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Article

Near-Infrared Fluorescence Probe for Visualizing Fluctuations of Peroxynitrite in Living Cells and Inflammatory Mouse Models

by
Shuchun Qin
1,†,
Yiming Ran
1,†,
Yitian He
1,
Xiaoyan Lu
1,
Jiamin Wang
2,
Weili Zhao
1 and
Jian Zhang
1,*
1
Key Laboratory for Special Functional Materials of Ministry of Education, School of Materials Science and Engineering, Henan University, Kaifeng 475004, China
2
Key Laboratory of Natural Medicine and Immuno-Engineering of Henan Province, Henan University, Kaifeng 475004, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Chemosensors 2023, 11(6), 316; https://doi.org/10.3390/chemosensors11060316
Submission received: 25 April 2023 / Revised: 15 May 2023 / Accepted: 22 May 2023 / Published: 24 May 2023
(This article belongs to the Special Issue A Theme Issue in Honor of Dr. Richard Horobin—Cell or Organelle Selective Fluorescent Probes: Their Design, Mechanism, Modeling and Application)
Browse Figures
Versions Notes

Abstract

:
Inflammation is a vital protective response in living systems and closely related to various diseases. As a member of the reactive oxygen species (ROS) family, peroxynitrite (ONOO−) is involved in the organism’s inflammatory process and considered as an important biomarker of inflammation. Therefore, the construction of a simple, rapid, and sensitive tool for detecting ONOO− is of great importance for the diagnosis of inflammation. In this study, we constructed the new near-infrared fluorescence probe BDP-ENE-S-Py+ based on BODIPY dye, which has the advantages of fast response speed (2 min), good selectivity, and a high signal-to-noise ratio. Moreover, the probe had a good linear relationship (LOD = 120 nM) when the ONOO− concentration was 10–35 µM. In addition, BDP-ENE-S-Py+ could detect exogenous ONOO− in liver cancer cells without interference from other reactive oxygen species and visualize the fluctuations in ONOO− concentrations in cells. More importantly, BDP-ENE-S-Py+ was able to track the upregulation of ONOO− content in a mouse model of peritonitis induced by LPS. This work demonstrated that the near-infrared fluorescent probe for visualizing ONOO− level fluctuations could provide a promising tool for inflammation-related studies.

1. Introduction

Inflammation is a kind of innate defense response of the biological body to tissue injury, which often occurs in the process of local or systemic inflammation after infection or injury [1,2,3]. In the process of inflammation, immune cells release a large number of soluble mediations, such as cytokines, reactive oxygen species (ROS), chemokines, reactive nitrogen species (RNS), etc., which can remove irritants by fighting pathogens or promoting tissue repair and healing [3,4,5,6]. However, the imbalance of inflammation regulation is closely related to a variety of diseases, such as atherosclerosis, rheumatoid arthritis, Alzheimer’s disease, tumors, and COVID-19 [7,8,9]. Therefore, it is of great importance to develop a reliable tool for detecting and diagnosing inflammation.
Peroxynitrite (ONOO−), an active molecule of ROS and RNS, is produced in biological systems by coupling the diffusion control of nitric oxide (NO) and superoxide anion (O2•−) [10,11]. Numerous studies have shown that ONOO− is more cytotoxic than NO and O2•− [10]. Excessive production of ONOO− will cause damage to key components in cells, such as proteins, lipids, mercaptans, and DNA, and eventually lead to cell death [12,13,14,15,16,17,18,19]. Therefore, ONOO− is concerned with various diseases, such as inflammation, diabetes, neurodegenerative diseases, and cancer [20,21,22]. In consequence, it is of great importance to construct a reliable, simple, and efficient method for the detection of ONOO− in vivo and develop full understanding of the relationship between ONOO− and inflammation. At present, the commonly used ONOO− detection methods include high-performance liquid chromatography, spectrophotometry, electron spin resonance, and the fluorescent probe method [23]. Among them, the fluorescence probe method is widely used due to its advantages of non-invasive and real-time imaging, good selectivity, and high sensitivity [24,25,26,27,28,29,30,31,32,33,34,35,36]. In the last few years, all kinds of fluorescent probes have been reported for the detection of ONOO−, and the detection groups commonly used in the construction of probes include phenyl borate [37,38,39], α-ketoamide [40,41,42], diphenyl phosphoric acid [43,44,45], hydrazine [46,47,48], and chalcogenide [49,50,51]. However, most of the probes are located in the visible region, with short emission wavelengths, large background interference, and poor tissue penetration ability; thus, they cannot perform in situ real-time in vivo imaging well. Therefore, it is imperative to construct a near-infrared fluorescence probe for in situ real-time in vivo imaging to visually detect ONOO− level fluctuations in living cells and inflammatory mouse models.
BODIPY dyes have been widely used in many aspects due to their excellent properties, such as good chemical and optical stability, high quantum yield, and pH insensitivity [52,53,54]. Therefore, by regulating the substituents at positions 3 and 5 and expanding their conjugated degree, we constructed a near-infrared BODIPY dye as a fluorophore and used benzeneboronic acid pinacol ester as the detection group. The near-infrared fluorescence probe BDP-ENE-S-Py+ was constructed by connecting the two parts with a benzylpyridinyl group (Scheme 1). The fluorescence of the probe was quenched thanks to photoinduced electron transfer (PET) between the electron-deficient benzyl quaternary pyridine salt and the electron-rich BDP-ENE-S-Me. Unsurprisingly, the probe displayed a fast response to ONOO− with high sensitivity, good selectivity, and a high signal-to-noise ratio. In addition, BDP-ENE-S-Py+ could detect exogenous ONOO− in liver cancer cells without interference from other reactive oxygen species and was also able to visually detect the fluctuations in ONOO− concentration levels in cells. More importantly, BDP-ENE-S-Py+ was able to detect upregulation of ONOO− in a mouse model of peritonitis induced by LPS. These results indicated that the probe had broad applicability and could be utilized as a powerful tool for the diagnosis of inflammation through detection of ONOO− fluctuations.

2. Experimental Section

2.1. Synthesis of Compounds

The details of synthesis are shown in Scheme S1.
Synthesis of BDP-Me: Under nitrogen, 2,4-dimethylpyrrole (1.03 mL, 10 mmol) and 4-pyridinecarboxaldehyde (471 mg, 5 mmol) were dissolved in 10 mL anhydrous dichloromethane, followed by addition of trifluoroacetic acid for overnight reaction at room temperature and pressure. After half an hour of reaction with chloranil (1.23 g, 5 mmol), triethylamine (10 mL, 7 mmol) and boron trifluoride diethyl etherate (10 mL, 8 mmol) were added in an ice bath. After stirring for three hours, the silica gel column was filtered. Orange crystals (200 mg, 25%) were purified by column chromatography.
Synthesis of BDP-ENE-S-Me: Under nitrogen, BDP-Me (98 mg, 0.3 mmol) and 5-methyl-2-thhene formaldehyde (0.13 mL, 1.2 mmol) were dissolved in 10 mL anhydrous acetonitrile, piperidine (0.3 mL, 10 mmol) was added, and the mixture was then stirred for 3 h at 80 °C reflux. After the reaction, the solvent was removed by using a rotary evaporator, and the green crystal (61 mg, 40% yield) was purified by column chromatography. And nuclear magnetic verification was carried out (Figures S7 and S8). 1H NMR (500 MHz, DMSO-d6) δ 8.784 (d, J = 6.0 Hz, 2H), 7.723 (s, 1H), 7.691 (s, 1H), 7.571 (d, J = 6.0 Hz, 2H), 7.168 (d, J = 4.0 Hz, 2H), 7.135 (s, 1H), 7.104 (s, 1H), 6.954 (s, 2H), 6.875 (d, J = 3.5 Hz, 2H), 2.520 (s, 6H), 1.147 (s, 6H). 13C NMR (125 MHz, CDCl3) δ 151.75, 149.50, 141.96, 139.90, 139.38, 132.31, 131.35, 128.75, 128.01, 125.49, 123.00, 117.12, 116.18, 14.89, 13.83. HRMS-ESI: calculated for [C30H26BF2N3S2 + H]+: 542.17075; found: 542.17086.
Synthesis of BDP-ENE-S-Py+: BDP-ENE-S-Me (30 mg, 0.055 mmol) and 4-bromomethyl benzeneboronic acid pinacol ester (49 mg, 0.165 mmol) were dissolved in 10 mL toluene and stirred for 24 h at 120 °C reflux. At the end of the reaction, the mixture was filtered and washed with toluene, which was followed by recrystallization with dichloromethanes and petroleum ether, finally yielding the green solid BDP-ENE-S-Py+ (15 mg, yield 36%). And nuclear magnetic verification was carried out (Figures S9 and S10). 1H NMR (500 MHz, DMSO-d6) δ 9.389 (d, J = 6.5 Hz, 2H), 8.521 (d, J = 6.5 Hz, 2H), 7.773 (d, J = 4 Hz, 2H), 7.749 (d, J = 4.5 Hz, 2H), 7.495 (d, J = 8.0 Hz, 2H), 7.202 (d, J = 3.5 Hz, 2H), 7.139 (s, 1H), 7.107 (s, 1H), 7.005 (s, 2H), 6.892 (d, J = 4.0 Hz, 2H), 6.017 (s, 2H), 2.526 (s, 6H), 1.423 (s, 6H), 1.304 (s, 12H). 13C NMR (125 MHz, DMSO-d6) δ 153.11, 152.26, 146.56, 143.96, 141.10, 137.88, 135.65, 135.35, 132.10, 131.66, 131.57, 130.33, 129.94, 128.32, 127.97, 127.74, 119.74, 116.20, 84.43, 63.98, 25.14, 24.96, 16.03, 16.00, 15.47. HRMS-ESI: calculated for [C43H44B2F2N3O2S2]+: 758.30236; found: 758.30254.

2.2. General Information

All the materials and reagents were purchased and used without further purification. 1H NMR spectra were taken using a Bruker AVANCE NEO 500 MHz spectrometer. 1H NMR data of chemical shifts (δ) are given in ppm (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) using CDCl3 (δ = 7.26 ppm) and dimethyl sulfoxide (δ = 2.5 ppm) as a reference. 13C NMR spectra were recorded on a Bruker AVANCE NEO 125 MHz spectrometer, and the chemical shifts (δ) were reported in ppm with CDCl3 and DMSO-d6 at δ 77.0 and 39.4 ppm as the internal standard. Absorption spectra and fluorescence emission spectra were recorded on a LengGuang Tech. UV 1920 UV–vis spectrometer and F97 Pro spectrofluorophotometer, respectively. High-resolution mass spectra were measured from an Agilent 7250&JEOL-JMS-T100LP AccuTOF. The cell images were taken using the Leica DMI8 inverted fluorescence microscope (Leica, Germany). λex = 405/660 nm, λem = 420–500/662–738 nm. The live imaging was monitored using a fluorescence imaging system (IVIS Spectrum Live Imaging System, PerkinElmer, Waltham, MA, USA).

2.3. Spectroscopic Measurements in Solution

A stock solution of BDP-ENE-S-Py+ (1 mM) was prepared in acetonitrile and was subsequently diluted to appropriate concentration in acetonitrile/PBS (1:1, v/v, 10 mM, pH 7.4). The stock solutions (10 mM) of analytes were prepared in PBS. The analytes used in the stock aqueous solutions of analytes were 0: blank, 1: ONOO−, 2: HClO, 3: H2O2, 4: OH, 5: 1O2, 6: NO, 7: Ca2+, 8: Cu2+, 9: Mg2+, 10: NO2−, 11: HS−, 12: SO32−, 13: SO42−, 14: Cys, 15: GSH, and 16: Hcy. All absorption and fluorescence measurements were performed in acetonitrile/PBS buffer (1:1, v/v, 10 mM, pH 7.4) at room temperature. In the selectivity studies, the test samples were prepared by adding the appropriate amount of the individual stock solution of analytes to 4 mL solution of BDP-ENE-S-Py+ (10 µM). In the anti-interference study, the sample was prepared by adding ONOO− to 4 mL BDP-ENE-S-Py+ (10 µM) solution and the analyte of the same concentration. In the titration experiment, solutions of BDP-ENE-S-Py+ (10 µM) were incubated with different concentrations of ONOO−. In the pH stability study, samples were prepared by adding BDP-ENE-S-Py+ (10 µM) and ONOO− to 4 mL of buffer solution at different pH values.
The fluorescence spectra were recorded with the excitation at 620 nm and the emission was collected at 640–900 nm.

2.4. Determination of the Detection Limit

The detection limit was calculated based on the fluorescence titration. In the absence of ONOO−, the fluorescence emission spectrum of BDP-ENE-S-Py+ was measured eight times and the standard deviation of blank measurement was obtained. To gain the slope, the fluorescence intensity at 694 nm was plotted to the concentration of ONOO−. The detection limit was calculated with the following equation:
Detection limit = 3σ/k
where σ is the standard deviation of blank measurement and k is the slope between the fluorescence intensities versus the concentrations of ONOO−.

2.5. Density Functional Theory

The density functional theory (DFT) calculations were performed in Materials Studio. M11-L meta-Generalized gradient approximation (GGA) was adopted. The structures were relaxed until the forces acting over all atoms were less than 0.05 eV/Å, and electronic energies converged in the range of 2 × 10−5 eV.
Prof. Changwei Gong from Taiyuan University of Science and Technology is thanked for his assistance in the theoretical calculations.

2.6. Cell Culture and Imaging

The HepG2 cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin and incubated under an atmosphere containing 5% CO2 in 37 °C humidified air for 24 h.

2.7. Cytotoxicity Assays

Cytotoxicity test: HepG2 cells were incubated in DMEM medium containing 10% fetal bovine serum in 96-well plates (5 × 103) for 24 h. BDP-ENE-S-Py+ was diluted into different concentrations (2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM) in medium and incubated cells. After HepG2 cells were placed in darkness for 24 h, the cytotoxicity of BDP-ENE-S-Py+ was evaluated using a CCK-8 assay. The cells were then cultured with CCK-8 (10 μL) for 1 h. After full mixing, the absorbance of the cells was measured at 450 nm using an enzyme label. Each result was averaged over the three wells and 100 percent survival was measured for untreated cells. Relative cell viability (%) was calculated as follows: cell viability = OD sample/OD control × 100%.

2.8. Cell Selectivity Experiment

Intracellular selective imaging ONOO− experiment: The HepG2 cells could be divided into four groups. The first group of cells was incubated with 10 µM BDP-ENE-S-Py+ for 30 min, was incubated with Hoechst 33342 (1 μg/mL) (Biyuntian Biotechnology Co., Ltd., Shanghai, China) for 10 min, and then washed with PBS 3 times for fluorescence imaging. In the second group, cells were preincubated with H2O2 (500 μM) for 1 h and incubated with 10 µM BDP-ENE-S-Py+ for 30 min, incubated with Hoechst 33342 (1 μg/mL) for 10 min, and then washed with PBS 3 times for fluorescence imaging. In the third group, cells were preincubated with HClO (500 μM) for 1 h and incubated with 10 µM BDP-ENE-S-Py+ for 30 min, incubated with Hoechst 33342 (1 μg/mL) for 10 min, and then washed with PBS 3 times for fluorescence imaging. In the fourth group, cells were preincubated with 500 µM 3-morpholine pyridine imine hydrochloride (SIN-1) for 1 h and incubated with 10 µM BDP-ENE-S-Py+ for 30 min, incubated with Hoechst 33342 (1 μg/mL) for 10 min, and then washed with PBS 3 times for fluorescence imaging.

2.9. Experiments with Different Concentrations of SIN-1

The HepG2 cells could be divided into three groups. The first group of cells was incubated with 10 µM BDP-ENE-S-Py+ for 30 min. In the second group, cells were preincubated with SIN-1 (300 μM) for 1 h and then added to 10 µM BDP-ENE-S-Py+ for 30 min. The final group of cells was treated with BDP-ENE-S-Py+ (10 μM) for 30 min after incubation with SIN-1 (500 μM) for 2 h. Finally, Hoechst 33342 (1 μg·mL−1) was added to the three cell groups and they were incubated for another 10 min. Cell imaging was performed using an inverted fluorescence microscope after washing the cells with PBS three times.

2.10. Fluorescence Imaging in Mice

All animal care and experimental protocols for this study were approved by the Animal Experiment Ethics Committee of Henan University.
Mouse peritonitis induced by lipopolysaccharide (LPS) was tested. The LPS experimental group of mice was intraperitoneally injected with LPS (100 µg·kg−1) for 24 h, and the BDP-ENE-S-Py+ probe (100 μL, 10 µM) was then injected in the same region. The probe control group of mice was injected only with BDP-ENE-S-Py+ (100 μL, 10 µM). Fluorescence images were taken at 0, 10, 30, and 60 min after the addition of the probe. Images were taken using an excitation laser at 630 nm and an emission filter of 700 nm. All groups within the study contained n = 3 mice.

3. Results and Discussion

3.1. Spectral Properties of BDP-ENE-S-Py+ Responding to ONOO−

Initially, we examined the response of the BDP-ENE-S-Py+ probe to ONOO− at room temperature in a buffer system of PBS/acetonitrile (1:1, v/v, 10 mM, pH 7.4). It can be seen from the figure that the maximum absorption of the BDP-ENE-S-Py+ probe (Φ = 0.0138) occurred at 683 nm, and the maximum absorption peak shifted to 667 nm after reacting with ONOO− (Figure 1a). However, before BDP-ENE-S-Py+ reacted with ONOO−, no fluorescence emission was observed. After the probe reacted with ONOO−, obvious fluorescence enhancement was observed at 694 nm, which increased by about 66 times (Figure S1). The high signal-to-noise ratio demonstrates BDP-ENE-S-Py+ could become an excellent tool for detecting ONOO−. For the sake of studying the response speed of BDP-ENE-S-Py+ in the presence of ONOO−, time-dependent spectral changes were measured. As shown in Figure 1b, when BDP-ENE-S-Py+ reacted with 100 µM ONOO−, the fluorescence intensity increased rapidly at 694 nm, and the reaction was complete within two minutes. When the probe reacted with 10 µM ONOO−, the fluorescence intensity slightly increased compared with the initial value and gradually stabilized.
Sensitivity is another important argument when estimating the feasibility of probe detection. Consequently, we investigated the response behavior of the probe at different concentrations of ONOO− by fluorescence titration. As shown in Figure 1c, with augmented ONOO− concentration (0–100 µM), the fluorescence strength at 694 nm gradually increased, showing a good linear relationship with 10–35 μM ONOO− (Figure 1d); the detection limit was calculated to be 120 nM (LOD = 3σ/k). When the concentration of ONOO− was small, it could not react quickly with the probe and the fluorescence intensity was low. Therefore, fluorescence intensity would be enhanced instantly when concentration increases from 50 µM to 60 µM. These results confirmed that the BDP-ENE-S-Py+ probe could respond to ONOO− rapidly and sensitively, which is advantageous for the detection of ONOO− in the biosystem.

3.2. The Selective Response of BDP-ENE-S-Py+ to ONOO− and the Effect of pH

On account of the complex environment of the biosystem, a probe with good capability needs to avert disturbance from other substances. To investigate this, we carried out a selectivity experiment with BDP-ENE-S-Py+. As shown in Figure 2a, various reactive nitrogen species (RNS), reactive sulfur species (RSS), reactive oxygen species (ROS), cations (Ca2+, Mg2+, Cu2+), biologically important amino acids (Cys, Hcy, GSH), and ONOO− were added into the probe solution to observe changes in their fluorescence signals. The fluorescence intensity of BDP-ENE-S-Py+ solution only increased significantly when ONOO− was added. In order to realize application in complex organisms, we further verified the anti-interference performance of the probe. Under the co-existence of various analytes, ONOO− still caused a nearly 66-fold fluorescence enhancement (Figure 2b). The experimental results showed that BDP-ENE-S-Py+ had good selectivity for ONOO− and could detect ONOO− with high selectivity. In addition, we also studied the stability of BDP-ENE-S-Py+ in relation to pH. It can be seen from the figure that the fluorescence signal of the BDP-ENE-S-Py+ probe hardly changed when the pH value was 5–10 and gradually increased along with the increasing pH value when ONOO− was added (Figure S2). We also measured the time stability of the probe within a 30 min time frame under the test system (Figure S3), and it can be seen from the experimental results that the probe was relatively stable under the system. The experimental results exhibited that BDP-ENE-S-Py+ had good pH stability and was appropriate for further application in the test of ONOO− in complex physiological environments.

3.3. Sensing Mechanism of BDP-ENE-S-Py+

As displayed in Scheme 1, the detection mechanism of BDP-ENE-S-Py+ for ONOO− is described as follows: the benzeneboronic acid pinacol ester is oxidized by ONOO− to phenoxide, and then a self-elimination reaction occurs, resulting in obstruction of the PET process and the fluorescence of BDP-ENE-S-Me being released. The results are confirmed by ESI-MS analysis (Figures S4 and S5). For the sake of a better interpretation of the fluorescence response mechanism of BDP-ENE-S-Py+, we performed density functional theory (DFT) calculations for BDP-ENE-S-Py+ and BDP-ENE-S-Me. As shown in Figure 3, in the lowest molecular unoccupied orbital (LUMO), the π electron was predominantly found in the benzylpyridine group of the BDP-ENE-S-Py+ probe, while in the highest molecular occupied orbital (HOMO), the π electron was predominantly found in the parent nucleus of the BODIPY dye. The results show that the fluorescence quenching of BDP-ENE-S-Py+ was due to the PET process from the electron-rich BODIPY parent nucleus to the electron-deficient benzylpyridine group. In contrast, in the LUMO and HOMO orbitals of BDP-ENE-S-Me, π electrons were focused on the parent nucleus of BODIPY, and thus emitted bright fluorescence. The calculated HOMO–LUMO energy levels of the BDP-ENE-S-Py+ probe were −5.362 eV and −3.867 eV, respectively. After reaction of the BDP-ENE-S-Py+ probe with ONOO−, the released BDP-ENE-S-Me dyes had energy levels of −4.074 eV and −5.853 eV, respectively. The energy gap between LUMO and HOMO increased from 1.479 eV to 1.779 eV, which was in accordance with the blue shift of the spectrum of absorption after BDP-ENE-S-Py+ reacted with ONOO−. The results of theoretical arithmetic inosculated with the laboratory results, which testified to the reasonableness of DFT computation.

3.4. BDP-ENE-S-Py+ Intracellular Imaging

Based on the excellent in vitro testing performance of BDP-ENE-S-Py+, we extended testing to intracellular detection of ONOO−, where we expected that BDP-ENE-S-Py+ could also show excellent detection performance for ONOO− in cells. We selected hepatoma cells (HepG2 cells) for the intracellular ONOO− selectivity experiment. Before cell imaging, the cytotoxicity of BDP-ENE-S-Py+ was verified by standard CCK-8 assays (Figure S6). After incubating the cells with different concentrations of BDP-ENE-S-Py+ for 24 h, cell livability still reached more than 90% when the probe concentration was 50 µM. The results showed that the probe had low cytotoxicity and good biocompatibility.
Subsequently, the intracellular selectivity of BDP-ENE-S-Py+ to ONOO− was assessed in HepG2 cells. It can be seen from the figure that only faint red fluorescence was observed in the red channel of the blank control group (Figure 4). HClO and H2O2 were selected as the typical interference ROS in this study. The experimental results obtained in the two experimental groups were the same as those in the blank control group. There were weak fluorescence signals in the red channel. Only in the experimental group with ONOO−-releasing agent 3-mollopyridinimine hydrochloride (SIN-1) was an obvious fluorescence signal observed in the red channel. The experimental results showed that the probe could achieve highly selective imaging of peroxynitrite in the complex cellular environment.
Since the BDP-ENE-S-Py+ probe could sensitively detect intracellular ONOO−, we further investigated whether the probe could detect changes in the concentration level of ONOO− in cells. We co-incubated the cells with different concentrations of SIN-1 (ONOO−-releasing agent) and then treated them with the probe to observe the fluorescence changes. We selected two concentrations of SIN-1: 300 µM and 500 µM. It can be seen from the figure that along with the increased SIN-1 concentration, the fluorescence released after incubation with the probe became stronger (Figure 5). In conclusion, the BDP-ENE-S-Py+ probe could detect changes in the ONOO− content of cells.

3.5. BDP-ENE-S-Py+ Imaging of ONOO− in Inflammatory Mice

Encouraged by these results, we expected that the BDP-ENE-S-Py+ probe would detect ONOO− in mice to achieve real-time imaging of inflammation induced by lipopolysaccharide (LPS). To verify this probe’s application in inflammation diagnosis, we established a mouse inflammatory model using LPS to induce upregulation of ONOO− content in mice. Mice were intraperitoneally injected with LPS (100 µg/kg) and incubated for 24 h. After injecting the BDP-ENE-S-Py+ probe into the belly of mice in the blank controller and inflammatory groups, fluorescence imaging was performed at different times (Figure 6). At 10 min, the obvious fluorescence signal of the treatment group injected with LPS was observed, and fluorescence intensity enhanced gradually along with increased time. Although the fluorescence intensity of the blank control group was also enhanced, it was significantly smaller than that of the treatment group. The results show that the BDP-ENE-S-Py+ probe could detect and image ONOO− in peritonitis mice, which would be helpful for the early diagnosis of inflammation.

4. Conclusions

In summary, we reasonably constructed the new near-infrared fluorescence probe BDP-ENE-S-Py+ by introducing thiophene methyl at positions 3 and 5 of the BODIPY dye to expand its conjugated structure. The probe has the advantage of a rapid, sensitive, and specific response to ONOO− with a high signal-to-noise ratio. In addition, the probe was successfully applied to detect exogenous ONOO− in hepatocellular carcinoma cells without disturbance from other ROS and was capable of visualizing the changes in ONOO− content in cells. More importantly, BDP-ENE-S-Py+ was able to detect upregulation of ONOO− in a mouse model of peritonitis induced by LPS. These results demonstrate that the BDP-ENE-S-Py+ probe has broad applicability and could be utilized as a powerful tool for the diagnosis of inflammation by detecting ONOO− fluctuations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/chemosensors11060316/s1, Scheme S1: The synthesis routine of BDP-ENE-S-Py+; Figure S1: The fluorescence emission spectra of the BDP-ENE-S-Py+ probe (10 µM) before and after reaction with ONOO− (100 µM); Figure S2: Fluorescence intensity spectra of the BDP-ENE-S-Py+ probe (10 µM) after reaction with ONOO− (100 µM) at different pH values; Figure S3: Probe time stability test; Figure S4: The ESI-MS of BDP-ENE-S-Py+; Figure S5: The ESI-MS of BDP-ENE-S-Py+ upon addition of ONOO−; Figure S6: Cytotoxicity assays of BDP-ENE-S-Py+ in living HepG2 cells; Figure S7: 1H NMR of BDP-ENE-S-Me; Figure S8: 13C NMR of BDP-ENE-S-Me; Figure S9: 1H NMR of BDP-ENE-S-Py+; Figure S10: 13C NMR of BDP-ENE-S-Py+.

Author Contributions

Conceptualization, J.Z.; methodology, J.Z.; software, J.W. and S.Q.; validation, S.Q. and Y.R.; formal analysis, S.Q., Y.H. and X.L.; investigation, S.Q., Y.R., Y.H. and X.L.; resources, W.Z. and J.Z.; data curation, X.L. and S.Q.; writing—original draft preparation, S.Q. and Y.R.; writing—review and editing, J.W. and J.Z.; visualization, S.Q. and Y.R.; supervision, J.Z.; project administration, J.Z.; funding acquisition, J.W. and J.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (No. 82030107, No. 21702046), the Key Scientific and Technological Project of Henan Province (No. 212102311064, No. 222102310692), and the Key Scientific Research Project of Colleges and Universities in Henan Province (No. 22A150005).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Review Board of the Animal Experiment Ethics Committee of Henan University (protocol code HUSOM2022-140 and date of approval 6 March 2022) for studies involving animals.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be shared upon reasonable request.

Acknowledgments

The authors thank Changwei Gong, School of Materials Science and Technology, Taiyuan University of Science and Technology, for his helpful discussions regarding density functional theory calculations.

Conflicts of Interest

The authors declare no conflict of interest.

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Scheme 1. The response mechanism of BDP-ENE-S-Py+ for the detection of ONOO−.
Scheme 1. The response mechanism of BDP-ENE-S-Py+ for the detection of ONOO−.
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Figure 1. (a) The absorption spectra of the BDP-ENE-S-Py+ probe (10 μM) with ONOO− (100 μM) in PBS/acetonitrile (1:1, v/v, 10 mM, pH 7.4) buffer solution at room temperature before and after reaction. (b) The BDP-ENE-S-Py+ probe’s time dynamic curve. (c) The fluorescence intensity of the BDP-ENE-S-Py+ probe changed with the concentration of ONOO−. (d) A linear relationship between fluorescence intensity changes at 694 nm of BDP-ENE-S-Py+ and ONOO− concentration, which can be linearly fitted by the equation Y = −3.492 + 0.597X, with R2 = 0.991 (n = 3).
Figure 1. (a) The absorption spectra of the BDP-ENE-S-Py+ probe (10 μM) with ONOO− (100 μM) in PBS/acetonitrile (1:1, v/v, 10 mM, pH 7.4) buffer solution at room temperature before and after reaction. (b) The BDP-ENE-S-Py+ probe’s time dynamic curve. (c) The fluorescence intensity of the BDP-ENE-S-Py+ probe changed with the concentration of ONOO−. (d) A linear relationship between fluorescence intensity changes at 694 nm of BDP-ENE-S-Py+ and ONOO− concentration, which can be linearly fitted by the equation Y = −3.492 + 0.597X, with R2 = 0.991 (n = 3).
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Figure 2. The (a) selectivity and (b) interference of the BDP-ENE-S-Py+ probe (10 µM). The concentration of each analyte was 100 µM. The buffer system was PBS/acetonitrile (1:1, v/v, 10 mM, pH 7.4) at room temperature and the reaction time was 2 min. λex = 630 nm and λem = 694 nm.
Figure 2. The (a) selectivity and (b) interference of the BDP-ENE-S-Py+ probe (10 µM). The concentration of each analyte was 100 µM. The buffer system was PBS/acetonitrile (1:1, v/v, 10 mM, pH 7.4) at room temperature and the reaction time was 2 min. λex = 630 nm and λem = 694 nm.
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Figure 3. Density functional theory (DFT)-optimized structures and frontier molecular orbitals (MOs) of BDP-ENE-S-Py+ and BDP-ENE-S-Me.
Figure 3. Density functional theory (DFT)-optimized structures and frontier molecular orbitals (MOs) of BDP-ENE-S-Py+ and BDP-ENE-S-Me.
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Figure 4. Intracellular selective imaging of ONOO− in HepG2 cells. (a1,a2) Incubation of the cells with BDP-ENE-S-Py+ (10 μM) for half an hour; (b1,b2) incubation of the cells with H2O2 (0.1 mM) for one hour followed by incubation with BDP-ENE-S-Py+ (10 µM) for half an hour; (c1,c2) incubation of the cells with HClO (0.1 mM) for one hour followed by incubation with BDP-ENE-S-Py+ (10 µM) for half an hour; (d1,d2) incubation of the cells with SIN-1 (0.1 mM) for one hour followed by incubation with BDP-ENE-S-Py+ (10 μM) for half an hour; (a3–d3) merged images of the blue channel and red channel. Hoechst 33342 (1 μg/mL); blue channel: λex = 405 nm, λem = 420–500 nm; red channel: λex = 660 nm, λem = 662–738 nm. Scale bar signifies 50 µm.
Figure 4. Intracellular selective imaging of ONOO− in HepG2 cells. (a1,a2) Incubation of the cells with BDP-ENE-S-Py+ (10 μM) for half an hour; (b1,b2) incubation of the cells with H2O2 (0.1 mM) for one hour followed by incubation with BDP-ENE-S-Py+ (10 µM) for half an hour; (c1,c2) incubation of the cells with HClO (0.1 mM) for one hour followed by incubation with BDP-ENE-S-Py+ (10 µM) for half an hour; (d1,d2) incubation of the cells with SIN-1 (0.1 mM) for one hour followed by incubation with BDP-ENE-S-Py+ (10 μM) for half an hour; (a3–d3) merged images of the blue channel and red channel. Hoechst 33342 (1 μg/mL); blue channel: λex = 405 nm, λem = 420–500 nm; red channel: λex = 660 nm, λem = 662–738 nm. Scale bar signifies 50 µm.
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Figure 5. ONOO− imaging in HepG2 cells with different concentrations of SIN-1. (a1–a3) Cells only incubated with BDP-ENE-S-Py+ (10 μM) for half an hour; (b1–b3) incubation of cells with SIN-1 (0.3 mM) for one hour followed by BDP-ENE-S-Py+ (10 μM) for half an hour; (c1–c3) incubation of cells with SIN-1 (0.5 mM) for one hour followed by BDP-ENE-S-Py+ (10 μM) for half an hour; (d) histogram of the fluorescence intensity ratio between the different concentrations of SIN-1 groups and the control group in the HepG2 cells experimental group. Hoechst 33342 (1 μg/mL) for ten minutes; blue channel: λex = 405 nm, λem = 420–500 nm; red channel: λex = 660 nm, λem = 662–738 nm. Scale bar signifies 50 µm.
Figure 5. ONOO− imaging in HepG2 cells with different concentrations of SIN-1. (a1–a3) Cells only incubated with BDP-ENE-S-Py+ (10 μM) for half an hour; (b1–b3) incubation of cells with SIN-1 (0.3 mM) for one hour followed by BDP-ENE-S-Py+ (10 μM) for half an hour; (c1–c3) incubation of cells with SIN-1 (0.5 mM) for one hour followed by BDP-ENE-S-Py+ (10 μM) for half an hour; (d) histogram of the fluorescence intensity ratio between the different concentrations of SIN-1 groups and the control group in the HepG2 cells experimental group. Hoechst 33342 (1 μg/mL) for ten minutes; blue channel: λex = 405 nm, λem = 420–500 nm; red channel: λex = 660 nm, λem = 662–738 nm. Scale bar signifies 50 µm.
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Figure 6. Fluorescence imaging of ONOO− in mouse peritonitis. (a1–a4) Mice were imaged at 0, 10, 30, and 60 min after abdominal injection of the BDP-ENE-S-Py+ probe (10 µM, 100 µL). (b1–b4) The mice were intraperitoneally injected with LPS (100 µg/mL, 200 µL) for incubation for 24 h and then injected with BDP-ENE-S-Py+ (10 μM, 100 µL) in the same location, with imaging performed after incubation at 0, 10, 30, and 60 min. (c) Fluorescence intensity collected from (a1–a4,b1–b4). Images were taken using an excitation laser at 630 nm and an emission filter of 700 nm.
Figure 6. Fluorescence imaging of ONOO− in mouse peritonitis. (a1–a4) Mice were imaged at 0, 10, 30, and 60 min after abdominal injection of the BDP-ENE-S-Py+ probe (10 µM, 100 µL). (b1–b4) The mice were intraperitoneally injected with LPS (100 µg/mL, 200 µL) for incubation for 24 h and then injected with BDP-ENE-S-Py+ (10 μM, 100 µL) in the same location, with imaging performed after incubation at 0, 10, 30, and 60 min. (c) Fluorescence intensity collected from (a1–a4,b1–b4). Images were taken using an excitation laser at 630 nm and an emission filter of 700 nm.
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Qin, S.; Ran, Y.; He, Y.; Lu, X.; Wang, J.; Zhao, W.; Zhang, J. Near-Infrared Fluorescence Probe for Visualizing Fluctuations of Peroxynitrite in Living Cells and Inflammatory Mouse Models. Chemosensors 2023, 11, 316. https://doi.org/10.3390/chemosensors11060316

AMA Style

Qin S, Ran Y, He Y, Lu X, Wang J, Zhao W, Zhang J. Near-Infrared Fluorescence Probe for Visualizing Fluctuations of Peroxynitrite in Living Cells and Inflammatory Mouse Models. Chemosensors. 2023; 11(6):316. https://doi.org/10.3390/chemosensors11060316

Chicago/Turabian Style

Qin, Shuchun, Yiming Ran, Yitian He, Xiaoyan Lu, Jiamin Wang, Weili Zhao, and Jian Zhang. 2023. "Near-Infrared Fluorescence Probe for Visualizing Fluctuations of Peroxynitrite in Living Cells and Inflammatory Mouse Models" Chemosensors 11, no. 6: 316. https://doi.org/10.3390/chemosensors11060316

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