An easily available ratiometric AIE probe for nitroxyl visualization in vitro and in vivo

Chunbin Li a, Guoyu Jiang a, Xiang Liu a, Qingfang Lai a, Miaomiao Kang b, Dong Wang b, Pengfei Zhang c, Jianguo Wang *a and Ben Zhong Tang *bd
aCollege of Chemistry and Chemical Engineering, Inner Mongolia Key Laboratory of Fine Organic Synthesis, Inner Mongolia University, Hohhot 010021, P. R. China. E-mail: wangjg@iccas.ac.cn
bCenter for AIE Research, College of Materials Science and Engineering, Shenzhen University, Shenzhen 518060, P. R. China
cGuangdong Key Laboratory of Nanomedicine, CAS-HK Joint Lab of Biomaterials, Shenzhen Engineering Laboratory of Nanomedicine and Nanoformulations, CAS Key Laboratory of Health Informatics, Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, P. R. China
dDepartment of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, P. R. China. E-mail: tangbenz@ust.hk

Received 28th November 2020 , Accepted 24th December 2020

First published on 28th December 2020


Abstract

Nitroxyl (HNO), as a reactive nitrogen species (RNS), has a pivotal role in many physiological and pathological processes, but the generation mechanism of endogeneous HNO has not been elucidated, because of the lack of highly sensitive and selective probes to achieve real-time visualization and monitoring of HNO in biosystems. Ratiometric fluorescence probes have emerged as powerful platforms for bioimaging applications due to their high sensitivity and anti-interference ability. In this work, we developed firstly a ratiometric fluorescent probe (TCFPB-HNO) with typical aggregation-induced emission (AIE) characteristics for the detection and visualization of HNO with excellent photostability, high specificity and sensitivity. Importantly, TCFPB-HNO was facilely fabricated in high yields via a two-step process, as a serviceable AIE toolbox, which could be employed for real-time monitoring of the HNO level in live samples.


Introduction

Nitroxyl (HNO), the one-electron reduced and protonated entity of nitric oxide (NO),1 plays important roles in a number of biological functions and pharmacological activities,2 and has received considerable interest in recent years. For instance, HNO has been found to inhibit the activities of thiol-containing enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldehyde dehydrogenase, and inducing the release of neurotransmitters, glutamate, and calcitonin-gene related peptides by modulation of calcium channels.3 HNO is implicated in modulating the functions of mammalian vascular systems by activating the TRPA1–CGRP signalling pathway, and repairing the vascular injuries caused by superoxide.4 Additionally, HNO also exhibits fascinating anti-tumor activity.5 Thus, the development of detection tools to selectively visualise biologically active HNO is of great importance for deep understanding of the physiological/pathological functions of HNO in biological systems.

Fluorescent probes6 are powerful tools for in situ monitoring of biologically important species in real time because of their high specificity, sensitivity, temporal and spatial resolutions, non-invasive detection and imaging ability.7,8 Pioneered by the study of Lippard and coworkers,9 a variety of reaction-based HNO fluorescent probes have been explored (Table S1, ESI). Nevertheless, the majority of the currently available probes for HNO detection depend mainly on the changes in fluorescence intensity of a single wavelength,10–12 which may suffer from severe disruption from various external factors, e.g. probe concentration and biotic environment (pH, protein, ions). Ratiometric fluorescent probes13 with built-in calibration for signal changes in two different emission wavelengths, are ideal candidates for HNO imaging in vivo due to high sensitivity and anti-interference ability. Yet so far, very limited ratiometric fluorescent probes for HNO have been reported (Table S1, ESI).14 In addition, a few ratiometric probes displayed poor or totally quenched luminescence properties after their accumulation in cells, which is known as the notorious aggregation-caused quenching (ACQ) effect. What's more, the tedious synthesis and poor photostability further limited their application in biological related systems. In 2001, Tang et al. reported the aggregation-induced emission (AIE) concept and thus opened a new window for anti-ACQ fluorescent probe design.15 Luminogens with AIE features (AIEgens)16 are almost non-emissive or weakly emissive in the single molecular state but emit intensely in the aggregated or solid state due to the restriction of intramolecular motions and prohibition of energy dissipation by way of the non-radiative transition.17 AIEgens have become promising candidates for bio-fluorescent probes18 owing to their intriguing advantages, including high photostability, sensitivity, low background noise and outstanding biocompatibility in living systems.19,20 However, to the best of our knowledge, there has not been any ratiometric AIE fluorescent probe reported for highly sensitive and selective detection of HNO.

Herein, we developed for the first time a ratiometric fluorescent probe (TCFPB-HNO, Scheme 1) with AIE features for HNO detection and real-time fluorescence imaging in vitro and in vivo. TCFPB-HNO was devised to detect HNO based on intramolecular charge transfer (ICT), and contained tricyanofuranyl iminobenzaldehyde as the luminescent platform and a 2-(diphenylphosphino)benzoyl group as the HNO recognition site. We anticipated that TCFPB-HNO possessed weak ICT effects due to the weak electro-donating ability of the ester group, which connected the luminescent moiety and the recognition moiety together. The reaction between the probe and HNO generated the corresponding phosphine oxide and azaylides, and further underwent a Staudinger ligation to give tricyanofuranyl iminosalicylaldehyde (TCFIS) with strong ICT effects. As expected, TCFPB-HNO showed weak emission at 670 nm in phosphate buffered saline (PBS) because of the weak ICT effects. The AIE curves have verified that TCFPB-HNO was AIE-active, and thus is favorable for averting the autofluorescence issue. After incubation with HNO, TCFPB-HNO exhibited typical ratiometric changes (I618/I670) with excellent sensitivity (detection limit of 157.6 nM), specificity and photostability in PBS solution. Importantly, the probe could also be used for HNO detection and selective imaging in vitro and in vivo with a high signal-to-noise ratio. These results have indicated that TCFPB-HNO was a ready-to-use tool for in situ detection of HNO in living systems and will provide a new prospective on the deep understanding of the physiological/pathological functions of HNO.


image file: d0qm00995d-s1.tif
Scheme 1 Recognition mechanism of ratiometric AIE-active fluorescent probe TCFPB-HNO toward HNO.

Results and discussion

Synthesis and optical properties

Initially, TCFIS and TCFPB-HNO were synthesized as shown in Fig. 1A, and their structures were confirmed by 1H NMR, 13C NMR and high resolution mass spectrometry (HRMS) (Fig. S1–S6, ESI). Subsequently, the photophysical properties of TCFPB-HNO (5 μM) were investigated under physiological conditions (PBS solution, pH = 7.4) to verify our initial design concept. As shown in Fig. 1B, the maximum absorption of TCFPB-HNO was located at 556 nm with the emission band covered from 600 to 850 nm with the maximum emission located at 670 nm in PBS solution. Interestingly, TCFPB-HNO demonstrated an excellent photostability unaltered with continuous exposure to exciting light for 60 min (Fig. S7, ESI). The AIE properties of TCFPB-HNO were confirmed by photoluminescence (PL) spectroscopy using toluene/DMSO mixtures with different toluene fraction (fT) (Fig. 1C and Fig. S8, ESI). The PL intensities of TCFPB-HNO were gradually enhanced with increased fT from 0% to 80%, accompanied by a spectral blue-shift attributed to the restricted motions of the C–C double bond and benzene rings in the aggregated state. These results indicated that TCFPB-HNO possesses typical AIE characteristics similar to other AIEgens.21 TCFIS was also verified to have AIE activity by PL spectroscopy in toluene/DMSO mixtures with varying fraction (Fig. S9, ESI).
image file: d0qm00995d-f1.tif
Fig. 1 (A) Synthetic route to probe TCFPB-HNO. (B) Absorption and emission spectra of TCFPB-HNO (5 μM) in PBS solution (pH = 7.4). (C) Plot of PL intensity of TCFPB-HNO at maximum emission wavelength vs. the toluene fraction in the toluene/DMSO mixtures.

To evaluate the responses of TCFPB-HNO (5 μM) toward HNO, the UV-vis and PL spectra of TCFPB-HNO treated with or without Angeli's salt (AS, a donor of HNO) were compared to those of TCFIS and shown in Fig. S10 (ESI). After treatment with 50 μM of AS, the absorption peak of TCFPB-HNO at 556 nm decreased with the increase of a new peak at 600 nm in accordance with that of TCFIS (Fig. S10A, ESI). The results of the PL spectra showed that a new emission peak similar to that of TCFIS at 618 nm appeared, and the maximum emission (670 nm) of TCFPB-HNO decreased upon the addition of AS (Fig. S10B, ESI). On the basis of our design concept displayed in Scheme 1, the HNO-promoted oxidation of TCFPB-HNO could generate phosphine oxide and azaylides, which would finally produce TCFIS through Staudinger ligation. To further verify the reaction mechanism, HRMS analysis was carried out. As displayed in Fig. S11 (ESI), a peak of m/z = 375.18164 similar to TCFIS (m/z = 375.18155) was observed after incubating TCFPB-HNO with HNO, confirming the reaction mechanism of TCFPB-HNO as depicted in Scheme 1.

In order to optimize the reaction conditions, time-dependent (0–50 min) PL intensities of TCFPB-HNO were recorded after incubation with various concentrations of AS (0, 10, 20, 30 μM) (Fig. S12, ESI). The PL intensity ratio (I618/I670) was observed to increase gradually with the reaction time and eventually reached a steady state after 40 min. Therefore, we selected 40 min as the appropriate incubation time for all the following experiments. Fluorescence titrations were implemented to rationalise the sensitivity of TCFPB-HNO towards HNO. As shown in Fig. 2A and B, the PL intensity of TCFPB-HNO at 618 nm increased gradually with the addition of AS, with a nearly 10-fold enhancement of the I618/I670 ratio. Besides, I618/I670 exhibited good linearity with the concentrations of AS ranging from 5 to 30 μM and the detection limit was calculated to be 157.6 nM (Fig. 2C). These results indicated that TCFPB-HNO had great potential for quantitative detection of HNO with high sensitivity.


image file: d0qm00995d-f2.tif
Fig. 2 (A) PL spectra of TCFNPB-HNO (5 μM in PBS solution, pH = 7.4) after incubation with AS (0–50 μM) for 40 min. (B) The I618/I670 ratio of TCFPB-HNO vs. the concentration of AS (0–50 μM). (C) The linear fitting of the I618/I670 ratio vs. the concentration of AS (5–30 μM). Y = 0.0491X + 0.3331, R2 = 0.998. Excitation wavelength (λex = 570 nm). (D) The I618/I670 ratio of TCFPB-HNO toward AS (50 μM) or various other biomolecules (100 μM).

To further evaluate the specificity and stability of TCFPB-HNO towards HNO, the probe was incubated with a modest variety of biologically relevant species. As shown in Fig. 2D and Fig. S13 (ESI), the I618/I670 ratio barely varied for TCFPB-HNO incubated with a series of anions, cations, reactive oxygen species, reactive nitrogen species, and amino acids in biological systems, while the I618/I670 ratio displayed over 10-fold enhancement after incubation with 50 μM of AS, indicating the high selectivity of TCFPB-HNO towards HNO. The influence of pH (4–10) on TCFPB-HNO responsiveness was then studied (Fig. S14, ESI). After treating with AS, a dramatic fluorescence intensity ratio change in HNO responsiveness was observed when the pH is 7.4, indicating the suitability of such systems for detecting HNO in living systems. Moreover, it's worth noting that the I618/I670 ratio changes partially decreased when the pH value exceeded 8, mainly attributed to the incomplete decomposition of AS in the alkaline environment.

Encouraged by the excellent properties, real-time monitoring of HNO in living cells was performed using TCFPB-HNO as the probe. Viabilities of MCF-7 cells were initially evaluated using a CCK-8 assay (24 h incubation) against the probe TCFPB-HNO at various concentrations (0, 1, 5, 15, 30, 50 μM) (Fig. S15, ESI). The unchanged cell viabilities revealed negligible cytotoxicities of TCFPB-HNO, indicating the excellent biocompatibility of the probe for biological applications. Since TCFPB-HNO was highly sensitive to HNO, AS-pretreated MCF-7 cells were incubated with the probe (15 μM) and then observed by a laser scanning confocal microscope. As shown in Fig. 3, a remarkably enhanced emission intensity was observed in the green channel with the increase of AS concentration, while the emission in the red channel displayed a slight decrease compared to the probe without AS preincubation. The merged images have demonstrated that the emission changed significantly in cells from red to green, indicating the excellent permeability and sensitivity of the probe TCFPB-HNO. Moreover, the ratio of Igreen/Ired showed over 10-fold enhancement (Fig. S16, ESI), which was in good agreement with the results in PBS, revealing the potential of TCFPB-HNO for fluorescence imaging of HNO in living systems.


image file: d0qm00995d-f3.tif
Fig. 3 Cell imaging of probe TCFPB-HNO (15 μM for 30 min at 37 °C) with different concentrations of AS (0, 30, 50 and 100 μM for 60 min). (Scale bar: 20 μm.) λex = 543 nm.

To further estimate the application potential of this probe, imaging of HNO in live mice was performed using TCFPB-HNO (subcutaneous injection). As seen in Fig. 4 and Fig. S17–S19 (ESI), slight fluorescence intensity changes (1.54-folds enhancement than injecting probe at 0 min) were detected on mice injected with the TCFPB-HNO probe alone (3.3 mg kg−1) over 60 min using a whole-body small-animal imaging system. In contrast, the fluorescence signals were observed with 10.38-fold enhancement when the mice (pretreated with TCFPB-HNO, 3.3 mg kg−1) were injected with AS solution, further confirming the potential application of TCFPB-HNO in vivo.


image file: d0qm00995d-f4.tif
Fig. 4 Time-dependent fluorescence images of probe TCFPB-HNO in live mice. (A) Control group: TCFPB-HNO (20 μL, 5 mM) was injected in a subcutaneous manner, followed by an injection of PBS (80 μL). (B) Experimental group: TCFPB-HNO (20 μL, 5 mM) was injected, followed by an injection of AS (80 μL, 10 mM). Fluorescence emissions were collected from 580 to 900 nm. λex = 523 nm.

Conclusions

In this work, a ratiometric AIE probe (TCFPB-HNO) was constructed for the first time for fluorescence detection and visualization of HNO in vitro and in vivo. TCFPB-HNO, which was comfirmed to be AIE-active, was easily synthesized using a two-step process. TCFPB-HNO exhibited high sensitivity and excellent selectivity for ratiometric sensing of HNO. Importantly, the probe could be employed for in situ visualization of HNO via a ratiometric response mode with high signal-to-background ratio in vitro and in vivo. This work opened up new avenues to obtain easy-to-handle AIE probes for real-time monitoring of the HNO level in living systems and to enrich the understanding of the physiological and pathological functions of HNO in biological systems.

Experimental section

Materials and methods

Solvents and other common reagents were available from Innochem, Sigma-Aldrich, TCI, and J&K, and were used without any purification unless otherwise specified. Angeli's salt (AS) as a water-soluble HNO donor was purchased from Santa Cruz Biotechnology. NMR spectra were measured on a Bruker ARX 500 MHz instrument and Bruker ARX 400 MHz instrument. A GCT Premier CAB 048 mass spectrometer was used to collect the high-resolution mass spectra (HRMS) with MALDI-TOF mode. UV-vis absorption spectra and fluorescence emission spectra were acquired by a Shimadzu UV-2600i UV-Visible spectrophotometer and an Edinburgh FS5 fluorescence spectrophotometer, respectively. Cellular imaging was captured on a Zeiss confocal laser scanning microscope (LSM880, Germany).

Cell viability

Cell viability was evaluated by CCK-8 assay. 100 μL of MCF-7 cell suspension (5000 cells per well) was seeded in a 96-well plate and then the plate was precultured at 37 °C in a humidified incubator with 5% CO2/air for 24 h. After that, the medium was removed and then fresh medium containing various concentrations of TCFPB-HNO (0, 1, 5, 15, 30 and 50 μM) was added to the plate. After co-culture for 24 h in the incubator, the culture medium was exchanged with fresh medium (100 μL) containing CCK-8 solution (10 μL) and the cells were further cultured for a period of 2 h. The absorption of each well was recorded at 450 nm via an ELISA Plate Reader (Biotek Synergy HT). Each group was carried out in sextuplicate with untreated cells serving as a control.

Cell treatment and cell imaging

For the imaging of HNO, MCF-7 cells were stained with TCFPB-HNO (15 μM) for 30 min under normal culture conditions, and then the medium was removed and the cells were rinsed with PBS solution, followed by the addition of various concentrations of AS (0, 30, 50 and 100 μM) for 60 min. The image was captured using a ZEISS LSM880. Before cell imaging, the cells were washed with PBS solution (three times) to remove background interference. A 543 nm laser was used as the excitation source and the fluorescence signal was acquired from 580 to 640 nm (green channel) and 660–720 nm (red channel).

In vivo animal imaging

All male BALB/c white mice (6 weeks old, weighted 20 g) for the imaging experiment in vivo were provided by Vital River Laboratory Animal Technology Co. Ltd (Beijing, China) and animals all received good care according to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were authorized by Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences Animal Care and Use Committee. The TCFPB-HNO solution (20 μL, 5 mM in DMSO) was injected into white mice by subcutaneous injection, followed by an injection of AS solution (80 μL, 10 mM). The AS solution was replaced by PBS solution (80 μL) as a control group. After that, images were collected from different points in time using the Maestro in vivo imaging system.

Synthesis of TCFIS

A mixture of 4-(diethylamino)salicylaldehyde (compound 1, 231.0 mg, 1.2 mmol), 2-(3-cyano-4,5,5-trimethylfuran-2(5H)-ylidene)malononitrile (compound 2, 199.0 mg, 1.0 mmol) and ammonium acetate (77 mg, 1 mmol) in ethanol was refluxed under a nitrogen atmosphere. The reaction was monitored until compound 2 was fully consumed (determined by TLC), then cooled to room temperature and purified by silica gel chromatography with the mixed solvent of petroleum ether/dichloromethane (1[thin space (1/6-em)]:[thin space (1/6-em)]1–1[thin space (1/6-em)]:[thin space (1/6-em)]5, v/v) to give TCFIS as a purple solid (280.0 mg, 75%). 1H NMR (400 MHz, DMSO-d6) δ 10.86 (br, 1H), 8.23 (s, 1H), 7.70 (d, J = 9.2 Hz, 1H), 6.95 (s, 1H), 6.45 (d, J = 9.6 Hz, 1H), 6.15 (s, 1H), 3.46 (q, J = 7.4, 6.7 Hz, 4H), 1.70 (s, 6H), 1.16 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 177.58, 175.30, 162.35, 154.11, 114.14, 113.26, 112.90, 112.07, 106.82, 97.24, 96.47, 48.71, 44.58, 25.95, 12.69. HRMS (MALDI-TOF): m/z: [M]+ calcd for C22H23N4O2: 375.18155; found: 375.18164.

Synthesis of TCFPB-HNO

A mixture of 2-(diphenylphosphino)benzoic acid (122 mg, 0.4 mmol), N,N′-dicyclohexylcarbodiimide (103 mg, 0.5 mmol), and 4-dimethylaminopyridine (12 mg, 0.1 mmol) in 20 mL CH2Cl2 was stirred at 0 °C under a nitrogen atmosphere for 1 h. TCFIS (112 mg, 0.3 mmol) was added to the mixture and reacted overnight at room temperature. The reaction mixture was purified by silica gel chromatography using a mixed solvent of petroleum ether/dichloromethane (2[thin space (1/6-em)]:[thin space (1/6-em)]1–1[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v) to collect as a blue to purple solid (150 mg, 78%). 1H NMR (500 MHz, chloroform-d) δ 8.38–8.32 (m, 1H), 7.71 (d, J = 16.0 Hz, 1H), 7.65 (d, J = 9.2 Hz, 1H), 7.55–7.50 (m, 2H), 7.36–7.29 (m, 6H), 7.27 (d, J = 2 Hz, 1H), 7.28–7.22 (m, 3H), 7.08–7.02 (m, 1H), 6.73 (d, J = 16 Hz, 1H), 6.59 (dd, J = 9.2, 2.6 Hz, 1H), 6.11 (d, J = 2.6 Hz, 1H), 3.38 (q, J = 7.1 Hz, 4H), 1.58 (s, 6H), 1.18 (t, J = 7.1 Hz, 6H). 13C NMR (125 MHz, chloroform-d) δ 176.22, 174.40, 164.52, 164.50, 153.29, 152.70, 142.13, 141.87, 137.30, 137.22, 134.84, 134.02, 133.86, 133.43, 131.71, 129.95, 128.91, 128.73, 128.66, 128.60, 114.33, 112.69, 111.85, 111.22, 110.50, 109.45, 105.25, 96.80, 94.64, 45.08, 26.71, 26.70, 12.62. HRMS (MALDI-TOF): m/z: [M]+ calcd for C41H36N4O2: 663.2446; found: 663.2520.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank the National Natural Science Foundation of China (21871060), Grassland Talent Program of Inner Mongolia Autonomous Region of China, the Natural Science Foundation of Inner Mongolia Autonomous Region of China [2020JQ02 and 2020MS02004], the Natural Science Foundation of Jiangxi Province (20192BCBL23013) and Science and Technology Plan of Shenzhen (JCYJ20170818113538482) for the financial support.

Notes and references

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/d0qm00995d
These authors contributed equally to this work.

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