A fast-response and highly specific Si-Rhodamine probe for endogenous peroxynitrite detection in living cells

Junma Tang , Qiang Li , Zhiqian Guo * and Weihong Zhu
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Functional Materials Chemistry, and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China. E-mail: guozq@ecust.edu.cn

Received 6th July 2018 , Accepted 30th July 2018

First published on 30th July 2018


Peroxynitrite (ONOO) is involved in a variety of physiological and pathological processes. We designed and synthesized a fluorescent probe SiNH based on Si-rhodamine. The nanoprobe SiNH encapsulated within the amphiphilic copolymer exhibited fast response within 10 s, and it was highly specific for ONOO in aqueous solution. It is demonstrated that the nanoprobe SiNH is applicable for real-time tracing of endogenous ONOO in living cells.


Introduction

Peroxynitrite (ONOO) possesses strong oxidizability, and it is formed by the reaction between nitric oxide (NO) and superoxide (O2˙) under diffusion control without the need of enzymatic catalysis. Peroxynitrite has been recognized as a central biological pathogenic agent, and its endogenous abnormal concentration results in a variety of diseases such as cardiovascular, neurodegenerative, and inflammatory disorders.1,2 Real-time monitoring of ONOO distribution is conducive to explore its origin, activities, and biological effects.3 However, due to the extremely short lifetime (<1 s) of ONOO in a physiological aqueous environment, it is difficult to separate or measure these bioactive species by conventional biology tools. Therefore, developing efficient and reliable tools to detect or visualize ONOO in real-time in biological systems is fairly essential for deeper insights of its origins, activities, and biological functions.

To be an excellent probe for the detection of ONOO, three characteristics are necessary: (1) the ability to function in an aqueous medium as metabolic processes require aqueous system to proceed; (2) fast response with ONOO due to the short half-life of ONOO (about 1 s at pH 7.4);4 (3) highly specific differentiation of ONOO from other similar reactive oxygen species (ROS) such as hypochlorite (ClO) and hydrogen peroxide (H2O2).5–8 Owing to the unique advantages of simplicity, sensitivity, and real-time and nondestructive detection, fluorescent probes have attracted great attention for tracing these bioactive ROS species in biochemistry. In fact, some reported fluorescent probes for ONOO were designed by the strategies that involved direct oxidation of sulfur groups, boronates, active ketone, selenium, and tellurium by ONOO.9–12 These significant studies help us better understand the biological effects of ONOO in living systems. However, most of these reported probes are not suitable for detecting ONOO in real biological systems because of their low reaction rates or poor selectivities. In particular, most of them suffered from poor solubility in water. Thus, it is urgent to develop high-performance fluorescent probes for selective and real-time detection of ONOO under physiological conditions.

To attain these goals, a promising silicon-substituted xanthene platform was directly conjugated with methyl(4-hydroxyphenyl)amino for the probe SiNH (Scheme 1). The probe is designed based on the following aspects: an Si-rhodamine fluorophore featuring excellent photophysical properties and easily modifiable structure;13–16 methyl(4-hydroxyphenyl)amino, which can be specifically oxidized by ONOO and then hydrolysed into a quinone product, enabling ONOO to be rapidly and selectively detected.17–19 The probe SiNH is supposed to detect ONOO with fast response and high specificity through the photo-induced electron transfer (PET) mechanism.


image file: c8ob01598h-s1.tif
Scheme 1 The formation of micelle encapsulated by mPEG-DSPE and the design structure and proposed sensing mechanism of SiNH.

However, poor water solubility of the probe SiNH limits its ability to detect ONOO under aqueous conditions of metabolic processes. The supramolecular assembly approach provides an opportunity to overcome this limitation. Recently, amphiphilic copolymers have attracted great interest due to their significant advantage of improving biocompatibility, and they are capable of capturing hydrophobic fluorescent probes inside the interior of micelles to form water-soluble nanoparticles. In this regard, amphiphilic copolymer mPEG-DSPE (1,2-dimyristoyl-sn-glycero-3-phospho-ethanolamine N(methoxy(polyethyleneglycol)-2000)) is chosen for encapsulating SiNH into the hydrophobic interior of the micelles to form hydrophilic nanoparticles (Scheme 1).20 As shown in Scheme 1, the hydrophilic PEG moieties of mPEG-DSPE are distributed in the exterior of the nanoparticles to improve water solubility and biocompatibility. In this way, the probe can show fast response, ultra-sensitivity, and favorable cellular uptake, and it can trace endogenous ONOO in living cells.

Experimental section

Materials and instruments

All reagents and solvents were purchased from commercial sources. Solvents were dried according to standard procedures. All reaction mixtures were magnetically stirred and monitored by thin-layer chromatography (TLC). Flash chromatography (FC) was performed using silica gel 60 (200–300 mesh). Absorption spectra were measured on Agilent Technologies Carry 60 UV-Vis spectrometer. Fluorescence spectra were taken on an F97pro fluorescence spectrometer. 1H NMR and 13C NMR spectra were recorded on a Bruker AV-400 spectrometer. Mass spectra were measured on an HP 1100 LC-MS spectrometer. The following abbreviations were used to explain the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; and br = broad.

Preparation of the test solution

Peroxynitrite (ONOO) solution was synthesized according to literature reports.21,22 Briefly, a mixture of sodium nitrite (0.6 M) and hydrogen peroxide (0.7 M) was acidified with hydrochloric acid (0.6 M), and sodium hydroxide (1.5 M) was added to make the solution alkaline. The excess hydrogen peroxide was removed by passing the solution through a short column of manganese dioxide. The resulting solution was divided into small aliquots and stored at −80 °C. The aliquots were thawed immediately before use, and the concentration of peroxynitrite was determined by measuring the absorbance change at 302 nm in solution. The extinction coefficient of peroxynitrite solution in 0.1 M NaOH was 1670 M−1 cm−1 at 302 nm.

Deionized water and spectroscopic grade CH3CN were used for spectroscopic studies. DEA·NONOate was purchased and used without further purification as the NO donor. H2O2 solution was purchased from Sigma-Aldrich and added into the probe solution directly. The source of NaOCl was commercial bleach. The aqueous solutions of NaNO2 were freshly prepared and were used as nitrite (NO2) sources. Various analyte solutions (100 equiv., 5 × 10−4 M) were prepared by adding Cys, GSH, Glu, and Pro to the solution of nanoprobe (5 μM) in HEPES buffer (50 mM, pH = 7.4). The resulting solution was kept at room temperature for 30 min and then, the fluorescence spectra were recorded.

Preparation of micelles

mPEG-DSPE (5 mg) was rapidly poured into a vial containing 9 mL distilled-deionized water under vigorous sonication for 20 min. SiNH (1 mg, 0.002 mmol) was then rapidly poured into the mixture under continuous sonication for another 20 min. Then, the aqueous solution was filtered through a polyvinylidene fluoride (PVDF) syringe-driven filter. Free dyes were removed through dialysis, and the micelle stock solution was obtained. The initial micelle solution was then diluted with HEPES buffer for further studies.20

Synthesis

The synthetic route is shown in Scheme 2.19
image file: c8ob01598h-s2.tif
Scheme 2 The synthetic route of probe SiNH and the reaction product SiMH.

Preparation of SiX

The key intermediate SiX was synthesized according to the reported methods.23–27 M.p. 136.6–137.3 °C; 1H NMR (400 MHz, CDCl3): δ 8.35 (d, 2H, J = 8.98 Hz), 6.79 (d, 2H, J = 9.00 Hz), 6.74 (s, 2H), 3.43 (q, 8H, J = 7.08 Hz), 1.23 (t, 12H, J = 7.08 Hz), 0.45 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 184.4, 150.0, 140.3, 132.3, 129.1, 115.1, 113.6, 44.9, 13.6, −0.1; MS (ESI): calcd for {M + H}+, 381.2362, found, 381.2368.

Preparation of SiCl

SiX (0.381 g, 1 mmol) was dissolved in dry CH3CN (10 mL) in a dried flask and then, oxalyl chloride (1.2 mmol) was added dropwise into the SiX solution. The reaction mixture was stirred for about 2 h at room temperature. The crude product was recrystallized from CH3CN/ether to obtain the chloride species SiCl (0.152 g, yield 36.8%). M.p. 148.3–149.1 °C; 1H NMR (400 Hz, CDCl3) δ 8.49 (s, 2H), 7.36 (d, J = 9.6 Hz, 2H), 7.14 (d, J = 2.4 Hz, 2H), 3.46 (q, J = 7.2 Hz, 8H), 1.27 (t, J = 7.2 Hz, 12H), 0.52 (s, 6H); MS (ESI): calcd for {M + H}+, 399.2023, found, 399.2025.

Preparation of SiNH

4-Methylaminophenol sulfate (0.344 g, 1 mmol) was dissolved in CH3CN (10 mL); then, with the addition of Et3N (0.242 g, 2.4 mmol), the reaction mixture was stirred for 30 min. Then, SiCl (0.042 g, 0.11 mmol) was added into the reaction mixture with further agitation for 5 min. The solvents were removed under reduced pressure, and the residue was purified by flash chromatography (CH2Cl2/MeOH = 15/1) to afford the pure product SiNH (0.012 g, yield 29%). M.p. 168.2–169.1 °C; 1H NMR (400 Hz, CDCl3) δ 7.46 (d, J = 9.6 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.02 (d, J = 9.0 Hz, 2H), 6.70 (dd, J1 = 2.4 Hz, J2 = 9.6 Hz, 2H), 6.52 (d, J = 2.4 Hz, 2H), 3.45 (q, J = 7.2 Hz, 11H), 1.27(t, J = 7.2 Hz, 12H), 0.52 (s, 6H). 13C NMR (100 Hz, CDCl3): δ 173.3, 149.7, 140.4, 138.2, 126.7, 125.8, 122.7, 121.2, 115.2, 112.1, 69.0, 45.6, 44.7, 30.9, 29.6, 14.6; MS (ESI): calcd for {M + H}+, 486.2941, found, 486.2941.

Preparation of SiMH

In an argon-flushed flask fitted with a septum cap, SiCl (20 mg, 0.05 mmol) was dissolved in CH3CN (10 mL) and then, the solution was cooled at 0 °C with constant agitation. Then, methylamine solution (400 μL, 4.1 mmol, 40%) was added into the flask dropwise over 1 min with a syringe with stirring for another 20 min. After evaporating the solvent, the residue was purified by silica gel column chromatography (CH2Cl2[thin space (1/6-em)]:[thin space (1/6-em)]MeOH = 15[thin space (1/6-em)]:[thin space (1/6-em)]1) to give SiMH (5 mg, yield 23%). M.p. 145.2–146.1 °C; 1H NMR (400 Hz, CDCl3) δ 8.48 (d, J = 9.0 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 6.89 (dd, J1 = 2.4 Hz, J2 = 9.6 Hz, 2H), 6.75 (s, 1H), 6.70 (s, 1H), 3.65 (s, 3H), 3.52 (q, J = 7.2 Hz, 8H), 0.97 (t, J = 7.2 Hz, 12H), 0.52 (s, 6H); MS (ESI): calcd for {M + H}+, 394.2679, found, 394.2674.

Results and discussion

To verify the above-mentioned concerns, the photophysical properties of SiNH toward ONOO were investigated in different solvent systems. The mixed HEPES buffer solution (50 mM, pH = 7.4, containing 30% CH3CN) was chosen as the optimum solvent due to the best performance of SiNH toward ONOO. As shown in Fig. 1A, SiNH initially showed the main absorption peak at 500 nm, but it revealed extremely weak fluorescence (Φfl = 0.0008), owing to the effective PET process from the methyl(4-hydroxyphenyl) amino group with high electron density to the excited fluorophore. When treated with 10 equiv. of ONOO, the initial absorbance of SiNH decreased quickly and simultaneously, there was a new absorbance peak at 470 nm, with the colour changing from red to yellow (inset in Fig. 1A). When it was excited at 470 nm, robust fluorescence enhancement peaking at 595 nm (Φfl = 0.324) was observed along with bright yellow fluorescence (Fig. 1B). The fluorescence turn-on process was mainly because the reaction group methyl(4-hydroxyphenyl)amino was oxidized by ONOO and then hydrolysed into a quinone product, blocking the PET progress.
image file: c8ob01598h-f1.tif
Fig. 1 Absorption spectra (A) and emission spectra (B) of SiNH (5 μM) in the presence of 10 equiv. ONOO. (C) Time-dependent emission spectra. (D) Fluorescence spectra of SiNH (5 μM) treated with 10 equiv. of ONOOvs. that of product SiMH (5 μM). Conditions: HEPES buffers (50 mM, pH = 7.4, H2O[thin space (1/6-em)]:[thin space (1/6-em)]CH3CN = 7[thin space (1/6-em)]:[thin space (1/6-em)]3).

The reaction mechanism was well validated by the titration experiment of high resolution mass spectrum (HRMS) (Fig. S1).14,15,28 Furthermore, to confirm the reaction mechanism, the proposed product SiMH was also obtained by the reaction of SiNH with ONOO. In fact, it was found that the emission spectrum of SiMH was consistent with those of SiNH and ONOO, indicating the validity of the reaction mechanism (Fig. 1D). In particular, only few seconds were required for SiNH to react with ONOO, indicating that the probe is very favourable for the real-time detection of ONOO (Fig. 1C). Meanwhile, the probe SiNH exhibited good selectivity towards ONOO over other potential competitive ROS substances (Fig. S2). The excellent selectivity of SiNH could be due to the electropositive Si-rhodamine fluorophore, which could partially decrease the electron density of the methyl(4-hydroxyphenyl)amino group to resist oxidation, significantly enhancing the selectivity of SiNH towards ONOO.19 Taken together, the probe SiNH could realize the selective and real-time detection of ONOO in non-aqueous systems. However, owing to the undesirable performance of SiNH in aqueous systems, the application of SiNH to detect ONOO in living systems was restricted.

To achieve the goal of detecting ONOO in aqueous conditions, we encapsulated SiNH into the hydrophobic interior of the micelles to form nanoparticles using amphiphilic copolymer mPEG-DSPE. The exterior of the mPEG-DSPE moiety is hydrophilic, dramatically improving the water solubility. First, the average size of the nanoprobe was measured by dynamic light scattering (DLS), and it was about 80 nm, making the nanoprobe suitable for detecting ONOO in living cells (Fig. S3). Then, we investigated the photophysical properties of this nanoprobe toward ONOO in aqueous system. After adding 10 equiv. of ONOO, we observed an intense emission peak at 595 nm (Fig. 2A). Also, compared with only hydrophobic probe SiNH, which requires longer reaction time (over 2 min) in an aqueous solution, the nanoprobe with the copolymer could completely react with ONOO within 10 s, thus satisfying the requirements for real-time detection in biological system (Fig. 2B). Moreover, when the nanoprobe was treated with ONOO under various concentrations (0 to 10 μM), the fluorescence intensity at 595 nm was linearly proportional to the ONOO concentration in the range of 0–10 μM, indicating that the nanoprobe can be employed for detecting ONOO quantitatively (Fig. S4). When the concentration of ONOO was over 10 μM, the fluorescence intensity reached a plateau, signifying the completion of the reaction (inset in Fig. S4).


image file: c8ob01598h-f2.tif
Fig. 2 Emission spectra (A) of micelles (5 μM) in the presence of 10 equiv. ONOO. (B) Time-dependent emission spectra of micelles and SiNH in the presence of 10 equiv. of ONOO in aqueous solution. Conditions: HEPES buffers (50 mM, pH = 7.4).

To test the specificity toward ONOO, we carried out an important procedure to determine whether other species could introduce signal noise. As illustrated in Fig. 3, biologically relevant species including reactive oxygen species (H2O2, HClO), various biothiols (GSH, Cys, Glu, Pro), and reactive nitrogen species (NO2, NO) were investigated. There were almost no fluorescent signals with the existence of all test species except ONOO. Moreover, the effect of pH was evaluated in the absence and presence of ONOO (Fig. S5). The normal physiological pH value is about 7.4. Meanwhile, the nanoprobe exhibited slight effect on the performance with a change in pH (from 6.5 to 8.5), indicating that the nanoprobe could function properly at a physiological pH. All these experiments demonstrated that the nanoprobe SiNH showed high selectivity toward ONOO in an aqueous solution.


image file: c8ob01598h-f3.tif
Fig. 3 Fluorescence intensity changes of micelles (5 μM) for ONOO over various species (100 equiv.) in HEPES buffers (1) Probe only (5 μM), (2) Cys, (3) NO, (4) HClO, (5) H2O2, (6) NaNO2, (7) GSH, (8) Glu, (9) Pro, (10) ONOO. λem = 595 nm.

All these aforementioned investigations confirmed that the nanoprobe can realize fast and selective detection of ONOO in aqueous solutions. Then, we investigated the capability of the nanoprobe to monitor ONOO in living cells. First, an MTT assay on HeLa cells was carried out with the concentrations of SiNH in the range from 0 μM to 10.0 μM. Clearly, even after incubating with nanoprobe (10.0 μM) for 8 h, over 80% of HeLa cells survived (Fig. S6), demonstrating the low toxicity of the nanoprobe toward cultured cell lines.12,25,29,30 Second, the ability of the nanoprobe for real-time monitoring of ONOO in living cells was investigated. As shown in Fig. 4A1–A3, after incubating the nanoprobe only with the HeLa cells for 30 min, there were almost no fluorescent signals. In contrast, when the nanoprobe-loaded cells were treated with ONOO donor SIN-1 (3-morpholinosydnonimine) for 30 min, significant fluorescence enhancement could be observed (Fig. 4B2), suggesting that the nanoprobe can detect exogenous ONOO.31,32 To monitor endogenous ONOO in living cells, robust signals could be obtained when the cells were pretreated with LPS (ONOO stimulation) for about 8 h and then incubated with the nanoprobe for 30 min (Fig. 4C2).33–36 With the stimulation of LPS, all types of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were generated in living cells. Thus, strong fluorescence originated from endogenous ONOO (Fig. 4C2). When the inhibitors of nitric oxide synthase aminoguanidine (AG) and superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) were added, there was almost no fluorescence signal from the nanoprobe-loaded cells in the presence of LPS (Fig. 4D2 and E2), suggesting the inhibition effects of TEMPO and AG on the cellular ONOO production. All these results indicated that the nanoprobe SiNH can be used for real-time monitoring of ONOO selectively in living cells.37–39


image file: c8ob01598h-f4.tif
Fig. 4 Fluorescent imaging of exogenous and endogenous ONOO with the nanoprobe SiNH (2 μM) in living HeLa cells. (A1–A3) Images from cells pre-incubated by the micelle for 30 min (B1–B3) then stained by SIN-1 (20 μM) for 30 min; (C1–C3) fluorescent images of cells stained by micelles incubated with the stimulants LPS (20 μg mL−1) for 8 h. (D1–D3) NOS inhibitor AG (5 mM) was co-incubated during LPS (20 μg mL−1) stimulation for 8 h and then with nanoprobe; (E1–E3) superoxide inhibitor TEMPO (300 mM) was co-incubated during LPS (20 μg mL−1) stimulation for 8 h and then stained by micelles. Fluorescent signals were collected at 550–650 nm for the red channel (λex = 488 nm).

Conclusions

In summary, we have designed and synthesized the fluorescent probe SiNH by combining methyl(4-hydroxyphenyl) amino as the reaction group and Si-rhodamine as a high-performance fluorophore. Using the supramolecular assembly approach, the probe SiNH is embedded into the amphiphilic copolymer mPEG-DSPE to overcome the limitation of poor water solubility, forming nanoparticles with the exterior of hydrophilic PEG unit. The nanoprobe exhibits not only fast response (within 10 s) but also high selectivity toward ONOO in both aqueous and biological environments. The excellent performance in cell imaging indicates that the nanoprobe can be employed to monitor endogenous ONOO with minimal cytotoxicity. The nanoprobe is well applicable for the detection and imaging of endogenous ONOO in living cells, which can result in better understanding of various pathophysiological roles of ONOO in cellular processes.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by National Key Research and Development Program (No. 2017YFC0906902), Excellent Young Scholars (21622602), Key Project (21636002), NSFC/China, Oriental Scholarship, Scientific Committee of Shanghai (14ZR1409700 and 15XD1501400), the Fundamental Research Funds for the Central Universities, Open Funding Project of the State Key Laboratory of Bioreactor Engineering.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ob01598h

This journal is © The Royal Society of Chemistry 2019