9-11 For example, under the disproportionation of superoxide dismutase (SOD), superoxide anion was conversed to H2O2,12 regulating the activity of NOX proteins, thus regulating cell proliferation.13,14 When excessive superoxide anions were produced, the autophagy or apoptotic signaling pathway are activated, leading to cell death and eventually triggering multiple diseases.15-17 For example, superoxide anion can also react with NO to generate ONOO-, excessive ONOO- can activate caspase apoptotic proteins or cause lipid peroxidation, leading to apoptosis or necrosis of cells. 18,19 Therefore, developing sensitive and selective technology for superoxide anion detection, monitoring the concentration change of superoxide anion, will reveal the variation rule and the molecular mechanism of the related diseases, thus help to discover the therapy target of diseases. Fluorescence imaging, as a new method for molecular detection, has attracted widely attentions because of the advantages of its good sensitivity, high spatial and temporal resolution, easy operation, etc.20-23 Recently, plenty of researches have been carried out mainly utilizing the oxidability or nucleophilicity of superoxide anion as recognition mechanisms, providing turn on fluorescent probes for superoxide anion detection and imaging.24-27 Compared with
traditional turn-on and turn-off fluorescent probes, ratiometric fluorescent probes utilize the ratio of the intensity at two different emission wavelengths as detecting signal, thus can improve the sensitivity, selectivity and the range of dynamic response. 28-34 Therefore, designing and synthesizing of new ratiometric fluorescent probes with high sensitivity has become one of the research topics of great concern. Based on this, a few single-dependent ratiometric probes for superoxide anion detection were reported. For example, a probe based on 2phenylbenzothiazole derivative with different emission change was designed.35 However, the maximum emission wavelength of the probe before and after reaction with superoxide anion were no more than 480 nm. In 2016, Tang group reported a long wavelength ratiometric fluorescent probe for superoxide anion detection and imaging in HepG2 cells.36 Recently, two new naphthalene imide probes for superoxide anion detection with ratiometric fluorescence change in live cells, as well as in vivo was also developed.37,38 A pity is that the maximum emission wavelength change of these probes were all low than 100 nm, as well as poor limit of detection (LOD), thus limiting their applications to some extent (Table S1). Therefore, more efforts should be made in constructing more ratiometric methods for the detection and imaging of superoxide anion with large emission change. In this report, we present a novel fluorescent probe for superoxide anion detection based on diketopyrrolopyrrole derivates (DPP-S, Scheme 1). Construction of pyridinium structure to the skeleton of diketopyrrolopyrrole can further increase the maximum emission of the compounds, 39,40 and phosphinate at side chain serves as a reaction site with superoxide anion. 41-43 The reaction between DPP-S and
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Scheme 1. Chemical structure of DPP-S and proposed mechanism of DPP-S toward KO2.
superoxide anion induced deprotection of diphenyl phosphinated fluorophore resulting in self-cleavage afterwards to produced 3, thus the emission wavelength was shift from 652 nm to 545 nm. Finally, DPP-S was acted as a ratiometric fluorescent probe for the detection and imaging of superoxide anion in vitro and in vivo with obvious emission shift (>100 nm), showing advantages than reported single-dependent fluorescent probes.
EXPERIMENTAL SECTION Synthesis of target DPP-S. Synthesis of 1. 1 was synthesized according to reported literature.44 In a 100 mL two-necked round bottom flask, 4-hydroxybenzyl alcohol (248.28 mg, 2.0 mmol) and triethylamine (1 mL) were dissolved in 20 mL ultradry tetrahydrofuran (THF), then diphenylphosphinyl chloride (709.89 mg, 3.0 mmol, dissolved in 20 mL ultra-dry THF) was added dropwise under an argon atmosphere. The mixture was stirred for 8 h at room temperature. Afterwards, 10 mL water were added to quench the reaction, the organic layer was combined and the solvent was removed. The crude product was purified by silica gel column with PE/EA (2/1, v/v) as the eluent to provide a white solid. Then the afforded solid (324.32 mg, 1.0 mmol), carbon tetrabromide (331.63 mg, 1.0 mmol) and triphenylphosphine (262.29 mg, 1.0 mmol) were dissolved in 20 mL dichloromethane (DCM) and the solution was stirred at room temperature for 12 h. Then 5 mL saturated NaHCO3 solution was added to quench the reaction. The crude product was obtained after removed the solvent, further purified by silica gel column using PE/EA (1/1, v/v) as eluent to get a white solid 2 (201.32 mg, 51.99% yield). Synthesis of 3. 3 was synthesized according to our previous work. 45 Sodium hydride (96 mg, 4.0 mmol) was added to a suspension of 2 (289.29 mg, 1.0 mmol) in 20 mL N,NDimethylformamide (DMF), the mixture was stirred at 0 °C for 1 h under an argon atmosphere. Then 1-bromohexane (0.5 mL, 3.5 mmol) was added dropwise heated to 80 oC for another 8 h. After completion of the reaction, the mixture was extracted with ethyl acetate (3×20 mL). The organic layer was combined and the solvent was removed under vacuum. The crude product was purified by column chromatography on silica gel DCM/EA (20/1, v/v) to afford 3 (72.36 mg, 15.81% yield) as an orangered solid.
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Synthesis of probe DPP-S. 1 (77.44 mg, 0.2 mmol) and 3 (91.53 mg, 0.2 mmol) were mixed in a 50 mL two-necked round bottom flask with 20 mL acetonitrile, the mixture was heated to 80 oC and stirred for 12 h, then cooled to room temperature. After the solvent was removed, the crude product was purified by the column chromatographic method using DCM/EtOH (20/1, v/v) as eluent to afford the desired DPP-S (125.99 mg, 82.34% yield) as a dark red solid. 1H NMR (400 MHz, CDCl3), δ (ppm): δ 9.64 (d, J = 8.0 Hz, 2H), 8.34 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 4.0 Hz, 1H), 7.88 – 7.86 (m, 2H), 7.85 (d, J = 4.0 Hz, 1H), 7.75 (dd, J = 7.4, 4.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.58 – 7.54 (m, 2H), 7.51 – 7.45 (m, 7H), 7.22 (d, J = 8.0 Hz, 2H), 6.40 (s, 2H), 3.82 – 3.73 (m, 4H), 1.66 (s, 8H), 1.26 (s, 4H), 1.18 – 1.15 (m, 4H), 0.82 (t, J = 8.0 Hz, 6H). 13C NMR (100 MHz, CDCl3), δ (ppm): 162.11, 161.29, 155.23, 155.02, 151.94, 145.17, 142.25, 138.08, 132.81, 132.79, 132.70, 131.74, 131.64, 131.56, 131.17, 129.80, 129.49, 129.01, 128.96, 128.86, 128.72, 126.70, 125.64, 122.02, 121.97, 116.10, 109.94, 62.78, 42.55, 42.17, 31.18, 31.01, 29.84, 29.70, 28.89, 26.39, 26.18, 22.46, 22.35, 13.95, 13.90. HRMS (ESI, m/z): [M]+ calcd. for C48H51N3O4P+ 764.3617, found 764.3629. Materials and measurements. Phorbol-12-myristate-13acetate (PMA), lipopolysaccharide (LPS) and all other reactants and reagents were purchased from Sigma-Aldrich and used directly if there is no specific declaration. Sample of KO2, hydroxyl radical, and single oxygen were prepared according to the reported literature,36,44 PBS buffer (pH = 7.4) was prepared with ultrapure water. 1H NMR, 13C NMR and 31P NMR spectra were all obtained on a Bruker AM-400 MHz spectrometer. Flow cytometry was conducted by Cytomics FC 500 MPL. Absorption spectra and fluorescence spectra were recorded on a Varian Cary 500 UV-vis spectrophotometer and an F97pro fluorescence spectrophotometer, respectively. MTT assay. MCF-7 cells were plated in 96 well plate (1×104 per well), and hatched in DMEM culture medium (contain 10% Fetal bovine serum) for 24 hours. Then DPP-S was diluted with culture medium to provide different concentration solution (1.25 μM, 2.5 μM, 5.0 μM, 10.0 μM, 20.0 μM) and replacing previous medium. The MCF-7 cells were placed in dark environment for 24 hours. Subsequently, the methyl thiazolyl tetrazolium (MTT) assay was carried out to evaluate the cytotoxicity of DPP-S. The cells were then cultured with MTT (25μL, 5 mg/mL) for 3 hours. The absorbance at 470 nm were measured after the medium was replaced with DMSO (100 μL per well) with a microplate reader. The relative cell viability (%) was calculated according to the following formula: cell viability = ODsample/ODcontrol × 100%. Cell culture. The MCF-7 and RAW 264.7 macrophage cell lines were propagated in T-75 flasks, cultured at 37 °C under a humidified 5% CO2 atmosphere in DMEM medium, which were supplemented with 10% fetal bovine serum, 1% penicillin and 1% streptomycin. Cell imaging. MCF-7 and RAW 264.7 macrophage cells were cultured with a density of 3×104 cells per dish and incubated for 24 h. In the control group, the cells were incubated with 10 μM DPP-S solution (diluted with culture medium) for 30 min at 37 °C. Then the culture medium was removed and the cells were washed with PBS (1 mL) twice. In the experimental group, the MCF-7 and RAW 264.7 cells were treated with LPS (5 μg/mL) for 60 min, PMA (1 μg/mL) for 30 min, then
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Analytical Chemistry incubated with DPP-S (10 μM) for 30 min. The culture medium was removed and washed with PBS twice. LPS-induced inflammation in mice.46 The animal procedures were carried out in agreement with the guidelines issued by the Institutional Animal Care and Use Committee. Lipopolysaccharide (LPS, 2 mg/mL) was injected into the tibiotarsal joints of 6-8 week-old mice to induce acute inflammation. The tibiotarsal joints of two mice were divided into four groups. (I) LPS (50 µL, 2 mg/mL) was injected into the left tibiotarsal joints of mouse. (II) LPS (50 µL, 2 mg/mL) was injected into the right tibiotarsal joints of mouse. 5 hours later, same amount of saline and DPP-S (30 µL, 0.5 mM) was injected into region I and II, respectively. (III) the left tibiotarsal joints of mouse without any pretreated. (IV) LPS (50 µL, 2 mg/mL) was injected into the right tibiotarsal joints of mouse, after 5 hours, Tiron (10 mΜ, 50 µL) was injected into region IV. 1 hour later, both of two tibiotarsal joints were injected with same amount of DPP-S (30 μL, 0.5 mM). (V) the left tibiotarsal joints of mouse without any treated. (VI) saline (50 µL) was injected into the right tibiotarsal joints of mouse, 5 hours later, DPP-S (30 µL, 0.5 mM) was injected in to the same region. The photographs were acquired by a Bruker In Vivo Xtreme imaging system. Scheme 2. The synthetic routes of DPP-S
Figure 1. (A) UV-vis absorption spectra of DPP-S (10 μM) before and after addition of 100 μM KO2 in EtOH-PBS solution (EtOH : PBS = 3:7, v/v, pH = 7.4) at 37 oC for 3 min. (B) Fluorescence emission spectra of DPP-S (10 μM) with 0-100 μM KO2 at 37 oC for 3 min. (C) The emission ratio (I545/I652) of DPP-S (10 μM) upon addition of 0-100 μM KO2. (D) Linear relationship between the fluorescence ratio (I545/I652) changes and the determination of the limit of detection (LOD) of DPP-S. λex = 490 nm.
with diphenylphosphinyl chloride to produce 1 in the presence of carbon tetrabromide and triphenylphosphine. Subsequently, 3 was obtained from 2 by introduced alkyl chain in the existence of sodium hydride. The target probe DPP-S was prepared through nucleophilic attack of pyridine on 3 to the methylene of 4-(bromomethyl)phenyl diphenylphosphinate in acetonitrile. All new compounds were well characterized by 1H NMR, 13C NMR, and HRMS (ESI). Reagents and conditions: (i) diphenylphosphinyl chloride, Et3N, dry THF. (ii) CBr4, PPh3, DCM. (iii) sodium hydride (NaH), 0 oC, 1-bromohexane, DMF, 80 oC. (iV) MeCN, 80 oC.
RESULTS AND DISCUSSION Synthesis of target DPP-S. The synthetic approach of DPPS was shown in Scheme 2. 4-Hydroxybenzyl alcohol reacted
Optical properties of DPP-S. The UV-vis and photoluminescence response of DPP-S to KO2 were tested in EtOH-PBS solution (3:7, v/v, pH = 7.4). The fluorescence response of DPP-S toward 50 μM KO2 from 0 to 3 min was studied. As shown in Figure S1, the reaction could almost complete within 2.5 min. Then fluorescence response of DPPS reacted with different equivalent KO2 were conducted within 3 min. As depicted in Figure 1, DPP-S showed a maximum
Figure 2. (A) 1H NMR titration of DPP-S without and with 8.0 eq KO2 in MeOH-d4. HPLC analysis of (B) diphenylphosphinic acid, (C) the reaction mixture of DPP-S by KO2, (D) compound 3 and (E) DPP-S.
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absorption peak at about 520 nm with violet color observed by naked eye, accompanying with red fluorescence with maximum emission peak at 652 nm. After reacted with 0 to 10 equivalent KO2, the maximum absorption peak was blue-shifted to 500 nm. While the red fluorescence at 652 nm decreased gradually and a new emission peak at 545 nm increased, providing an obvious ratiometric signal of fluorescence intensity at 545 nm and 652 nm. These results suggested the cleavage of diphenyl phosphoryl of DPP-S in the presence of KO2 and further removing the pyridinium cation by self-cleavage. Thus the ICT process was broken. Moreover, the intensity ratio I545/I652 was increased by 80-fold after treated with 100 μM KO2, demonstrating that DPP-S was advantageous for the detection of KO2 based on ratiometric fluorescence change. Investigation of the detecting mechanism. To verify the proposed mechanism of the reaction between DPP-S and KO2, the 1H NMR analysis were conducted. As shown in Figure 2A when 8.0 equiv. KO2 (dissolved in DMSO-d6) were added, the proton Ha at 9.09 ppm (doublet) and Hb at 8.51 ppm (doublet) belong to the signal of pyridine were shifted to a higher field at about 7.83 and 7.73 ppm, respectively. Furthermore, the proton signal at 5.80 ppm (single) which assigned to Hc in methylene attached with pyridine was disappeared, attributing to the selfcleavage triggered by KO2. The 31P NMR and MS titrations were also conducted (Figure S2), the results showed that DPPS could reacted with KO2 to produced 3. Furthermore, high performance liquid chromatography (HPLC) were performed for intermediate 3, DPP-S, diphenylphosphinic acid as well as the mixture of DPP-S after reaction with KO2. As seen from D and E in Figure 2, the solution of 3 and DPP-S gave a single peak at 23.7 min and 6.3 min, respectively. For the solution of diphenylphosphinic acid, a clear single peak at 7.2 min can be observed (Figure 2B). After addition of KO2 into DPP-S solution and reacted for 3 min, a clearly single peak at 7.2 min and 23.7 min were emerged, matching well with the retention time of diphenylphosphinic acid and 3, respectively. Furthermore, the peak at 6.3 min of DPP-S was almost disappeared after reaction (Figure 2C). All these results further proved the proposed mechanism. Effect of pH. To prove the wide application of DPP-S as a ratiometric probe for detecting superoxide anion, the
Figure 3. (A) Fluorescence ratio (I545/I652) of DPP-S (10 μM) before (red circles) and after (blue squares) the addition of 80 μM KO2 for 3 min at different pH values. (B) Fluorescence ratio variation (I545/I652) of DPP-S (10 μM) before and after addition of 8.0 equiv. KO2 in the presence of 20 equiv. other additives in EtOH-PBS solution (EtOH : PBS = 3:7, v/v, pH = 7.4). λex = 490 nm.
Figure 4. Confocal fluorescence images of MCF-7 cells incubated with (A) 10 μM DPP-S (red channel) at 37 oC for 30 min; (B) 200 nM Mito Tracker Green FM (green channel) at 37 oC for 30 min; (C) 8 μg/mL Hoechst 33258 (blue channel) at 37 oC for 30 min; (D) Merged image of (A), (B), (C) and bright field; (E) Merged image of (A), (B) and (C); (F) Intensity profiles of ROI across MCF-7 cells, Red lines represent the intensity of DPP-S, green lines represent the intensity of Mito Tracker Green FM and blue lines represent the intensity of Hoechst 33258; Correlation plot of (G) Mito Tracker Green FM and DPP-S intensities, R = 0.93; (H) Hoechst 33258 and DPP-S intensities, R = 0.14; (I) Hoechst 33258 and Mito Tracker Green FM, R = 0.13. Red channel: 630-680 nm, Green channel: 500-540 nm. λex = 490 nm. Blue channel: 440-480 nm. λex = 350 nm. Bar = 7.5 μm.
fluorescence characteristics of DPP-S towards KO2 was studied in solutions with different pH buffers. As shown in Figure 3A, the probe performed obviously ratio (I545/I652) increased after reacted with KO2 in a wide range of pH from 3.8 to 9.3, indicating that DPP-S is suitable for biological applications. Selectivity. To investigate the specific reaction of the probe toward superoxide anion, we tested some biological species, including some ions (Na+, K+, Fe2+, SO32-), amino acids (cysteine, glutathione and homocysteine), ATP, ADP, and some oxidizing species (TBHP, OH., 1O2, H2O2, ClO-, ONOO-). As shown in Figure 3B, with the different interfering species added to the probe solutions, the fluorescence ratios (I545/I652) of DPPS were very low, indicating that DPP-S did not react with these species. While after add KO2, the ratios (I545/I652) were obviously increased, demonstrating that DPP-S can transformed to 3 after reaction with KO2. These results indicated that DPP-S could serve as a specifically fluorescent probe for the detection of superoxide anion, because the cleavage of the recognition moiety from DPP-S can be triggered selectively by superoxide anion. Subcellular localization study of DPP-S. Firstly, MTT assays were studied and revealed that the low toxicity of DPPS to cells after incubation for 24 h (Figure S3). To study if the DPP-S probe can load in mitochondria specifically, thus
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Analytical Chemistry
Figure 5. Confocal fluorescence images of MCF-7 cells with different treatments. (A-D) MCF-7 cells treated with DPP-S (10 μM) for 60 min at 37 °C; (E-H) MCF-7 cells pretreated with PMA (1 μg/mL) for 30 min, then incubated with probe (10 μM) for another 60 min; (I-L) MCF-7 cells pretreated with LPS (5 μg/mL) for 60 min, then incubated with probe (10 μM) for another 60 min; (M-P) MCF-7 cells pretreated with LPS (5 μg/mL) for 60 min, then treated with Tiron (300 μM) for 30 min, then incubated with probe (10 μM) for another 60 min. Columns from left to right: yellow channel, red channel, bright field and ratio image, respectively. Red channel: 630-680 nm, yellow channel: 520-560 nm. λex = 490 nm. Bar = 25 μm.
Figure 6. Confocal fluorescence images of RAW264.7 cells with different treatments. (A-D) RAW264.7 cells treated with DPP-S (10 μM) for 60 min at 37 °C; (E-H) RAW264.7 cells pretreated with PMA (1 μg/mL) for 30 min, then incubated with probe (10 μM) for another 60 min; (I-L) RAW264.7 cells pretreated with LPS (5 μg/mL) for 60 min, then incubated with probe (10 μM) for another 60 min; (M-P) RAW264.7 cells pretreated with LPS (5 μg/mL) for 60 min, then treated with Tiron (300 μM) for 30 min, then incubated with probe (10 μM) for another 60 min. Columns from left to right: yellow channel, red channel, bright field and ratio image, respectively. Red channel: 630-680 nm, yellow channel: 520-560 nm. λex = 490 nm. Bar = 25 μm.
imaging of superoxide anion in situ,47,48 Mito Tracker Green FM, a mitochondrial dye, was applied to study the colocalization ability of DPP-S (Figure 4). Furthermore, Hoechst 33258 was used to stain the nucleus of cells. MCF-7 cells were co-incubated with DPP-S (10 μM), Hoechst 33258 (8 μg/mL) and Mito Tracker Green FM (200 nM) at 37 oC for 30 min. As depicted in Figure 4, the red channel (630-680 nm) belongs to DPP-S, and the green channel (500-540 nm) and blue channel (440-460 nm) attributes to mitochondrial and nuclear imaging, respectively, which stained by Mito Tracker Green FM and Hoechst 33258. It was clear that the image of red channel matched well with green channel (Figure 4E), and the changes in the intensity profiles of linear regions of interest (ROIs) (DPP-S and Mito Tracker Green FM co-staining) were synchronous (Figure 4F), demonstrating that the fluorescence imaging of DPP-S matched well with the imaging of Mito Tracker Green FM. Furthermore, the co-localization was also quantified through Pearson's correlation factors (R). The correlation plot of Mito Tracker Green FM and DPP-S intensities (Figure 4G) displayed a high Pearson coefficient (0.93), while the Pearson coefficient of Hoechst 33258 and DPP-S, Hoechst 33258 and Mito Tracker Green FM was only 0.14 and 0.13, respectively (Figure 4H, 4I).
DPP-S was well located and suitable for the mitochondria imaging in living MCF-7 cells.
In order to further study the targeted ability of DPP-S. Lyso Tracker green DND-26 and ER-Tracker Blue-White DPX were also co-incubated with DPP-S in MCF-7 cells. As shown in Figure S4 and S5, the Pearson coefficient of Lyso Tracker green DND-26 and DPP-S, ER-Tracker Blue-White DPX and DPPS was 0.43 and 0.62, respectively. All the results revealing that
Imaging of intracellular endogenous superoxide anion. With the favorable fluorescence properties of DPP-S for superoxide anion, DPP-S was then utilized for the endogenous mitochondrial superoxide anion detection in MCF-7 cells. Endogenous superoxide anion may produce when stimulated by lipopolysaccharide (LPS) or phorbol-12-myristate-13-acetate (PMA). As shown in Figure 5, in the control group (Figure 5AD), MCF-7 cells were only incubated with 10 μM DPP-S in culture medium for 60 min without any other treated, very weak yellow fluorescence in the range of 520-560 nm could be detected (Figure S6), which may attribute to the superoxide anion produced in cells. While in the experimental group (Figure 5E-H), MCF-7 cells were pretreated with 1.0 μg/mL PMA for 30 min and then incubated with DPP-S (10 μM), it could see that fluorescence of the red channel disappeared, accompanying with obvious fluorescence in the yellow channel emerged. Furthermore, in the other experimental group, MCF7 cells were pretreated with LPS (5.0 μg/mL) for 60 min to study the inflammation enhanced superoxide anion level, and similar results were obtained, as the cells only exhibited yellow fluorescence instead of red. Meanwhile, Tiron, a superoxide scavenger was used to further study the imaging ability. As shown in Figure 5M-P, the yellow channel signal was obviously blocked and red fluorescence could be detected, indicating that DPP-S could imaging endogenous superoxide anion in MCF-7 cells. To further demonstrate the ability of DPP-S to detect
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Figure 7. Fluorescence images of a nude mouse model with acute inflammation. The tibiotarsal joints of mouse was injection of LPS (50 μL, 2 mg/mL) for 5 h, then DPP-S (30 μL, 0.5 mM) was inject into the right tibiotarsal joint (right ankle), and the left tibiotarsal joint (left ankle) was used as a control. Images were taken after incubation for 7, 12, 17, 22, 27, 32 min, respectively. Images were taken using an excitation laser of 490 nm and an emission filter of the (A-F) red channel and (G-L) green channel. Green channel: emission-band pass filter with centre wavelength at 560 nm; Red channel: emission-band pass filter with centre wavelength at 655 nm. Band width: ± 15 nm.
endogenous superoxide anion, the imaging of DPP-S in RAW264.7 macrophage cells were also conducted. As shown in Figure 6, when the cells without any stimulated or treated with LPS and Tiron, only red fluorescence could detect in the red channel, and strong yellow fluorescence was observed obviously while cells pretreated with LPS (5 μg/mL) or PMA (1 μg/mL). All these results demonstrated that DPP-S was suitable for endogenous superoxide anion imaging both in MCF-7 and RAW264.7 macrophage cells. Imaging endogenous superoxide anion in vivo. Then, the ability of DPP-S for endogenous superoxide anion imaging in living animals was tested. The superoxide anion was generated in vivo through lipopolysaccharide (LPS) induced acute inflammation in a nude mice model. A solution of LPS (50 μL, 2 mg/mL) was injected into the tibiotarsal joints of the mouse for 5 hours. Then DPP-S (30 μL, 0.5 mM) was injected into the same region of the ritht tibiotarsal joints, and the left tibiotarsal joints was used as a control with injection of 30 μL saline. The photographs were taken and collected after treated for 7, 12, 17, 22, 27, 32 min, respectively. As shown depicted in Figure 7AF (region II), the signal intensity of DPP-S injected ankle obtained in red channel decreased gradually with the time (7-32 min) went by, while the intensity of the fluorescence signal from the green channel increased gradually (Figure7G-L, region II). Meanwhile, the left ankle injection with saline showed almost no fluorescence signal both in red and green channel (Figure 7, region I), suggesting the reaction between DPP-S and the endogenous superoxide anion in the inflammated nude mouse. In control, the tibiotarsal joint injected with saline and DPP-S only exhibited fluorescence signal in red channel with ignorable change (Figure S7A-F, region VI), and no fluorescence could be detected both in green and red channel from the tibiotarsal joint without any treatment (Figure S7, region V). Tiron, a superoxide anion inhibiter, was used to study the detection and imaging ability of DPP-S. As displayed in Figure S8, the right tibiotarsal joints of the mouse
was pretreated with LPS (50 μL, 2 mg/mL) for 5 hours, then treated with Tiron (50 μL, 10 mM) for another 1 hour, then DPP-S was injected into the same region. With the time went by, fluorescence signal of the right ankle showed no obvious change in red channel (Figure S8A-E, region IV), while no signal could be detected from green channel (Figure S8F-J, region IV). Furthermore, the left ankle without any treated except injection of DPP-S (30 μL, 0.5 mM), only showed signal in the red channel as the time went by (Figure S8A-E, region III), accompanying with no fluorescence in green channel (Figure S8F-J, region III). All these results demonstrating that DPP-S is a desired probe for detection and imaging of endogenous superoxide anion in vivo.
CONCLUSIONS In summary, a new diketopyrrolopyrrole derivate (DPP-S) for superoxide anion detection was developed. DPP-S showed ratiometric fluorescent characteristics toward superoxide anion with large emission wavelength shifts over 100 nm. DPP-S displayed a red fluorescence emission at 652 nm, and shifted to 545 nm after reacted with superoxide anion. The use of DPP-S for the detection of superoxide anion with ratiometric fluorescent detection was thus carried out. The probe has high sensitivity for superoxide anion with low LOD up to 2.05×10-8 M. Due to the existence of pyridine cation unit, DPP-S could target mitochondria and was successfully applied for detecting and imaging endogenous superoxide anion generated in mitochondria in live cells like MCF-7 and Raw264.7 cells. Furthermore, DPP-S could trace in vivo stimulated superoxide anion in inflamed mouse. The strategy will be benefit to accomplish more ICT-based probes as reaction-activated biosensors.
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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Figures showing time-dependent fluorescent spectra of DPP-S, cytotoxicity of DPP-S, 31P NMR and MS titrations of DPP-S toward KO2, fluorescence images in vivo of mouse cells treated with DPP-S, and original spectral copy of target DPP-S.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected], (T. Yi) *E-mail:
[email protected], (J. L. Hua)
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21772040, 21421004, 21788102 and 21572062), the Fundamental Research Funds for the Central Universities (222201717003) and the Programme of Introducing Talents of Discipline to Universities (B16017). The authors thank Research Center of Analysis and Test of East China University of Science and Technology for the help on the characterization.
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