An easily available ratiometric AIE probe for peroxynitrite in vitro and in vivo imaging

https://doi.org/10.1016/j.snb.2020.129223Get rights and content

Highlights

  • The first ratiometric fluorescent probe (BTCV-PN) with AIE feature for ONOO was reported.

  • It displayed superior photostability, remarkable sensitivity and outstanding specificity toward ONOO.

  • Furthermore, probe BTCV-PN was adopted for real-time imaging of ONOO in live HeLa cells.

  • Notably, real-time visualization of ONOO in live mice was also accomplished by using BTCV-PN.

Abstract

Peroxynitrite (ONOO), a potent oxidative and nitrating reagent that can damage a variety of molecules in cells is reported to be closely related to various diseases. To determine the physiological or pathophysiological concentrations of ONOO and to deeply understand its pathophysiological processes strongly rely on the development of more powerful detection methods. Hereof, we fabricated the first ratiometric fluorescent probe by using aggregation-induced emission luminogens (AIEgens) with superior photostability for efficient and reliable visualization of ONOO both in vitro and in vivo. Probe BTCV-PN, obtained via simple two-step process, displayed superior photostability, remarkable sensitivity and outstanding specificity toward ONOO through a ratiometric response mode. Furthermore, BTCV-PN was successfully applied for imaging of exogenous and endogenous ONOO in live cells. Notably, in vivo imaging of ONOO in live mice was also accomplished. The excellent performances plus the superior parameters of BTCV-PN including reliable ratiometric response and superior stability against photobleaching would not only make BTCV-PN a powerful tool-box for facilely investigating the cellular concentrations of ONOO, but also open up an updated way for straightforward visualization of the pathophysiological process of ONOO in live cells and animals. Elsevier B.V. All rights reserved.

Introduction

Peroxynitrite (ONOO) is a potent oxidative and nitrating reagent generated via the coupling of nitric oxide (Nradical dotO) and superoxide (O2radical dot--) radicals, and can damage a wide array of molecules in cells, including DNA, thiols, phospholipids and proteins [[1], [2], [3], [4]]. It is reported to be a significant contributor to several cardiovascular, inflammatory and neurodegenerative diseases [[4], [5], [6], [7]]. Thus the sensitive and reliable detection and estimation of the concentration of ONOO is very meaningful for the diagnosis and prevention of ONOO-related diseases. However, it is extremely difficult to measure or evaluate the physiological or pathophysiological contents of ONOO due to the various sources, complex reactions, short-lived (~10 ms) feature and the shortage of efficient and reliable approaches to estimate it in vivo [4]. Thereafter, the exploitation of reliable strategies for real-time and in situ visualization of ONOO is of high demand yet very challenging for deeply comprehending the physiological and pathophysiological roles of ONOO.

The facility, sensitivity, costless and outstanding non-invassive imaging capability of fluorescent probes make them come into focus recently [[8], [9], [10], [11], [12], [13], [14]]. Although various fluorescent probes based on rhodamine, dicyanomethelene, hemicyanine, naphthalimide derivatives have been developed [[15], [16], [17], [18], [19], [20], [21], [22], [23], [24]], the aggregation-caused quenching (ACQ) phenomena and the severe photobleaching impeded the practical applications for long-term tracking of ONOO in biological systems. To circumvent the ACQ phenomena, AIEgens are emerging as the ideal candidates for efficient and reliable bioimaging of various biologically relevant species [[25], [26], [27], [28], [29], [30], [31], [32]]. AIEgens exhibit no emission or emit faintly when dissolved, whereas luminescent strongly in the aggregated or the solid state. AIEgens have commonly good photo-stability and large Stokes shift in aggregate or solid state, which can efficiently avoid photobleaching problem and the effect of excitation light. Thanks to these unique features, AIEgens have shown great promises not only in the visualization of physiological process, but also in disease diagnosis and therapy in clinic [[33], [34], [35], [36], [37], [38], [39], [40]]. Unfortunately, only three fluorescent probes for ONOO based on AIEgens are reported [[41], [42], [43]] (Table S1). What’s worse, these probes rely on single emission wavelength to boost OFF-ON responses, which is susceptible to probe concentration, temperature, pH and polarity. In contrary, ratiometric probes which use two independent emission wavelengths to give ratiometric responses exhibit self-calibration ability and thus are superior in sensitivity and accuracy [[44], [45], [46], [47]]. However, to the best of our knowledge, ratiometric fluorescent probe for ONOO with AIE feature is scarcely ever recorded so far. Thus, developing novel AIE-active ratiometric fluorescent probe with high photostability for real-time and in situ visualization of ONOO is of high demand.

Hereof, with these aspects in mind, we fabricated the first AIE-active ratiometric fluorescent probe (BTCV-PN) with high photostability for direct visualization of ONOO in vitro and in vivo. As displayed in Scheme 1, BTCV-PN was fabricated with benzothiazolyl derivative as the AIE-active luminogen and diphenylphosphinate group as the reaction site of ONOO. BTCV-PN is expected to emit faintly in good solvent such as DMSO due to the fast Cdouble bondC cis-trans isomerization. The increase of water fraction will promote BTCV-PN to form aggregates, restrict the Cdouble bondC isomerization, and subsequentially light up its emission. The reaction with ONOO will cleave the diphenylphosphinate group, result in phenate intermediate, and finally give benzithiazolyl iminocoumarin (BTIC) through fast cyclization. As a result, Cdouble bondC isomerization was further restricted and the strong intramolecular donor-acceptor (Dsingle bondA) interactions in BTCV-PN was altered to weak D-A interactions in BTIC, making the emission blue shifted. By this means, a ratiometric response mode can be realized. The AIE-active probe BTCV-PN exhibited superior photostability and excellent sensitivity (detection limit: 30 nM) and selectivity toward ONOO. It was successfully applied in fluorescent imaging of both exogenous and endogenous ONOO in live cells. Additionally, direct observation of ONOO in live mice also demonstrated its great usefulness for efficient and reliable visualization of ONOO in live animals. This is the first ratiometric fluorescent probes based on AIEgens with high photostability for sensitive and reliable detection of ONOO, which will certainly shed new light not only on the deep understanding of the pathophysiological effect of ONOO, but also for the clinical application in precise theranostics.

Section snippets

Experimental

Synthesis of compound 1. 4-(Diethylamino)salicylaldehyde (231.9 mg, 1.2 mmol) and K2CO3 (109.2 mg, 1.3 mmol) was mixed in 10 mL CH3CN, followed by adding diphenylphosphinic chloride (236.6 mg, 1.0 mmol) dropwise. The mixture was refluxed under N2 atmosphere and monitored by TLC. When diphenylphosphinic chloride disappeared, filtration was conducted to get rid of any solids. Solution as obtained was concentrated, and subjected to silica gel chromatography with PE/EA (4:1~1:1, v/v) to give

Results and discussion

Probe BTCV-PN was easily obtained via a facile two-step synthetic route [47]. As displayed in Scheme S1 in supplementary materials, compound 1 was prepared as a white solid by esterification reaction between 4-(diethylamino)salicylaldehyde and diphenylphosphinic chloride (yield: 81 %). Compound 1 was further reacted with benzothiazole-2-yl-acetonitrile through knoevenagel reaction to give BTCV-PN with a yield of 85 %. According to previous report [47], the knoevenagel reaction between

Conclusion

An AIEgen-based ratiometric fluorescent probe for efficient and reliable visualization of ONOO in live cells and live mice was facilely synthesized for the first time. Probe BTCV-PN exhibited superior stability against photobleaching, outstanding sensitivity and remarkable specificity for ONOO in PBS. These remarkable parameters ensured the successful use of probe BTCV-PN in real-time and in situ visualization of ONOO in live cells. Additionally, imaging of ONOO in live mice also

CRediT authorship contribution statement

Guoyu Jiang: Investigation, Conceptualization, Data curation, Project administration, Writing - original draft. Chunbin Li: Data curation, Conceptualization, Methodology. Qingfang Lai: Data curation, Methodology. Xiang Liu: Data curation, Methodology. Qingqing Chen: Methodology. Pengfei Zhang: Investigation, Supervision. Jianguo Wang: Conceptualization, Validation, Resources, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration, Funding

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgments

We acknowledge the National Natural Science Foundation of China [21663005 and 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] and the Natural Science Foundation of Jiangxi Province [2018ACB21009 and 20192BCBL23013].

Guoyu jiang was born in 1984. She earned her bachelor’s degree at Beijing Normal University and her PhD at Prof. Xuesong Wang’s group in Technical Institute of Physics and Chemistry, Chinese Academy of Sciences (CAS). She is now an associate professor at Inner Mongolia University. Her research interest is the design of novel AIE based fluorescent sensors for the detection of chemical or biological important species. She is also interested in the design and synthesis of novel photosensitizers

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    Guoyu jiang was born in 1984. She earned her bachelor’s degree at Beijing Normal University and her PhD at Prof. Xuesong Wang’s group in Technical Institute of Physics and Chemistry, Chinese Academy of Sciences (CAS). She is now an associate professor at Inner Mongolia University. Her research interest is the design of novel AIE based fluorescent sensors for the detection of chemical or biological important species. She is also interested in the design and synthesis of novel photosensitizers with the aim of their applications in antitumor and antibacterial.

    Chunbin Li was born in 1992. He earned his master’s degree under the supervision of Prof. Guoyu Jiang and Jianguo Wang. He is now working for his doctor’s degree under the supervision of Prof. Jianguo Wang. His research interest is the design of novel AIE based fluorescent sensors for the detection of chemical or biological important species.

    Qingfang Lai was born in 1994. He earned his master’s degree under the supervision of Prof. Guoyu Jiang and Jianguo Wang. His research interest is the design of novel AIE based fluorescent sensors.

    Xiang Liu was born in 1994. He earned his master’s degree under the supervision of Prof. Jianguo Wang. His research interest is the design of novel AIE based fluorescent sensors.

    Qingqing Chen was born in 1993. He earned his master’s degree under the supervision of Prof. Guoyu Jiang. Her research interest is the design of novel AIE based fluorescent sensors for the detection of chemical or biological important species.

    Pengfei Zhang was born in 1983. Zhang received B.S. and Ph.D. degrees from Hunan University and The Hong Kong University of Science and Technology (HKUST), respectively. Now, he is an associate professor of nanomedicine at Shenzhen Institute of Advanced Technology, CAS. His research interest is bioorthogonal chemistry‬, multifunctional nanoprobes and nanomedicine.‬‬‬‬‬‬‬‬

    Jianguo Wang was born in 1982. Wang received Ph.D. degrees from the Institute of Chemistry, CAS. He conducted postdoctoral research at Prof. Ben Zhong Tang’s group in HKUST. Now, he is a professor at Inner Mongolia University. His research interest is the synthesis of novel organic functional materials and their application as novel chemical sensors and biosensors.

    Ben Zhong Tang is Stephen K. C. Cheong Professor of Science, Chair Professor of Chemistry, and Chair Professor of Chemical and Biological Engineering at HKUST. His research interests include macromolecular chemistry, materials science, and biomedical theranostics. Tang received B.S. and Ph.D. degrees from South China University of Technology and Kyoto University, respectively. He conducted postdoctoral research at University of Toronto. He joined HKUST as an assistant professor in 1994 and was promoted to chair professor in 2008. He was elected to the Chinese Academy of Sciences (CAS) and the Royal Society of Chemistry (RSC) in 2009 and 2013, respectively. He is currently serving as Dean of the SCUT-HKUST Joint Research Institute.

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