An Easily Available Ratiometric AIE Probe for Nitroxyl Visualization in Vitro and in Vivo† Cite This: Mater

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An easily available ratiometric AIE probe for nitroxyl visualization in vitro and in vivo† Cite this: Mater. Chem. Front., 2021, 5, 1817 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

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 Received 28th November 2020, developed firstly a ratiometric fluorescent probe (TCFPB-HNO) with typical aggregation-induced Accepted 24th December 2020 emission (AIE) characteristics for the detection and visualization of HNO with excellent photostability, DOI: 10.1039/d0qm00995d 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 rsc.li/frontiers-materials the HNO level in live samples.

Introduction biologically active HNO is of great importance for deep understanding of the physiological/pathological functions of HNO in Nitroxyl (HNO), the one-electron reduced and protonated entity biological systems. of nitric oxide (NO),1 plays important roles in a number of Fluorescent probes6 are powerful tools for in situ monitoring biological functions and pharmacological activities,2 and has of biologically important species in real time because of their received considerable interest in recent years. For instance, high specificity, sensitivity, temporal and spatial resolutions, HNO has been found to inhibit the activities of thiol-containing non-invasive detection and imaging ability.7,8 Pioneered by the 9 Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM. enzymes including glyceraldehyde-3-phosphate dehydrogenase study of Lippard and coworkers, a variety of reaction-based (GAPDH) and aldehyde dehydrogenase, and inducing the HNO fluorescent probes have been explored (Table S1, ESI†). release of neurotransmitters, glutamate, and calcitonin-gene Nevertheless, the majority of the currently available probes for related peptides by modulation of calcium channels.3 HNO is HNO detection depend mainly on the changes in fluorescence implicated in modulating the functions of mammalian vascular intensity of a single wavelength,10–12 which may suffer from systems by activating the TRPA1–CGRP signalling pathway, and severe disruption from various external factors, e.g. probe repairing the vascular injuries caused by superoxide.4 Additionally, concentration and biotic environment (pH, protein, ions). HNO also exhibits fascinating anti-tumor activity.5 Thus, the Ratiometric fluorescent probes13 with built-in calibration for development of detection tools to selectively visualise signal changes in two different emission wavelengths, are ideal candidates for HNO imaging in vivo due to high sensitivity and

a anti-interference ability. Yet so far, very limited ratiometric College of Chemistry and Chemical Engineering, Inner Mongolia Key Laboratory of Fine Organic Synthesis, Inner Mongolia University, Hohhot 010021, P. R. China. fluorescent probes for HNO have been reported (Table S1, 14 E-mail: [email protected] ESI†). In addition, a few ratiometric probes displayed b Center for AIE Research, College of Materials Science and Engineering, poor or totally quenched luminescence properties after their Shenzhen University, Shenzhen 518060, P. R. China accumulation in cells, which is known as the notorious c Guangdong Key Laboratory of Nanomedicine, CAS-HK Joint Lab of Biomaterials, Shenzhen Engineering Laboratory of Nanomedicine and Nanoformulations, aggregation-caused quenching (ACQ) effect. What’s more, the CAS Key Laboratory of Health Informatics, Institute of Biomedicine and tedious synthesis and poor photostability further limited their Biotechnology, Shenzhen Institutes of Advanced Technology, application in biological related systems. In 2001, Tang et al. Chinese Academy of Sciences, Shenzhen 518055, P. R. China reported the aggregation-induced emission (AIE) concept and d Department of Chemistry, The Hong Kong University of Science and Technology, thus opened a new window for anti-ACQ fluorescent probe Clear Water Bay, Kowloon, Hong Kong, P. R. China. E-mail: [email protected] 15 16 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ design. Luminogens with AIE features (AIEgens) are almost d0qm00995d non-emissive or weakly emissive in the single molecular state ‡ These authors contributed equally to this work. but emit intensely in the aggregated or solid state due to the

This journal is The Royal Society of Chemistry and the Chinese Chemical Society 2021 Mater. Chem. Front.,2021,5,18171823 | 1817 View Article Online Research Article Materials Chemistry Frontiers

Scheme 1 Recognition mechanism of ratiometric AIE-active fluorescent probe TCFPB-HNO toward HNO.

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 Fig. 1 (A) Synthetic route to probe TCFPB-HNO. (B) Absorption and 19,20 biocompatibility in living systems. However, to the best emission spectra of TCFPB-HNO (5 mM) in PBS solution (pH = 7.4). of our knowledge, there has not been any ratiometric AIE (C) Plot of PL intensity of TCFPB-HNO at maximum emission wavelength fluorescent probe reported for highly sensitive and selective vs. the toluene fraction in the toluene/DMSO mixtures. detection of HNO. Herein, we developed for the first time a ratiometric fluorescent probe (TCFPB-HNO, Scheme 1) with AIE features for HNO 13C NMR and high resolution mass spectrometry (HRMS) detection and real-time fluorescence imaging in vitro and in vivo. (Fig.S1–S6,ESI†). Subsequently, the photophysical properties TCFPB-HNO was devised to detect HNO based on intramolecular of TCFPB-HNO (5 mM) were investigated under physiological charge transfer (ICT), and contained tricyanofuranyl iminoben- conditions (PBS solution, pH = 7.4) to verify our initial design zaldehyde as the luminescent platform and a 2-(diphenylpho- concept. As shown in Fig. 1B, the maximum absorption of sphino)benzoyl group as the HNO recognition site. We TCFPB-HNO was located at 556 nm with the emission band anticipated that TCFPB-HNO possessed weak ICT eﬀects due covered from 600 to 850 nm with the maximum emission to the weak electro-donating ability of the ester group, which located at 670 nm in PBS solution. Interestingly, TCFPB-HNO connected the luminescent moiety and the recognition moiety demonstrated an excellent photostability unaltered with con- together. The reaction between the probe and HNO generated tinuous exposure to exciting light for 60 min (Fig. S7, ESI†). The

Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM. the corresponding phosphine oxide and azaylides, and further AIE properties of TCFPB-HNO were confirmed by photolumi- underwent a Staudinger ligation to give tricyanofuranyl imino- nescence (PL) spectroscopy using toluene/DMSO mixtures with

salicylaldehyde (TCFIS) with strong ICT eﬀects. As expected, different toluene fraction ( fT) (Fig. 1C and Fig. S8, ESI†). The PL TCFPB-HNO showed weak emission at 670 nm in phosphate intensities of TCFPB-HNO were gradually enhanced with

buﬀered saline (PBS) because of the weak ICT eﬀects. The AIE increased fT from 0% to 80%, accompanied by a spectral curves have verified that TCFPB-HNO was AIE-active, and thus is blue-shift attributed to the restricted motions of the C–C favorableforavertingtheautofluorescenceissue.Afterincubation double bond and benzene rings in the aggregated state. These with HNO, TCFPB-HNO exhibited typical ratiometric changes results indicated that TCFPB-HNO possesses typical AIE char- 21 (I618/I670) with excellent sensitivity (detection limit of 157.6 nM), acteristics similar to other AIEgens. TCFIS was also verified to specificity and photostability in PBS solution. Importantly, the have AIE activity by PL spectroscopy in toluene/DMSO mixtures probe could also be used for HNO detection and selective imaging with varying fraction (Fig. S9, ESI†). in vitro and in vivo with a high signal-to-noise ratio. These results To evaluate the responses of TCFPB-HNO (5 mM) toward have indicated that TCFPB-HNO was a ready-to-use tool for in situ HNO, the UV-vis and PL spectra of TCFPB-HNO treated with or detection of HNO in living systems and will provide a new without Angeli’s salt (AS, a donor of HNO) were compared to prospective on the deep understanding of the physiological/ those of TCFIS and shown in Fig. S10 (ESI†). After treatment pathological functions of HNO. with 50 mM 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 Results and discussion of the PL spectra showed that a new emission peak similar to that of TCFIS at 618 nm appeared, and the maximum emission Synthesis and optical properties (670 nm) of TCFPB-HNO decreased upon the addition of AS Initially, TCFIS and TCFPB-HNO were synthesized as shown (Fig. S10B, ESI†). On the basis of our design concept displayed in Fig. 1A, and their structures were confirmed by 1H NMR, in Scheme 1, the HNO-promoted oxidation of TCFPB-HNO

1818 | Mater. Chem. Front.,2021,5,18171823 This journal is The Royal Society of Chemistry and the Chinese Chemical Society 2021 View Article Online Materials Chemistry Frontiers Research Article

could generate phosphine oxide and azaylides, which would Fig. S13 (ESI†), the I618/I670 ratio barely varied for TCFPB-HNO finally produce TCFIS through Staudinger ligation. To further incubated with a series of anions, cations, reactive oxygen verify the reaction mechanism, HRMS analysis was carried out. species, reactive nitrogen species, and amino acids in biological

As displayed in Fig. S11 (ESI†), a peak of m/z = 375.18164 similar systems, while the I618/I670 ratio displayed over 10-fold enhancement to TCFIS (m/z = 375.18155) was observed after incubating TCFPB- after incubation with 50 mM of AS, indicating the high selectivity HNO with HNO, confirming the reaction mechanism of TCFPB- of TCFPB-HNO towards HNO. The influence of pH (4–10) on HNO as depicted in Scheme 1. TCFPB-HNO responsiveness was then studied (Fig. S14, ESI†). In order to optimize the reaction conditions, time-dependent After treating with AS, a dramatic fluorescence intensity ratio (0–50 min) PL intensities of TCFPB-HNO were recorded after change in HNO responsiveness was observed when the pH is 7.4, incubation with various concentrations of AS (0, 10, 20, 30 mM) indicating the suitability of such systems for detecting HNO in

(Fig. S12, ESI†). The PL intensity ratio (I618/I670) was observed to living systems. Moreover, it’s worth noting that the I618/I670 ratio increase gradually with the reaction time and eventually changes partially decreased when the pH value exceeded 8, reached a steady state after 40 min. Therefore, we selected mainly attributed to the incomplete decomposition of AS in 40 min as the appropriate incubation time for all the following the alkaline environment. experiments. Fluorescence titrations were implemented to ratio- Encouraged by the excellent properties, real-time monitoring nalise the sensitivity of TCFPB-HNO towards HNO. As shown in of HNO in living cells was performed using TCFPB-HNO as the Fig. 2A and B, the PL intensity of TCFPB-HNO at 618 nm probe. Viabilities of MCF-7 cells were initially evaluated using a increased gradually with the addition of AS, with a nearly CCK-8 assay (24 h incubation) against the probe TCFPB-HNO at

10-fold enhancement of the I618/I670 ratio. Besides, I618/I670 various concentrations (0, 1, 5, 15, 30, 50 mM) (Fig. S15, ESI†). exhibited good linearity with the concentrations of AS ranging The unchanged cell viabilities revealed negligible cytotoxicities from 5 to 30 mM and the detection limit was calculated to be of TCFPB-HNO, indicating the excellent biocompatibility of the 157.6 nM (Fig. 2C). These results indicated that TCFPB-HNO had probe for biological applications. Since TCFPB-HNO was highly great potential for quantitative detection of HNO with high sensitive to HNO, AS-pretreated MCF-7 cells were incubated with sensitivity. the probe (15 mM) and then observed by a laser scanning To further evaluate the specificity and stability of TCFPB- confocal microscope. As shown in Fig. 3, a remarkably enhanced HNO towards HNO, the probe was incubated with a modest emission intensity was observed in the green channel with the variety of biologically relevant species. As shown in Fig. 2D and 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 Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM.

Fig. 2 (A) PL spectra of TCFNPB-HNO (5 mM in PBS solution, pH = 7.4)

after incubation with AS (0–50 mM) for 40 min. (B) The I618/I670 ratio of TCFPB-HNO vs. the concentration of AS (0–50 mM). (C) The linear fitting of

the I618/I670 ratio vs. the concentration of AS (5–30 mM). Y = 0.0491X + 2 0.3331, R = 0.998. Excitation wavelength (lex = 570 nm). (D) The I618/I670 Fig. 3 Cell imaging of probe TCFPB-HNO (15 mM for 30 min at 37 1C) ratio of TCFPB-HNO toward AS (50 mM) or various other biomolecules with diﬀerent concentrations of AS (0, 30, 50 and 100 mM for 60 min).

(100 mM). (Scale bar: 20 mm.) lex = 543 nm.

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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).

Fig. 4 Time-dependent fluorescence images of probe TCFPB-HNO in Cell viability live mice. (A) Control group: TCFPB-HNO (20 mL, 5 mM) was injected Cell viability was evaluated by CCK-8 assay. 100 mL of MCF-7 cell in a subcutaneous manner, followed by an injection of PBS (80 mL). (B) Experimental group: TCFPB-HNO (20 mL, 5 mM) was injected, followed suspension (5000 cells per well) was seeded in a 96-well plate by an injection of AS (80 mL, 10 mM). Fluorescence emissions were and then the plate was precultured at 37 1C in a humidified

collected from 580 to 900 nm. lex = 523 nm. 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 mM) was added to showed over 10-fold enhancement (Fig. S16, ESI†), which was in the plate. After co-culture for 24 h in the incubator, the culture good agreement with the results in PBS, revealing the potential medium was exchanged with fresh medium (100 mL) containing of TCFPB-HNO for fluorescence imaging of HNO in living CCK-8 solution (10 mL) and the cells were further cultured for a systems. period of 2 h. The absorption of each well was recorded at To further estimate the application potential of this probe, 450 nm via an ELISA Plate Reader (Biotek Synergy HT). Each imaging of HNO in live mice was performed using TCFPB-HNO group was carried out in sextuplicate with untreated cells (subcutaneous injection). As seen in Fig. 4 and Fig. S17–S19 serving as a control. (ESI†), slight fluorescence intensity changes (1.54-folds enhancement than injecting probe at 0 min) were detected on Cell treatment and cell imaging mice injected with the TCFPB-HNO probe alone (3.3 mg kg1) For the imaging of HNO, MCF-7 cells were stained with TCFPB- over 60 min using a whole-body small-animal imaging system. HNO (15 mM) for 30 min under normal culture conditions, and In contrast, the fluorescence signals were observed with 10.38- then the medium was removed and the cells were rinsed with fold enhancement when the mice (pretreated with TCFPB-HNO, PBS solution, followed by the addition of various concentrations 3.3 mg kg1) were injected with AS solution, further confirming of AS (0, 30, 50 and 100 mM) for 60 min. The image was captured the potential application of TCFPB-HNO in vivo. using a ZEISS LSM880. Before cell imaging, the cells were washed with PBS solution (three times) to remove background Conclusions interference. A 543 nm laser was used as the excitation source and the fluorescence signal was acquired from 580 to 640 nm

Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM. In this work, a ratiometric AIE probe (TCFPB-HNO) was con- (green channel) and 660–720 nm (red channel). structed for the first time for fluorescence detection and visualization of HNO in vitro and in vivo. TCFPB-HNO, which In vivo animal imaging was comfirmed to be AIE-active, was easily synthesized using a All male BALB/c white mice (6 weeks old, weighted 20 g) for the two-step process. TCFPB-HNO exhibited high sensitivity and imaging experiment in vivo were provided by Vital River Labora- excellent selectivity for ratiometric sensing of HNO. Importantly, tory Animal Technology Co. Ltd (Beijing, China) and animals the probe could be employed for in situ visualization of HNO via all received good care according to the guidelines outlined in a ratiometric response mode with high signal-to-background the Guide for the Care and Use of Laboratory Animals. The ratio in vitro and in vivo. This work opened up new avenues to procedures were authorized by Shenzhen Institutes of obtain easy-to-handle AIE probes for real-time monitoring of the Advanced Technology, Chinese Academy of Sciences Animal HNO level in living systems and to enrich the understanding Care and Use Committee. The TCFPB-HNO solution (20 mL, 5 mM of the physiological and pathological functions of HNO in in DMSO) was injected into white mice by subcutaneous injection, biological systems. followed by an injection of AS solution (80 mL, 10 mM). The AS solution was replaced by PBS solution (80 mL) as a control group. Experimental section After that, images were collected from diﬀerent points in time using the Maestro in vivo imaging system. Materials and methods Solvents and other common reagents were available from Synthesis of TCFIS Innochem, Sigma-Aldrich, TCI, and J&K, and were used without A mixture of 4-(diethylamino)salicylaldehyde (compound 1, any purification unless otherwise specified. Angeli’s salt (AS) as 231.0 mg, 1.2 mmol), 2-(3-cyano-4,5,5-trimethylfuran-2(5H)- a water-soluble HNO donor was purchased from Santa Cruz ylidene)malononitrile (compound 2, 199.0 mg, 1.0 mmol) and Biotechnology. NMR spectra were measured on a Bruker ARX ammonium acetate (77 mg, 1 mmol) in ethanol was refluxed 500 MHz instrument and Bruker ARX 400 MHz instrument. under a nitrogen atmosphere. The reaction was monitored

1820 | Mater. Chem. Front.,2021,5,18171823 This journal is The Royal Society of Chemistry and the Chinese Chemical Society 2021 View Article Online Materials Chemistry Frontiers Research Article

until compound 2 was fully consumed (determined by TLC), Rev., 2011, 255, 2764; (c) J. F. DuMond, M. W. Wright and then cooled to room temperature and purified by silica gel S. B. King, Water soluble acyloxy nitroso compounds: HNO chromatography with the mixed solvent of petroleum ether/ release and reactions with heme and thiol containing dichloromethane (1 : 1–1 : 5, v/v) to give TCFIS as a purple solid proteins, J. Inorg. Biochem., 2013, 118, 140; (d)C.H. 1 (280.0 mg, 75%). H NMR (400 MHz, DMSO-d6) d 10.86 (br, 1H), Switzer, W. Flores-Santana, D. Mancardi, S. Donzelli, 8.23 (s, 1H), 7.70 (d, J = 9.2 Hz, 1H), 6.95 (s, 1H), 6.45 (d, J = D. Basudhar, L. A. Ridnour, K. M. Miranda, J. M. Fukuto, 9.6 Hz, 1H), 6.15 (s, 1H), 3.46 (q, J = 7.4, 6.7 Hz, 4H), 1.70 (s, 6H), N. Paolocci and D. A. Wink, The emergence of nitroxyl 13 1.16 (t, J = 7.2 Hz, 6H). C NMR (101 MHz, DMSO-d6) d 177.58, (HNO) as a pharmacological agent, Biochim. Biophys. Acta, 175.30, 162.35, 154.11, 114.14, 113.26, 112.90, 112.07, 106.82, 2009, 1787, 835. 97.24, 96.47, 48.71, 44.58, 25.95, 12.69. HRMS (MALDI-TOF): 2 J. M. Fukuto, C. J. Cisneros and R. L. Kinkade, A comparison + m/z: [M] calcd for C22H23N4O2: 375.18155; found: 375.18164. of the chemistry associated with the biological signaling and actions of nitroxyl (HNO) and nitric oxide (NO), J. Inorg. Synthesis of TCFPB-HNO Biochem., 2013, 118, 201. A mixture of 2-(diphenylphosphino)benzoic acid (122 mg, 3(a) N. Paolocci, T. Katori, H. C. Champion, M. E. St. John, 0.4 mmol), N,N0-dicyclohexylcarbodiimide (103 mg, 0.5 mmol), K. M. Miranda, J. M. Fukuto, D. A. Wink and D. A. Kass, and 4-dimethylaminopyridine (12 mg, 0.1 mmol) in 20 mL Positive inotropic and lusitropic eﬀects of HNO/NO in

CH2Cl2 was stirred at 0 1C under a nitrogen atmosphere for failing hearts: Independence from b-adrenergic signaling, 1 h. TCFIS (112 mg, 0.3 mmol) was added to the mixture and Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 5537; (b) D. Steinritz, reacted overnight at room temperature. The reaction mixture J. Weber, F. Balszuweit, H. Thiermann and A. Schmidt, was purified by silica gel chromatography using a mixed solvent Sulfur mustard induced nuclear translocation of glyceralde- of petroleum ether/dichloromethane (2 : 1–1 : 2, v/v) to collect as hyde 3-phosphate-dehydrogenase (GAPDH), Chem. -Biol. a blue to purple solid (150 mg, 78%). 1H NMR (500 MHz, Interact., 2013, 206, 529; (c) G. Keceli and J. P. Toscano, chloroform-d) d 8.38–8.32 (m, 1H), 7.71 (d, J = 16.0 Hz, 1H), 7.65 Reactivity of C-terminal cysteines with HNO, Biochemistry, (d, J = 9.2 Hz, 1H), 7.55–7.50 (m, 2H), 7.36–7.29 (m, 6H), 7.27 2014, 53, 3689; (d) A. T. Wrobel, T. C. Johnstone, A. Deliz (d, J = 2 Hz, 1H), 7.28–7.22 (m, 3H), 7.08–7.02 (m, 1H), 6.73 Liang, S. J. Lippard and P. Rivera-Fuentes, A fast and (d, J = 16 Hz, 1H), 6.59 (dd, J = 9.2, 2.6 Hz, 1H), 6.11 (d, J = 2.6 Hz, selective near-infrared fluorescent sensor for multicolor 1H), 3.38 (q, J = 7.1 Hz, 4H), 1.58 (s, 6H), 1.18 (t, J = 7.1 Hz, 6H). imaging of biological nitroxyl (HNO), J. Am. Chem. Soc., 13C NMR (125 MHz, chloroform-d) d 176.22, 174.40, 164.52, 2014, 136, 4697. 164.50, 153.29, 152.70, 142.13, 141.87, 137.30, 137.22, 134.84, 4(a) A. J. Vaananen, P. Salmenpera, M. Hukkanen, P. Rauhala 134.02, 133.86, 133.43, 131.71, 129.95, 128.91, 128.73, 128.66, and E. Kankuri, Cathepsin B is a diﬀerentiation-resistant 128.60, 114.33, 112.69, 111.85, 111.22, 110.50, 109.45, 105.25, target for nitroxyl (HNO) in THP-1 monocyte/macrophages, 96.80, 94.64, 45.08, 26.71, 26.70, 12.62. HRMS (MALDI-TOF): m/z: Free Radical Biol. Med., 2006, 41, 120; (b) D. A. Wink, + [M] calcd for C41H36N4O2: 663.2446; found: 663.2520. K. M. Miranda, T. Katori, D. Mancardi, D. D. Thomas,

Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM. L. Ridnour, M. G. Espey, M. Feelisch, C. A. Colton, J. M. Fukuto, P. Pagliaro, D. A. Kass and N. Paolocci, Conflicts of interest Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: a novel redox There are no conflicts to declare. paradigm, Am. J. Physiol., 2003, 285, H2264. 5 M. E. Shoman and O. M. Aly, Nitroxyl (HNO): a possible Acknowledgements strategy for fighting cancer, Curr. Top. Med. Chem., 2016, 16, 2464. We thank the National Natural Science Foundation of China 6(a) J. Wu, B. Kwon, W. Liu, E. V. Anslyn, P. Wang and (21871060), Grassland Talent Program of Inner Mongolia Auto- J. S. Kim, Chromogenic/fluorogenic ensemble chemosen- nomous Region of China, the Natural Science Foundation of sing systems, Chem. Rev., 2015, 115, 7893; (b) L. Zhou, Inner Mongolia Autonomous Region of China [2020JQ02 and X. Zhang, Y. Lv, C. Yang, D. Lu, Y. Wu, Z. Chen, Q. Liu 2020MS02004], the Natural Science Foundation of Jiangxi Province and W. Tan, Localizable and photoactivatable fluorophore (20192BCBL23013) and Science and Technology Plan of Shenzhen for spatiotemporal two-photon bioimaging, Anal. Chem., (JCYJ20170818113538482) for the financial support. 2015, 87, 5626; (c) X. Han, Y. Ma, Y. Chen, X. Wang and Z. Wang, Enhancement of the aggregation-induced emis- Notes and references sion by hydrogen bond for visualizing hypochlorous acid in an inflammation model and a hepatocellular carcinoma 1(a) H. Nakagawa, Photocontrollable nitric oxide (NO) and model, Anal. Chem., 2020, 92, 2830. nitroxyl (HNO) donors and their release mechanisms, Nitric 7(a) X. Wang, P. Li, Q. Ding, C. Wu, W. Zhang and B. Tang, oxide, 2011, 25, 195; (b) F. Doctorovich, D. Bikiel, Observation of acetylcholinesterase in stress-induced J. Pellegrino, S. A. Sua´rez, A. Larsen and M. A. Martı´, Nitroxyl depression phenotypes by two-photon fluorescence imaging (azanone) trapping by metalloporphyrins, Coord. Chem. in the mouse brain, J. Am. Chem. Soc., 2019, 141, 2061;

This journal is The Royal Society of Chemistry and the Chinese Chemical Society 2021 Mater. Chem. Front.,2021,5,18171823 | 1821 View Article Online Research Article Materials Chemistry Frontiers

(b) D. Ma, C. Huang, J. Zheng, W. Zhou, J. Tang, W. Chen, 5, 3557; (b) H. Li, Q. Yao, F. Xu, N. Xu, X. Ma, J. Fan, S. Long, J. Li and R. Yang, Azoreductase-responsive nanoprobe for J. Du, J. Wang and X. Peng, Recognition of exogenous and hypoxia-induced mitophagy imaging, Anal. Chem., 2019, endogenous nitroxyl in living cells via a two-photon fluor- 91, 1360; (c) D. Xu, Y. Li, C. Y. Poon, H. N. Chan, H. W. Li escent probe, Anal. Chem., 2018, 90, 4641; (c) W. Li, X. Wang, and M. S. Wong, A zero cross-talk ratiometric two-photon Y.-M. Zhang and S. X.-A. Zhang, Single probe giving diﬀer- probe for imaging of acid pH in living cells and tissues and ent signals towards reactive oxygen species and nitroxyl, early detection of tumor in mouse model, Anal. Chem., 2018, Dyes Pigm., 2018, 148, 348; (d) L. Nie, C. Gao, T. Shen, J. Jing, 90, 8800; (d) F. Hu, G. Qi, Kenry, D. Mao, S. Zhou, M. Wu, S. Zhang and X. Zhang, Dual-site fluorescent probe to W. Wu and B. Liu, Visualization and in situ ablation of monitor intracellular nitroxyl and GSH-GSSG oscillations, intracellular bacterial pathogens through metabolic label- Anal. Chem., 2019, 91, 4451. ing, Angew. Chem., Int. Ed., 2020, 59, 9288. 12 (a) J. Wang, W. Zhu, C. Li, P. Zhang, G. Jiang, G. Niu and 8(a) Y. Huang, X. You, L. Wang, G. Zhang, S. Gui, Y. Jin, B. Z. Tang, Mitochondria-targeting NIR fluorescent probe R. Zhao and D. Zhang, Pyridinium-substituted tetrapheny- for rapid, highly sensitive and selective visualization of lethylenes functionalized with alkyl chains as autophagy nitroxyl in live cells, tissues and mice, Sci. China: Chem., modulators for cancer therapy, Angew. Chem., Int. Ed., 2020, 2019, 63, 282; (b) M. Yang, J. Fan, W. Sun, J. Du, S. Long, 59, 10042; (b) A. C. Sedgwick, J. T. Brewster, P. Harvey, K. Shao and X. Peng, A nitroxyl-responsive near-infrared

D. A. Iovan, G. Smith, X. P. He, H. Tian, J. L. Sessler and fluorescent chemosensor for visualizing H2S/NO crosstalk T. D. James, Metal-based imaging agents: progress towards in biological systems, Chem. Commun., 2019, 55, 8583; interrogating neurodegenerative disease, Chem. Soc. Rev., (c) J. B. Li, Q. Wang, H. W. Liu, X. Yin, X. X. Hu, L. Yuan 2020, 49, 2886; (c) X. Teng, F. Li and C. Lu, Visualization of and X. B. Zhang, Engineering of a bioluminescent probe for materials using the confocal laser scanning microscopy imaging nitroxyl in live cells and mice, Chem. Commun., technique, Chem. Soc. Rev., 2020, 49, 2408; (d) G. Wang, 2019, 55, 1758; (d) S. Palanisamy, L.-F. Chen, S.-C. Tzou and L. Zhou, P. Zhang, E. Zhao, L. Zhou, D. Chen, J. Sun, X. Gu, Y.-M. Wang, Near-infrared templated fluorescent probe for W. Yang and B. Z. Tang, Fluorescence self-reporting pre- nitroxyl: Selective and sensitive turn-on detection in living cipitation polymerization based on aggregation-induced cells, Sens. Actuators, B, 2020, 310, 127839; (e) S. Peng, Z. Li, emission for constructing optical nanoagents, Angew. Y. Zhang, W. Cao, J. Liu, W. Zhu and Y. Ye, A two-photon Chem., Int. Ed., 2020, 59, 10122; (e) H. Yang, M. Li, C. Li, fluorescent probe for HNO rapid visualization in endoplas- Q. Luo, M. Q. Zhu, H. Tian and W. H. Zhu, Unraveling dual mic reticulum, Sens. Actuators, B, 2020, 317, 128211. aggregation-induced emission behavior in steric-hindrance 13 (a) W. Fu, C. Yan, Z. Guo, J. Zhang, H. Zhang, H. Tian and photochromic system for super resolution imaging, Angew. W. H. Zhu, Rational design of near-infrared aggregation- Chem., Int. Ed., 2020, 59, 8560. induced-emission-active probes: In situ mapping of amy- 9 J. Rosenthal and S. J. Lippard, Direct detection of nitroxyl in loidbeta plaques with ultrasensitivity and high-fidelity, aqueous solution using a tripodal copper(II) BODIPY J. Am. Chem. Soc., 2019, 141, 3171; (b) R. Wang, X. Gu,

Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM. complex, J. Am. Chem. Soc., 2010, 132, 5536. Q. Li, J. Gao, B. Shi, G. Xu, T. Zhu, H. Tian and C. Zhao, 10 (a) K. Kawai, N. Ieda, K. Aizawa, T. Suzuki, N. Miyata and Aggregation enhanced responsiveness of rationally H. Nakagawa, A reductant-resistant and metal-free fluores- designed probes to hydrogen sulfide for targeted cancer cent probe for nitroxyl applicable to living cells, J. Am. Chem. imaging, J. Am. Chem. Soc., 2020, 142, 15084. Soc., 2013, 135, 12690; (b) G. J. Mao, X. B. Zhang, X. L. Shi, 14 (a) C. Zhang, F. He, F. Huang, J. Xu and Y. Hu, A fluorescent H. W. Liu, Y. X. Wu, L. Y. Zhou, W. Tan and R. Q. Yu, A probe with low-background for visualization of nitroxyl in highly sensitive and reductant-resistant fluorescent probe living cells and zebrafish, Dyes Pigm., 2021, 185, 108889; for nitroxyl in aqueous solution and serum, Chem. Com- (b) H. Zhang, R. Liu, Y. Tan, W. H. Xie, H. Lei, H. Y. Cheung mun., 2014, 50, 5790; (c) K. N. Bobba, Y. Zhou, L. E. Guo, and H. Sun, A FRET-based ratiometric fluorescent probe for T. N. Zang, J. F. Zhang and S. Bhuniya, Resorufin based nitroxyl detection in living cells, ACS Appl. Mater. Interfaces, fluorescence ‘turn-on’ chemodosimeter probe for nitroxyl 2015, 7, 5438; (c) X. Zhu, M. Xiong, H. W. Liu, G. J. Mao, (HNO), RSC Adv., 2015, 5, 84543; (d) F. Ali, S. Sreedharan, L. Zhou, J. Zhang, X. Hu, X. B. Zhang and W. Tan, A FRET- A. H. Ashoka, H. K. Saeed, C. G. W. Smythe, J. A. Thomas based ratiometric two-photon fluorescent probe for dual- and A. Das, A super-resolution probe to monitor HNO levels channel imaging of nitroxyl in living cells and tissues, in the endoplasmic reticulum of cells, Anal. Chem., 2017, Chem. Commun., 2016, 52, 733; (d) Y. Zhou, X. Zhang, 89, 12087; (e) B. Dong, X. Song, X. Kong, C. Wang, N. Zhang S. Yang, Y. Li, Z. Qing, J. Zheng, J. Li and R. Yang, Ratio- and W. Lin, Two-photon red-emissive fluorescent probe for metric visualization of NO/H2S cross-talk in living cells and imaging nitroxyl (HNO) in living cells and tissues, J. Mater. tissues using a nitroxyl-responsive two-photon fluorescence Chem. B, 2017, 5, 5218. probe, Anal. Chem., 2017, 89, 4587; (e) S. Yuan, F. Wang, 11 (a) C. Liu, Y. Wang, C. Tang, F. Liu, Z. Ma, Q. Zhao, Z. Wang, G. Yang, C. Lu, J. Nie, Z. Chen, J. Ren, Y. Qiu, Q. Sun, B. Zhu and X. Zhang, A reductant-resistant ratiometric, C. Zhao and W.-H. Zhu, Highly sensitive ratiometric self- colorimetric and far-red fluorescent probe for rapid and assembled micellar nanoprobe for nitroxyl and its applica- ultrasensitive detection of nitroxyl, J. Mater. Chem. B, 2017, tion in vivo, Anal. Chem., 2018, 90, 3914.

1822 | Mater. Chem. Front.,2021,5,18171823 This journal is The Royal Society of Chemistry and the Chinese Chemical Society 2021 View Article Online Materials Chemistry Frontiers Research Article

15 (a) F. Hu, S. Xu and B. Liu, Photosensitizers with Semiconducting nanocomposite with AIEgen-triggered aggregation-induced emission: Materials and biomedical enhanced photoluminescence and photodegradation for applications, Adv. Mater., 2018, 30, e1801350; dual-modality tumor imaging and therapy, Adv. Funct. (b) J. Tavakoli, S. Pye, A. H. M. M. Reza, N. Xie, J. Qin, Mater., 2019, 29, 1903733; (c) G. Niu, X. Zheng, Z. Zhao, C. L. Raston, B. Z. Tang and Y. Tang, Tuning aggregation- H. Zhang, J. Wang, X. He, Y. Chen, X. Shi, C. Ma, induced emission nanoparticle properties under thin film R. T. K. Kwok, J. W. Y. Lam, H. H. Y. Sung, I. D. Williams, formation, Mater. Chem. Front., 2020, 4, 537; (c) H. Zhang, K. S. Wong, P. Wang and B. Z. Tang, Functionalized Z. Zhao, A. T. Turley, L. Wang, P. R. McGonigal, Y. Tu, Y. Li, acrylonitriles with aggregation-induced emission: Structure Z. Wang, R. T. K. Kwok, J. W. Y. Lam and B. Z. Tang, tuning by simple reaction-condition variation, efficient red Aggregate science: From structures to properties, Adv. emission, and two-photon bioimaging, J. Am. Chem. Soc., Mater., 2020, 32, 2001457. 2019, 141, 15111; (d) H. Tian, D. Li, X. Tang, Y. Zhang, 16 (a) M. Chen, A. Qin, J. W. Y. Lam and B. Z. Tang, Multifaceted Z. Yang, J. Qian, Y. Q. Dong and M. Han, Efficient red functionalities constructed from pyrazine-based AIEgen sys- luminogen with aggregation-induced emission for in vivo tem, Coord. Chem. Rev., 2020, 422, 213472; (b)H.T.Feng, three-photon brain vascular imaging, Mater. Chem. Front., Y. Li, X. Duan, X. Wang, C. Qi, J. W. Y. Lam, D. Ding and 2020, 4, 1634; (e) W. Wu and Z. Li, Nanoprobes with B. Z. Tang, Substitution activated precise phototheranostics aggregation-induced emission for theranostics, Mater. through supramolecular assembly of AIEgen and calixarene, Chem. Front., 2020, DOI: 10.1039/d0qm00617c. J. Am. Chem. Soc.,2020,142,15966;(c) Z. Guo, C. Yan and 20 (a) W. Zhang, J. Liu, X. Jin, X. Gu, X. C. Zeng, X. He and W. H. Zhu, High-performance quinoline-malononitrile core H. Li, Quantitative prediction of aggregation-induced emis- as a building block for the diversity-oriented synthesis of sion: A full quantum mechanical approach to the optical AIEgens, Angew. Chem., Int. Ed., 2020, 59, 9812; (d)D.Yan, spectra, Angew. Chem., Int. Ed., 2020, 59, 11550; (b) Y. Zhou, Q. Wu, D. Wang and B. Z. Tang, Innovative synthetic proto- J. Hua, B. Z. Tang and Y. Tang, AIEgens in cell-based cols for aggregation-induced emission luminogens: Recent multiplex fluorescence imaging, Sci. China: Chem., 2019, advances and prospects, Angew. Chem., Int. Ed., 2020, DOI: 62, 1312; (c) R. Wang, X. Gu, Q. Li, J. Gao, B. Shi, G. Xu, 10.1002/anie.202006191. T. Zhu, H. Tian and C. Zhao, Aggregation enhanced respon- 17 (a) X. Gu and D. Zhang, Reaction: the impact of the AIE siveness of rationally designed probes to hydrogen sulfide concept, Chem, 2020, 6, 1199; (b) J. Li, X. Ni, J. Zhang, for targeted cancer imaging, J. Am. Chem. Soc., 2020, Y. Liang, Z. Gao, X. Zhang, D. Zheng and D. Ding, A 142, 15084; (d) J. Wang, X. Gu, P. Zhang, X. Huang, fluorescence and photoactivity dual-activatable prodrug X. Zheng, M. Chen, H. Feng, R. T. K. Kwok, J. W. Y. Lam with self-synergistic magnification of the anticancer effect, and B. Z. Tang, Ionization and anion-p+ interaction: A new Mater. Chem. Front., 2019, 3, 1349; (c) Z. Zhao, H. Zhang, strategy for structural design of aggregation-induced emis- J. W. Y. Lam and B. Z. Tang, Aggregation-induced emission: sion luminogens, J. Am. Chem. Soc., 2017, 139, 16974; New vistas at the aggregate level, Angew. Chem., Int. Ed., (e) J. Qi, C. Chen, X. Zhang, X. Hu, S. Ji, R. T. K. Kwok,

Published on 28 December 2020. Downloaded 9/30/2021 8:18:19 PM. 2020, 59, 9888; (d) Y. Xie and Z. Li, Approaching aggregated J. W. Y. Lam, D. Ding and B. Z. Tang, Light-driven trans- state chemistry accelerated by aggregation-induced emis- formable optical agent with adaptive functions for boosting sion, Natl. Sci. Rev., 2020, DOI: 10.1093/nsr/nwaa199. cancer surgery outcomes, Nat. Commun., 2018, 9, 1848; 18 (a) X. Chen, H. Gao, Y. Deng, Q. Jin, J. Ji and D. Ding, ( f ) J. Wang, C. Li, Q. Chen, H. Li, L. Zhou, X. Jiang, Supramolecular aggregation-induced emission nanodots M. Shi, P. Zhang, G. Jiang and B. Z. Tang, An easily available with programmed tumor microenvironment responsiveness ratiometric reaction-based AIE probe for carbon monoxide for image-guided orthotopic pancreatic cancer therapy, ACS light-up imaging, Anal. Chem., 2019, 91, 9388. Nano, 2020, 14, 5121; (b) G. Niu, R. Zhang, X. Shi, H. Park, 21 (a) W. Qin, N. Alifu, J. W. Y. Lam, Y. Cui, H. Su, G. Liang, S. Xie, R. T. K. Kwok, J. W. Y. Lam and B. Z. Tang, AIE J. Qian and B. Z. Tang, Facile synthesis of eﬃcient lumino- luminogens as fluorescent bioprobes, Trends Anal. Chem., gens with AIE features for three-photon fluorescence ima- 2020, 123, 115769. ging of the brain through the intact skull, Adv. Mater., 2020, 19 (a) C. Chen, X. Ni, H. W. Tian, Q. Liu, D. S. Guo and D. Ding, 32, 2000364; (b) Z. Zhang, W. Xu, M. Kang, H. Wen, H. Guo, Calixarene-based supramolecular AIE dots with highly P. Zhang, L. Xi, K. Li, L. Wang, D. Wang and B. Z. Tang, An inhibited nonradiative decay and intersystem crossing for all-round athlete on the track of phototheranostics: subtly ultrasensitive fluorescence image-guided cancer surgery, regulating the balance between radiative and nonradiative Angew. Chem., Int. Ed., 2020, 59, 10008; (b) Y. Li, Z. Liu, decays for multimodal imaging-guided synergistic therapy, Y. Ma, Y. Chen, K. Ma, X. Wang, D. Zhang and Z. Wang, Adv. Mater., 2020, 32, 2003210.

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