A novel fluorescent probe for the detection of peroxynitrite and its application in acute liver injury model.

Realizing the early diagnosis of acute liver injury has become a hotspot in recent years. Since the current indexes are not specific, developing novel probes with new recognition group remains a necessity. In this work, we developed a novel fluorescent probe, NNP, for the detection of ONOO- in acute liver injury (ALI). The in vitro evaluation of NNP indicated the advantages including high sensitivity (LOD = 0.13 μM), rapid response (<25 s), naked-eye (red to yellow), and stability under various pH (4-10) and time (>48 h) conditions. Accordingly, NNP achieved the dose-dependent detection of ONOO- in human hepatic stellate LX-2 cells. Further, in the ALI model mice, NNP could monitor the ONOO- level in the occurrence of ALI in situ (in the liver region) with a steady performance. Subsequent immunohistochemical and imaging studies confirmed that NNP could achieve the detection of endogenous ONOO- in ALI liver tissues. This work introduced a practical implement for the ONOO- detection in ALI as well as a referable example for the establishment of molecular indexes in pre-clinical diagnosis.

Introduction

In all medicinal and therapeutic theories, liver acts as one of the five major organs in human body, and participates deeply in the procedure of metabolism [1,2]. Since the pace of modern life increases, the liver is also facing the threat of damage induced by ethanol, drugs and toxic materials [[3], [4], [5]]. Consequently, acute injury occurred more frequently in liver tissues. As a typical clinical symptom, acute liver injury (ALI) usually exhibited a dramatic failure of hepatocyte function with no previously disease-suffering in liver [6,7]. Actually, with the accurate early assessment, ALI is reversible under rational treatment or even simply getting rid of the injury sources. Therefore the challenging problem is how to realize the early diagnosis of ALI, which has become a hotspot in recent years [8,9].

For ALI, common clinical index of diagnosis includes alanine aminotransferase (ALT) [10], aspartate aminotransferase (AST) [11], alkaline phosphatase (ALP) [12], and γ-glutamyltranspeptidase (GGT) [13]. It is a pity that the blood tests based on the evaluation of these enzymes can be affected by various other diseases, that is, the tests are not specific [14,15]. Moreover, these manifestations are all post-symptom, which cannot fulfill the real-time detection in the physiological and pathological events with constant fluctuations. On the contrary, optical probes are practical implements for the above mentioned requirements with advantages including high sensitivity, high resolution, as well as noninvasive and rapid imaging [16,17]. Accompanied with good permeability and biocompatibility, they can achieve the real-time and in situ monitoring. Specifically for ALI, potential biomarkers which can be detected by optical probes include reactive nitrogen species (eg. ONOO−) [18,19], reactive sulfur species (H2S and H2Sn) [19], enzymes (LAP, MAO, and ALP) [[20], [21], [22]]. Among them, peroxynitrite (ONOO−) seems the most frequently associated one. It is a stronger oxidant than hydrogen peroxide, and consists of a superoxide anion and nitric oxide combination with a short half-life (~10 ms) [23,24]. Although peroxynitrite also inferred correlation with a variety of diseases [25,26], by in situ monitoring, it could be closely related to ALI. Till now, many ONOO− probes have been designed with the recognition moieties including borate ester [[27], [28], [29], [30], [31]], isatin [32], rhodamine [33], xanthene [[34], [35], [36]], diphenylphosphine ester [37,38] and nitrobenzoate [39]. However, an ideal probe for ONOO− should contain the reaction mechanism which is more specific than redox. Accordingly, developing novel probes with new recognition group remains a necessity [40].

In this work, we exploited a novel fluorescent probe, NNP, for the detection of ONOO− with a newly involved sensing group (Fig. 1). After the evaluation of optical properties in vitro, NNP was applied in the ALI mice model. We expect that this novel implement for liver injury detection could excite more inspiration on this topic, and help the medical researches as well as clinical trials.

Fig. 1

The illustration of the probe NNP for the detection of peroxynitrite.

Experimental section

Material and methods

All reagents used for synthesis were obtained from Aladdin without further purification. Column chromatography was performed on silica gel (200–300 mesh). 1H and 13C NMR spectra were recorded in CDCl3, DMSO-d6 at room temperature on Bruker DRX-600 (600 MHz). Mass spectra were obtained at the Mass Spectrometry Facility at Nanjing University. Fluorescence studies were carried out with F-7000 Fluorescence Spectrophotometer. UV spectrum was treated in UV-2550 ultraviolet spectrophotometer. Ordinary section was imaging on microscope (Olympus IX73). Confocal fluorescence images were recorded on TCS SP8 MP two-photon confocal fluorescent microscope (Leica). In vivo imaging was carried out on PE IVIS Lumina XR III. All pH measurements were carried out on pH meter of PHS-25. The flow cytometer model was CytoFlex.

The stock solution of the probe NNP was diluted at 1 mM in DMSO for further testing. Other testing analytes were prepared from the H2O2, NaClO, Cysteine, Glutathione, Homocysteine, CH3COONa, NaCl, Na2CO3, NaNO2, NaNO3, Na2SO3, Na2S, CaCl2, MgCl2, FeSO4, AlCl3, CuCl2 in the doubly distilled water. The reactive oxygen species (ROS) were prepared following the methods of reported literature [41]. Ultrapure water was produced from a Milli-Q reference system (Millipore Elix 5). Slight changes in pH variations of the PBS buffer were achieved by adding the minimum volumes of HCl or NaOH (1 M). All spectroscopic measurements of the probe NNP (10 μM) were performed in pH 7.4, 10 mM phosphate buffer with 1% DMSO and 2 mM CTAB. All the spectroscopic reactions were tested at 37 °C. Unless otherwise noted, for all the measurements, the excitation wavelength was 432 nm, the both excitation and emission slit widths were 10 nm and the voltage was 600 V. The emission spectrum was measured and scanned from 450 nm to 650 nm at 1200 nm/min.

Synthesis of the compounds

6-((4-methoxyphenyl)(methyl)amino)-2-naphthaldehyde (compound 3)

A solution of Cs2CO3 (815 mg, 2.5 mmol), Pd(OAc)2 (18 mg, 0.08 mmol), and BINAP (87 mg, 0.14 mmol) were added in 20 mL dry toluene was stirring at 110 °C for 10 min under N2 protection. Then the compound 1 (235 mg, 1 mmol) and compound 2 (137 mg, 1 mmol) were added immediately. The mixed contain all the ingredients was reacted another 18 h at 110 °C for 10 min under N2 protection. After the reaction was over, the solids in the reaction solution were filtered out upon cooling to room temperature. After removal of the solvent under reduced pressure by evaporation, the crude product was purified by silica column chromatography using PE/EtOAc (v/v 80:1) as eluent, obtaining 195 mg light-yellow compound 3 (Yield 67%). 1H NMR (600 MHz, DMSO) δ 9.98 (s, 1H), 8.31 (s, 1H), 7.85 (d, J = 9.1 Hz, 1H), 7.80–7.68 (m, 2H), 7.27–7.17 (m, 2H), 7.11 (d, J = 2.3 Hz, 1H), 7.06–6.90 (m, 3H), 3.79 (s, 3H), 3.35–3.28 (m, 3H). 13C NMR (151 MHz, DMSO) δ 192.46, 157.54, 150.17, 140.70, 138.60, 135.02, 131.04, 130.73, 128.07, 127.33, 125.94, 123.41, 118.80, 115.62, 107.25, 55.75, 41.02.

(E)-2-(3-(2-(6-((4-methoxyphenyl)(methyl)amino)naphthalen-2-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (compound 5)

A solution of compound 3 (170 mmg, 0.6 mmol) and compound 4 (167 mg, 0.9 mmol, the synthesis scheme referring to the literature of our group [42]) were added in 10 mL dry ethyl alcohol was stirring at 80 °C for 2 min. Then the mixture was dripped into piperidine and reflux for 12 h. After the reaction was over, the solid was separated upon cooling to room temperature to obtain 136 mg dark red compound 5 without further purification (Yield 48%). 1H NMR (600 MHz, DMSO) δ 7.96 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.66 (dd, J = 17.5, 8.6 Hz, 2H), 7.42 (d, J = 14.4 Hz, 2H), 7.19 (d, J = 7.0 Hz, 2H), 7.13–6.84 (m, 5H), 3.78 (s, 3H), 2.60 (d, J = 21.9 Hz, 4H), 1.03 (s, 6H). 13C NMR (151 MHz, DMSO) δ 192.45, 192.42, 157.54, 150.17, 140.69, 138.58, 134.99, 131.02, 130.70, 128.06, 128.03, 127.31, 125.92, 123.38, 118.78, 115.61, 115.57, 115.48, 107.23, 107.19, 56.50, 55.74, 41.02, 18.99.

2.2.3(E)-2-(3-(2-(6-((4-hydroxyphenyl)(methyl)amino)naphthalen-2-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (NNP)

Dissolve the compound 5 (95 mg, 0.21 mmol) into 15 mL dry DCM under ice bath conditions. Then BBr3 (525 mg, 200 μL, 1.05 mmol) was added drop by drop and stirred at 0 °C for 1 h. The mixture was kept reacting at room temperature for 12 h. After quenched by saturated NaHCO3, the solution was extracted by DCM and dried with anhydrous Na2SO4. After removal of the solvent under reduced pressure by evaporation, the crude product was purified by silica column chromatography using PE/EtOAc (v/v 6:1) as eluent, obtaining 66.7 mg black solid (NNP, Yield 70%). 1H NMR (600 MHz, DMSO-d6) δ 9.50 (s, 1H), 7.94 (d, J = 1.7 Hz, 1H), 7.76 (dd, J = 8.7, 1.8 Hz, 1H), 7.63 (dd, J = 17.9, 8.9 Hz, 2H), 7.45–7.33 (m, 2H), 7.07 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 2.5 Hz, 1H), 6.93 (dd, J = 9.0, 2.5 Hz, 1H), 6.87–6.81 (m, 3H), 3.29 (s, 3H), 2.59 (s, 2H), 2.55 (s, 2H), 1.02 (s, 6H). 13C NMR (151 MHz, DMSO) δ 169.53, 155.81, 154.52, 147.82, 138.62, 137.93, 134.82, 129.22, 128.51, 128.39, 127.09, 126.88, 126.09, 125.84, 123.85, 121.27, 117.62, 115.72, 113.53, 112.70, 106.37, 74.40, 41.65, 31.05, 26.84.

Determination of the fluorescence quantum yield

Fluorescence quantum yield (FQY) was determined using aqueous solution of Rhodamine B as a standard (Φ = 0.69, 10 μM in ethyl alcohol, λex = 365 nm). By measuring absorption and fluorescence spectra of NNP according to equation, calculated and finally get the quantum yield. The quantum yield Ф as a function solvent polarity is calculated using equation as follows:ΦWhere Φs is the quantum yield, Fs and Fu is the areas' integral values of the corrected fluorescence spectra of the reference and testing solution, As and Au stand for the absorbance of the reference and testing solution, nu and ns are the solvent refractive indexes of sample and reference, respectively.Quantum yield: Φ = 0.27.

The limit of detection (LOD) of NNP

The emission spectrum of free NNP in PBS buffer (10 mM, pH = 7.40, CTAB 2 mM, containing 1% DMSO) was collected for 30 times to confirm the background noise σ. The linear regression curve was then fitted according to the data in the range of ONOO− from 0 to 10 μM and obtained the slope of the curve. The detection limit (3σ/slope) was then determined to be 0.13 μM, which facilitate the quantitative detection of ONOO− in complex living body environment.

Cell culture, flow cytometry assays and imaging

LX-2 cells were cultured in Dulbecco's Modified Eagle Medium media (DMEM, Gibco) supplemented with 10% special grade fetal bovine serum (FBS, Gibco) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Vicente) at 37 °C and 5% CO2. The LX-2 cells were seeded in the 6-well plates at the density of 1 × 105 cells per well. After adherent, the cells were cultured for 0.5 h with NNP (10 μM). After washing three times with DPBS, the cells were digested with trypsin (without EDTA), beaten evenly with 500 mL DPBS and added into the flow tube. Then added different concentrations of ONOO− (0–50 μM) for 0.5 h. The procedures were treated in accordance with the manufacturer's protocol, and the data were processed using Flow Jo software.

Before the imaging experiments, 1 mL of LX-2 cells were subcultured and seeded in the glass bottom culture dishes (Corning) at a density of 1 × 105 cells per well. After adherent, the cells were cultured for 0.5 h with NNP (10 μM). After washing with PBS buffer to wipe out the residual probe in the surface, the LX-2 cell were further incubated with different concentrations of ONOO− (0–50 μM) for 0.5 h at 37 °C. The cells were imaged respectively by two-photon confocal microscope. For monitoring the endogenous ONOO−, the cells were pretreated with or without NAC (1 × 10−3 M, 12 h), FeTMpyp (100 × 10−6 M, 0.5 h), SIN-1 (200 × 10−6 M, 0.5 h), LPS (0.1 μg mL−1, 12 h), PMA (100 × 10−6 M, 1 h) and the cells were pretreated with 1 mM CCl4 for 4 h at 37 °C, then incubated with NNP (10 μM) for another 0.5 h and imaged after washing with PBS three times. The cell imaging detected by a Lecia Two-photon fluorescence microscope equipped with 40× objective lens. The excited wavelength was set at 405 nm, and emission wavelengths were collected from 530 nm to 600 nm.

Cytotoxicity assay

Cell cytotoxicity was assessed with a standard CCK-8 method. LX-2 cells were seeded into 96-well plates, the diluted cell suspension (5 × 103 per well) 100 μL was added to each well of 96-well culture plates. After adherent overnight, several concentrations of NNP (0, 5, 10, 15, 20, 25, 30, 35, 40 μM) were added. After 24 h exposure period, the culture solution was replaced with 10 μL of CCK-8 reagent for each well, and the cell samples were further incubated for 1 h. The mixture solution was measured with an ELISA reader (ELx800, BioTek, USA) at 450 nm. In all experiments, 10 replication wells were used for each probe concentration. The experiments were repeated six times.

Establishment of animal models

BAIB/c Nude mice were purchased from Institute of Zoology, Nanjing University. All animal care and experimental protocols complied with the Animal Management Rules of the Ministry of Health of the People's Republic of China and were approved by the Institute of Process Engineering, Chinese Academy of Sciences. The mice were divided into two groups. One group of mice used it directly without any other treatment as wide-type mice, and one group created model mice.

For CCl4-induced ALI model mice, a mixture of CCl4 (10 mL/kg bodyweight) and olive oil (1:9 (w/v)) was used to induce acute liver injury. Model group were i.p. injected with CCl4 for 24 h. At the end of the experiment, mice were sacrificed after anesthetization with Isoflurane gas anesthesia. The abdominal cavity was then opened to separate the liver, and a camera was used to take pictures of the complete liver of the mouse. A small portion of the liver was removed and immobilized in 10% formalin solution for histopathological, immunohistochemical and imaging studies.

In acetaminophen (APAP)-induced hepatotoxicity model, the prepared APAP (20 mg/mL in normal saline (NS)) solution was used to induce hepatotoxicity model. Model group were i.p. injected with APAP solution (dosage of administration was 200 mg/kg, 400 mg/kg) for 1 h. The model of alcohol-induced liver injury was established by gavage of 50% alcohol (150 μL). At the end of the experiment, mice were sacrificed after anesthetization with Isoflurane gas anesthesia. The abdominal cavity was then opened to separate the liver, and immobilized in 10% formalin solution for imaging studies.

Detection of serum liver injury indexes

Both the control group and model group mice were selected, rapid extraction of blood from orbit with a capillary of 0.05 mm inner diameter. The collected serum samples were left standing at room temperature for 2 h, centrifuged at 3500 r/min for 15 min and collecting supernatant. The supernatant was re-centrifuged for 10 min at 3500 r/min.

Serum liver injury indexes were detected using the ALT, AKP, LDH, TBIL Detection Kit (YI FEI XUE BIO TECH). In addition, liver injury indexes were further confirmed by the Mice IV-C ELISA kit, Mice HYP ELISA kit (YI FEI XUE BIO TECH). All experiments were measured with an ELISA reader (ELx800, BioTek, USA), and set the parameters according to the kit instructions. The procedures were treated in accordance with the manufacturer's protocol.

In vivo imaging of mice

The mice were intraperitoneally injected with NNP 100 μM in saline (10 mL/kg) and imaging with small animal in vivo imaging system. After, the mice were intraperitoneally injected with ONOO− 400 μM (500 μL in saline). The fluorescent images of each group were measured at the different time interval within 5 min. The excited wavelength was set at 440 nm.

For all the CCl4-induced model mice, 10% CCl4 (in olive oil, 10 mL/kg) was intraperitoneally injected for 24 h. The wild type and model mice were intraperitoneally injected with NNP 100 μM in saline (10 mL/kg), respectively. Fluorescent images of mice were captured at different time interval within 1 h. The excited wavelength was set at 440 nm.

In acetaminophen (APAP)-induced hepatotoxicity model, Balb/c nude mice were administrated (i.p.) with 0 mg/kg, 200 mg/kg, 400 mg/kg APAP for 1.0 h. Babl/c nude mice were first administrated (i.p.) with 400 mg/kg NAC for 1.0 h, and then 400 mg/kg APAP (i.p.) for another 1.0 h. Balb/c nude mice were treated with 150 μL 50% EtOH solution by oral gavage and pretreated with or without 200 mg/kg APAP by intraperitoneal injection. In vivo mapping of the liver to model mice. Fluorescent images of mice were captured at different time interval within 3 h. The excited wavelength was set at 440 nm.

Pathological examination of liver tissue

The fixed liver tissue (contain wide-type group and model group) will be entrusted to the company (Wuhan Seville Biological Co., Ltd.) to cut into blank paraffin tissue sections of 4 μm for histopathological analysis Thin sections (4 μm) were stained with Siriu Red, and Masson for histopathological study. Sirius Red and Masson-stained areas from 6 random fields were monitored by microscopy (Olympus IX73).

After two groups of blank slices were treated by de-waxing, and staining with probe NNP for further divided by fluorescence intensity. The pre-treated slices were washed with PBS buffer for 3 times, and 5 min each time. Then the liver biopsy were stained with NNP (10 μM) for 30 min, and washed with PBS buffer for three times. After treated with DAPI for another 30 min. After sealing, images detected by a Lecia Two-photon fluorescence microscope equipped with 20× objective lens. The excited wavelength was set at 405 nm, and emission wavelengths were collected from 530 nm to 600 nm.

Results and discussion

Synthesis of the probe NNP

The synthesis approach of the probe NNP was illustrated in Fig. 2. The structure was confirmed by satisfactory spectroscopic data including 1H NMR, 13C NMR and HRMS (Figs. S1–S9 in Supplement Materials).

Fig. 2

The synthesis approach of the probe NNP.

Optical properties of the probe NNP

Initially, the absorbance spectra of the probe NNP with and without ONOO− were scanned (Fig. 3a). The probe NNP itself showed a peak at 505 nm, whereas the mixture of NPP and ONOO− indicated two obvious peaks at 299 nm and 459 nm. This result inferred the significant response of NNP towards ONOO−. Accordingly, under the excitation at 432 nm, the fluorescence spectrum of the probe NNP suggested no obvious peaks, whereas the mixture of NPP and ONOO− exhibited a remarkable fluorescence signal at 560 nm, which was a typical “turn-on” probe (Fig. 3b). Therefore the parameters were basically set (λex = 432 nm, slit width: dex = dem = 10 nm, voltage = 600 V, 37 °C). The fluorescence quantum yield (FQY) of NNP was determined as 0.27 via the reference method. To check the working conditions, we subsequently involved the factors including pH and incubation time. As shown in Fig. S10, NNP itself kept stable in the pH range of 4–11, whereas the detecting system indicated steady fluorescence intensity in the range of 4–10. Such wide pH ranges ensured the application of the probe. Meanwhile, in Fig. 3c, we could see that the response completed within 25 s, accompanied by the color change from red to yellow. That was to say, NNP inferred a rapid response towards ONOO−, which was beneficial for detecting such a transient target. As for the stability, both NNP itself and the detection system could retain unchanged after 48 h (Fig. S11).

Fig. 3

(a) Absorption and (b) fluorescence of NNP (10 μM) in the absence and presence of ONOO− (100 μM) in PBS buffer. (c) Time-scan of NNP upon addition with ONOO− (100 μM) in PBS buffer at 37 °C. Inset: The visual colors of probe NNP (50 μM) in the absence and presence of ONOO- (500 μM). (d) Fluorescence intensity of NNP at 560 nm upon addition with ONOO− (100 μM) in PBS buffer. (e) The linear ship between the fluorescence intensity of NNP at 560 nm and the concentration of ONOO−. (f) Fluorescence intensity of NNP at 560 nm upon addition with varies analytes (100 μM) in PBS buffer. Numbers 1–24 represent: 1. ·OH; 2. 1O2; 3. NO; 4. O2−;5. ClO; 6. H2O2; 7. Ac−; 8. Cl−; 9. CO32−; 10. NO2−; 11. NO3−; 12. SO32−; 13. S2−; 14. Ca2+; 15. K+; 16. Na+; 17. Mg2+; 18. Fe2+; 19. Al3+; 20. Cu2+; 21.GSH; 22. Cys; 23. Hcy; 24. ONOO−. All spectra were monitored 5 min after mixing in PBS buffer (10 mM, pH 7.4, with 1% DMSO, CTAB 2 mM) at 37 °C. λex = 432 nm, slit width: dex = dem = 10 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

In Fig. 3d, when the concentration of ONOO− increased from 0 to 100 μM (10 equiv.), the fluorescence of the detection system indicated an enhancement accordingly. The plateau could be reached at about 50 μM (5 equiv.) (Fig. S12). According to the linear correlation (Fig. 3e), NNP could monitor ONOO− linearly in the range of 0–10 μM (0–1 equiv.). Further, the Limit of Detection (LOD) was calculated as 0.13 μM. The results above suggested that NNP could realize the quantitative detection of ONOO− with high sensitivity.

One step further, the selectivity of NNP for ONOO− from other analytes was evaluated. As shown in Fig. 3f, only ONOO− could cause the obvious fluorescence enhancement. Other analytes, no matter common ions, anions or ROS, caused no notable variation in fluorescence intensity. In the coexistence systems containing ONOO− and other analytes, the fluorescence response to ONOO− seemed not interfered, except for the reductive species such as SO32−, S2− and so on (Fig. S13). Anyway, in real cases, the selectivity of NNP could be maintained basically.

Intracellular imaging

Since the research object was ALI, in the intracellular experiments, we used LX-2 cells (Human hepatic stellate cell line). Before the application in cellular imaging, the cell cytotoxicity was assessed with a standard CCK-8 method (Fig. S14). After the treatment with 40 μM of NNP for 24 h, the survival rate of LX-2 cells was still over 80%. Subsequently, we attempt to check the correlation between the fluorescence signals and the concentration of ONOO− in living cells. In Fig. 4a&b, the NNP concentration was set as 10 μM while the incubation time was set as 30 min for insurance. The fluorescence signals showed a dose-dependent enhancement with various concentrations of ONOO− (0–50 μM). Commonly, Chlorine-related metabolites were nonnegligible interferences in detecting ONOO−. In Fig. S15, we checked the response of our probe towards ClO− and ClO4−. The results showed that NNP could exclude the interferences from these metabolites. Moreover, in the flow cytometry analysis, the result agreed with that on confocal imaging (Fig. 4c&f). As one notable point, the intracellular microenvironment in living cells was quite different from the PBS buffer, thus the incubation process should be evaluated for a second time. Besides, in Fig. S16, the buffer containing 1% DMSO and 0.1% DMSO were compared, these two buffer systems did not show obvious difference in fluorescence intensity during the imaging of exogenous ONOO− in LX-2 cells. In Fig. S17, when incubated with ONOO− (50 μM), the fluorescence of NNP (10 μM) exhibited gradually stronger fluorescence intensity along with the increase of incubation time from 0 to 30 min. Although the incubation time was prolonged, the detection of ONOO− was relatively stable, which inferred that NNP could work well in cellular microenvironment when the incubation time was set as 30 min. Therefore in the in vivo experiment, we consulted this result as a referable parameter. Further, in Fig. 4d&e, in the LX-2 cells treated with NAC(N-acetylcysteine, the ONOO− scavenger), FeTMPyP (an ONOO− deconstruction reagent), SIN-1 (a potent vasorelaxant and inhibitor of platelet aggregation, also an exogenous ONOO− donor) and PMA (phorbol-12-myristate-13-acetate, an ONOO− activator), the fluorescence intensity varied correspondingly. Accordingly, the regulated ONOO− level could be reflected by the fluorescence intensity by using our probe NNP.

Fig. 4

Microscopy images of exogenous ONOO− in LX-2 cells. (a) The cells were stained with 10 μM NNP for 30 min and washed with DPBS for three times, and then pretreated with different concentration of ONOO− (0–50 μM) for 30 min. (b) Quantification of imaging data (a), n = 3, error bars were ±SD. (c) Flow cytometry assays of NNP activation by ONOO− (LX-2) cancer cells. Assays were performed by counting 10,000 cells that had been exposed to 10 μM probe for 30 min. (d) LX-2 cells were pretreated with or without NAC (1 × 10−3 M, 12 h), FeTMpyp (100 × 10−6 M, 0.5 h), SIN-1 (200 × 10−6 M, 0.5 h), LPS (0.1 μg mL−1, 12 h), PMA (100 × 10−6 M, 1 h). Imaging after incubation with NNP (10 μM) for another 30 min. NAC(N-acetylcysteine) is the ONOO− scavenger, FeTMPyP is an ONOO− deconstruction reagent, SIN-1 is a potent vasorelaxant and inhibitor of platelet aggregation, also is an exogenous ONOO− donor, PMA (phorbol-12-myristate-13-acetate) is an ONOO− activator. (e) Quantification of imaging data (d), n = 3, error bars were ±SD. The images were obtained by collecting emissions at 530–600 nm nm upon excitation at 405 nm. Scale bar: 25 μm. (f) Quantification of flow cytometry data at median. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.

In vivo application in mice acute liver injury model

Upon the results in Human hepatic stellate cell line, we further constructed the ALI model in BALB/c mice to evaluate the performances of the probe NNP. Two independent models were checked. The acute liver injury in this work was initially induced by CCl4/olive oil via intraperitoneal injection. Then the in vivo fluorescence imaging of BALB/c mice was performed and the results were exhibited in Fig. 5. As shown in Fig. 5a and b, in normal mice, the pre-injected probe NNP (100 μM in saline) could response to the injected ONOO− (500 μM in saline) in a time-dependent manner. As the incubation time prolonged, the fluorescence signals enhanced and reached the plateau within 15 min. Meanwhile, in the control group with wild-type mice, no obvious fluorescence signal could be observed in 2 h, whereas in the ALI group, a dramatic fluorescence enhancement could be observed, in both the pictures taken in 1 h and 2 h (Fig. 5a&c). A majority of the signals were around the liver region of the ALI mice.

Fig. 5

(a) In vivo fluorescence imaging of BALB/c mice. The mice were intraperitoneal injected with 200 μL of probe (100 μM in saline) and then injected 400 μL ONOO− solution (500 μM in saline) was the time-course imaging at 0, 5 10, 15 min. In vivo mapping of ONOO− deposition in ALI model mice. Comparison of the fluorescent image at 60 min after intravenous injection of 100 μM of NNP into wild-type mice and ALI mice. (b) Quantification of imaging data from (a). (c) Quantification of imaging data from (a). (d) In vivo mapping of APAP-induced liver injury model mice. Balb/c nude mice were administrated (i.p.) with 0 mg/kg, 200 mg/kg, 400 mg/kg APAP for 1.0 h. Babl/c nude mice were first administrated (i.p.) with 400 mg/kg NAC for 1.0 h, and then 400 mg/kg APAP (i.p.) for another 1.0 h. Balb/c nude mice were treated with 150 μL 50% EtOH solution by oral gavage and pretreated with or without 200 mg/kg APAP by intraperitoneal injection. In vivo mapping of the liver to model mice after intravenous injection of 100 μM of NNP into mice. (e) Quantification of imaging data from image of model mice. (f) Quantification of imaging data from image of liver. n = 3, error bars were ±SD. Statistical analysis was performed with a two-tailed Student's t-test with unequal variance, *p value < 0.05, **p value < 0.001, ***p value < 0.001.

To avoid the interference of elemental chlorine and hypochlorous acid, we built the acetaminophen (APAP)-induced hepatotoxicity model. In Fig. S18, the fluorescence signal in normal mice showed the peak value at 15 min and was then eliminated gradually. In Fig. 5 d-f, the fluorescence intensity of the mice and the liver were measured. As a result, the probe NNP could reflect the dose-dependent accumulation of ONOO− by APAP and the elimination of ONOO− by NAC. Notably, 50% EtOH could induce the generation of ONOO−, and this kind of injury could also be detected by our probe. The above results indicated that NNP could monitor the ONOO− level in the occurrence of ALI in situ with a steady performance.

The following experiments including histopathological, immunohistochemical and imaging studies were conducted by using a small portion of the liver tissues in the above imaging procedure. Initially, the serum liver injury indexes were tested by using corresponding ELISA kits (Fig. S15). The tested indexes included ALT (Alanine aminotransferase), AKP (Alkaline phosphatase), LDH (Lactate dehydrogenase), TBIL (Total bilirubin), IV-C (Collage Type IV) and HYP (Hydroxyproline). All the tested indexes were much higher in the ALI model liver than in the control, which inferred that the model group was suffering ALI in diagnostics. Afterwards, the immunohistological analysis of the liver tissues were performed by using Masson and Sirius red staining (Fig. 6a–c). In appearance, the normal liver tissue (Fig. 6a1) was in dark red with luster, whereas the ALI model liver tissue (Fig. 6a2) was pale. After Masson staining, the normal group was in bright rose red (Fig. 6b1), whereas the ALI group exhibited the blue patches due to the progress of fibrosis (Fig. 6b2). Meanwhile, after Sirius red staining, the normal group indicated yellowish red color (Fig. 6c1), whereas the ALI group with fibrosis showed faint yellow color (Fig. 6c2). Besides, we used CCl4 to induce the ALI event in LX-2 cells, and the endogenous ONOO− was then detected by the probe NNP (10 μM). In the control group, the green fluorescence was weak (Fig. 6d1), which indicated the low endogenous ONOO− level in living LX-2 cells. Remarkably, in the induced ALI group, the green fluorescence was much stronger (Fig. 6d2), which inferred the high endogenous ONOO− level due to ALI. The quantification analysis from the imaging results also convinced the significant increase of endogenous ONOO− level (Fig. 6e). Furthermore, the liver biopsy was conducted under fluorescent microscopy. The general position of the nucleus was labeled by DAPI (Fig. 6f1, f2). In the green channel, it was easy to conclude that low ONOO− concentration in control (Fig. 6g1) and high ONOO− concentration in the ALI liver tissue (Fig. 6g2) could be significantly distinguished by NNP through the comparison the fluorescence intensity. The merged pictures (Fig. 6h1, h2), the zoomed images (Fig. 6i1, i2) and the quantification analysis (Fig. 6d&e) all confirmed the capability of NNP for the detection of endogenous ONOO− in ALI liver tissues. The similar analysis was conducted on the liver tissues of the APAP-induced ALI model (Fig. 6c&f). Both the visual pictures and the quantification of imaging data agreed with the results of the in vivo imaging above.

Fig. 6

(a) Livers, normal liver, ALI model liver. Immunohistological analysis with Masson and Sirius red, normal liver, ALI model liver. Scale bars: 50 μm. Microscopy images of endogenous ONOO− in LX-2 cells. The cells were treated with PBS and 1 mM CCl4 for 4 h. Then the cells were stained with 10 μM NNP for 30 min. Scale bar: 25 μm. (b) Microscopy images of liver biopsy, normal liver biopsy, ALI model liver biopsy. The liver biopsy were stained with NNP (10 μM) for 30 min, and then treated with DAPI for another 30 min. (c) Microscopy images of liver biopsy, the liver of Balb/c nude mice were administrated (i.p.) with 0 mg/kg, 200 mg/kg, 400 mg/kg APAP for 1.0 h. Babl/c nude mice were first administrated (i.p.) with 400 mg/kg NAC for 1.0 h, and then 400 mg/kg APAP (i.p.) for another 1.0 h. The liver of Balb/c nude mice were treated with 150 μL 50% EtOH solution by oral gavage and pretreated with or without 200 mg/kg APAP by intraperitoneal injection. The liver biopsy were stained with NNP (10 μM) for 30 min, and then treated with DAPI for another 30 min. (d) Quantification of imaging data (a). n = 3, error bars were ±SD. Statistical analysis was performed with a two-tailed Student's t-test with unequal variance, ***p value < 0.001. The images were obtained by collecting emissions at 530–600 nm upon excitation at 405 nm. Scale bar: 25 μm. (e) Quantification of imaging data (b). (f) Quantification of imaging data (c). n = 3, error bars were ±SD. Statistical analysis was performed with a two-tailed Student's t-test with unequal variance, *p value < 0.05, ***p value < 0.001. The images were obtained by collecting emissions at 530–600 nm upon excitation at 405 nm. Scale bar: 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Conclusion

To sum up, we developed a novel fluorescent probe, NNP, for the detection of ONOO− in acute liver injury. Under the excitation at 432 nm, NNP could response to ONOO− with a remarkable fluorescence enhancement at 560 nm. The fluorescence quantum yield of NNP was determined as 0.27. The in vitro evaluation of NNP indicated the advantages including high sensitivity (LOD = 0.13 μM), rapid response (<25 s), naked-eye (red to yellow), and stability under various pH (4–10) and time (>48 h) conditions. In Human hepatic stellate LX-2 cells, NNP could realize the dose-dependent detection of ONOO−, while the cytotoxicity was relatively low. In the ALI model mice, NNP could monitor the ONOO− level in the occurrence of ALI in situ with a steady performance. A majority of the signals were around the liver region of the ALI mice, and the fluorescence intensity generated in 1 h did not show obvious decay in 2 h. Further immunohistochemical and imaging studies confirmed the diagnostic characteristics of the constructed ALI model as well as the imaging capability of NNP in the liver tissues. As a result, NNP could achieve the detection of endogenous ONOO− in ALI liver tissues. More than introducing a practical implement for the ONOO− detection in ALI, this work provided a referable example for the establishment of molecular indexes in pre-clinical diagnosis.

Declaration of competing interest

There is no conflict of interest to declare.