A fluorinated bihydrazide conjugate for activatable sensing and imaging of hypochlorous acid by 19F NMR/MRI

Ao Li , Xiaoxue Tang , Xuanqing Gong , Hongming Chen , Hongyu Lin * and Jinhao Gao *
State Key Laboratory of Physical Chemistry of Solid Surfaces, The Key Laboratory for Chemical Biology of Fujian Province and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China. E-mail: hylin007@xmu.edu.cn; jhgao@xmu.edu.cn

Received 26th August 2019 , Accepted 23rd September 2019

First published on 23rd September 2019

Hypochlorous acid (HClO) is one of the most important reactive oxygen species (ROS) and plays a vital role in many physiological and pathological processes. The comprehensive exploration of mechanistic details and the potential clinical translation necessitate the development of reliable probes for prompt and accurate detection of HClO in complex biological environments. Herein we report a fluorinated bihydrazide conjugate as a 19F NMR/MRI probe with a “turn-on” character for the detection of HClO. This probe could selectively respond to HClO, leading to a significant recovery of 19F signals for 19F NMR/MRI. Activatable sensing and imaging of HClO were achieved with SMMC-7721 cells and nude mice, which demonstrates that this small molecular conjugate could serve as a selective probe for real-time sensing and imaging of HClO in biological systems.

Reactive oxygen species (ROS) are one of the most important chemical species in biological systems, and include oxygen radicals such as superoxide (O2), hydroxyl radicals (HO˙), peroxyl radicals (ROO˙), and non-radical derivatives of oxygen (O2) like singlet oxygen (1O2), ozone (O3), hydrogen peroxide (H2O2), and hypochlorous acid (HClO).1 They are generated either exogenously or endogenously (inflammation, metabolism, etc.) and deeply involved in many physiological and pathological processes.1–5 Among them, hypochlorous acid (HClO) is mostly produced by the reaction between hydrogen peroxide (H2O2) and chloride (Cl) catalyzed by myeloperoxidase (MPO).6 In living bodies, the HClO produced by activated neutrophils through the MPO/H2O2/Cl system is within 20–400 μM.7 Recent research has established the close association between uncontrolled upregulation of HClO and various diseases, such as cardiovascular diseases,8 cancer,9 neuron degeneration,10 and atherosclerosis.11 The elaboration of mechanistic details and further applications for diagnosis and therapy rely on real-time and accurate detection of HClO in complex physiological environments, which underscores the great demand for HClO sensing and imaging probes. Many efforts have been dedicated to this field and a variety of probes have been developed for the detection of HClO in cells and animal models.12–18 However, most of the probes are based on fluorescent organic dyes that are susceptible to photobleaching or oxidation. Furthermore, fluorogenic methods are often restricted by shallow tissue penetration or autofluorescence interference. Several approaches trying to overcome these obstacles have been reported, in spite of their own limitations.19–21

Magnetic resonance imaging (MRI) is a non-invasive imaging technique with high spatial resolution and deep tissue penetration which is widely used in clinics and research.22–27 Over the years, protons (1H) are the only class of nuclei used for MRI because of their abundance and high sensitivity. As an emerging complement to conventional 1H MRI, 19F MRI has several advantages including a comparable sensitivity (83% of 1H), a broad chemical shift (>350 ppm), and a negligible background (<10−6 M in the human body).28,29 As a result, 19F MRI is considered to be a promising method for imaging bioactive chemical species. Recently, Kikuchi and co-workers have developed a few activatable 19F MRI probes for assessing enzymatic activity.30–32 Several 19F MRI probes aimed at the detection of ROS, biothiols and cell hypoxia have also been reported.33–37 All these reports render it appealing to detect and visualize HClO in organisms with 19F MRI. Unfortunately, to the best of our knowledge, there has been no study reported on selective sensing and imaging of HClO with 19F MRI probes.

In this report, we designed a 19F NMR/MRI probe (Gd-DOTA-BTFPH) small molecular conjugate (Probe 1, as shown in Fig. 1) for selective detection and imaging of HClO. Probe 1 consists of a Gd-DOTA complex and a fluorine-containing moiety, 3,5-bis(trifluoromethyl)benzoic acid, which are connected with a bihydrazide linker. We envisioned that due to the paramagnetic relaxation enhancement (PRE) effect of Gd-DOTA, the T1 and T2 relaxation times of fluorine atoms in intact Probe 1 should be significantly reduced, leading to a substantial decrease in the intensity of 19F signals.30,38–40 Upon reaction with HClO, which could selectively oxidize and split the bihydrazide linker,41 the PRE effect would be markedly undermined, causing the recovery of the T1 and T2 of the fluorine atoms. As a result, their signal intensity would be remarkably increased, giving rise to a “turn-on” effect responsive to HClO. According to the above design, we successfully synthesized Probe 1 and demonstrated its feasibility and selectivity for the detection of HClO. Activatable sensing and imaging of HClO were accomplished with SMMC-7721 cells and nude mice, revealing the great potential of Probe 1 for real-time detection of HClO in biological systems.

image file: c9cc06622e-f1.tif
Fig. 1 Schematic illustration showing the functioning mechanism of the HClO-activated 19F NMR/MRI probe Gd-DOTA-BTFBH small molecular conjugate (Probe 1 or 1).

Construction of Probe 1 was started by preparation of a partially protected DOTA derivative,42 which was coupled to 3,5-bis(trifluoromethyl)benzoic acid via a bihydrazide linker (Scheme S1, ESI). Deprotection and chelation with Gd3+ furnished the final product Probe 1. Detailed synthesis and characterization of 1 and intermediates are included in the ESI.

To achieve a significant “turn-on” effect, the 19F signal of Probe 1 needs to be sufficiently quenched. We first studied the 19F NMR signals of DOTA-BTFBH with and without the chelation of Gd3+ ions. As expected, the 19F NMR spectrum of DOTA-BTFBH itself showed a single sharp peak at −62.7 ppm, while that of DOTA-BTFBH with Gd3+ (Probe 1) exhibited a very broad peak that could barely be seen (Fig. 2a). A further investigation reveals that the fluorine atoms in Probe 1 have a remarkably shorter transverse relaxation time (T2 = 1.2 ms) than those in DOTA-BTFBH (915 ms) (Table S1, ESI), which causes severe line broadening and results in the broad peak in the 19F NMR spectrum.39 These results demonstrate that the presence of Gd3+ in close proximity could successfully have the PRE effect on fluorine atoms, significantly shorten their relaxation time, and considerably abate the 19F signal intensity.30,38–40 Meanwhile, phantom imaging of Probe 1 and Gd-DOTA, a typical clinical MRI contrast agent, was conducted at 0.5 T (Fig. S1, ESI). Their longitudinal relaxivities (r1) were calculated to be 6.15 and 4.78 mM−1 s−1, respectively (Fig. S2, ESI).

image file: c9cc06622e-f2.tif
Fig. 2 (a) 19F NMR spectra of DOTA-BTFPH and Probe 1 in PBS buffer (pH 7.4). (b) 19F NMR spectra of Probe 1 (150 μM) treated with various analytes (0.45 mM) in PBS buffer for 5 min at 25 °C. CF3COONa (at −75.4 ppm) was used as an internal reference of chemical shift. (c) 19F “hot spot” MRI phantom images of PBS, Probe 1 (3 mM), Probe 1 with HClO, and Probe 1 with HClO and taurine acquired on a 9.4 T MRI scanner. (d) HPLC chromatograms of Probe 1 (1 mM), Probe 1 incubated with HClO and taurine for 15 min, Probe 1 incubated with HClO for 15 min, and 3,5-bis-trifluoromethyl-benzoic acid.

We then investigated the “turn-on” property of Probe 1 in the presence of HClO. Upon treatment with HClO, the 19F NMR signal of Probe 1 was considerably recovered (Fig. 2b). The measurement of relaxation times indicates that after reacting with HClO, which cleaves the linkage between the Gd3+ complex and the fluorine atoms, the PRE effect is substantially weakened and the T2 of the fluorine atoms is recovered (197 ms, Table S1, ESI). Though the T2 is still shorter than that of the fluorine atoms in DOTA-BTFBH, which is probably due to the presence of Gd-DOTA resulting from the cleavage, it is sufficient to lead to a single sharp peak in the 19F NMR spectrum.30 The specificity of Probe 1 was then assessed. No appreciable recovery of the 19F NMR signal was observed with a variety of other ROS (H2O2, ROO˙, HO˙, O2) and common biological molecules/macromolecules (GSH, FBS). Peroxynitrite (ONOO) did react with Probe 1. However, the recovered 19F NMR signal was far from comparable to that of HClO treated Probe 1, which was also supported by quantitative analysis of 19F NMR signals (Fig. S3, ESI). This “turn-on” property of Probe 1 was also validated with preliminary 19F MRI experiments. The 19F MRI “hot spot” signal was remarkably enhanced after treatment with HClO, while in the presence of both HClO and its scavenger taurine, no detectable 19F MRI signal was observed (Fig. 2c and Fig. S4, ESI).

To further confirm the cleavage induced by HClO, high performance liquid chromatography (HPLC) was used to track this process (Fig. 2d). After treating Probe 1 with HClO, the original peak for Probe 1 at 30 min shrank distinctly and an intense peak of 3,5-bis-trifluoromethyl-benzoic acid appeared at 60 min. In contrast, the treatment of Probe 1 with both HClO and taurine resulted in the appearance of a little peak for 3,5-bis-trifluoromethyl-benzoic acid. Collectively, these results demonstrate that Probe 1 could specifically respond to HClO without the interference from other ROS, leading to a significant signal recovery for 19F NMR/MRI due to the cleavage of the bihydrazide, which could be exploited for the visualization of HClO in organisms.

image file: c9cc06622e-f3.tif
Fig. 3 (a) 19F MRI phantom images and (b) the corresponding SNRs of Probe 1 at different concentrations (with respect to Gd) before and after being treated with HClO (1 equiv.) for 1 h. (c) 19F MRI phantom images acquired with different TE values and (d) the corresponding SNRs of Probe 1 (5.5 mM), Probe 1 treated with HClO (1 equiv.), and Probe 1 treated with HClO (1 equiv.) and taurine (1.2 equiv.).

The behavior of the 19F MRI signal recovery of Probe 1 in the presence of HClO was further investigated. The concentration dependence of the signal recovery was first studied. A series of solutions containing Probe 1 at different concentrations in the presence and absence of HClO were subjected to 19F MRI. The phantom images show that the intensity of the recovered signal increased as the concentration of Probe 1 was increased (Fig. 3a). Quantitative analysis confirms the increasing trend of the signal-to-noise ratio (SNR) of Probe 1 with the increase of the concentration, either with or without HClO. Though at high concentrations the SNRs of Probe 1 with and without HClO were both increased, the enhancement of the SNR (SNRwith[thin space (1/6-em)]HClO/SNRwithout[thin space (1/6-em)]HClO) was decreased (Fig. 3b). The influence of the signal acquiring parameter TE on the signal recovery was also evaluated. Probe 1 treated with HClO showed strong signals with TE ranging from 1.3 to 3.9 ms, whereas Probe 1 alone and Probe 1 treated with HClO and taurine exhibited weak signals (Fig. 3c). It is noted that the signals of the three formulations all decreased with the increase of TE. For most common pulse sequences used for 19F MRI, approximately,

image file: c9cc06622e-t1.tif
where Savg represents the average signal intensity, T2 is the transverse relaxation time, and T2* is the time constant for the relaxation caused by the transverse relaxation and the inhomogeneity of an external magnetic field.29 In general, T2* < T2. If TE is shorter than or close to T2 (T2*) of the fluorine atoms, strong or moderate signals can be observed; on the other hand, if TE is much longer than T2 (T2*) of the fluorine atoms, the signals become very weak due to the exponential decay relationship between Savg and TE. Since the T2 (T2*) of fluorine atoms in Probe 1 alone or Probe 1 treated with HClO and taurine is very short (T2 = 1.2 ms < TE) due to the PRE effect, the corresponding 19F MRI signals are weak. In contrast, the T2 (T2*) of fluorine atoms in Probe 1 treated with HClO is prolonged because of the cleavage of the bihydrazide and the extinction of the PRE effect, resulting in T2 (T2*) ≫ TE and strong 19F MRI signals. The exponential decay relationship between Savg and TE also explains the abatement of the signal intensities with the extension of TE (Fig. 3d).

The detection of HClO by 19F NMR at the cellular level was evaluated with hepatocellular carcinoma SMMC-7721 cells. After being treated with Probe 1, SMMC-7721 cells were washed with PBS, and exposed to various stimuli including HClO, excess H2O2, and HClO + taurine, after which the cells were collected by centrifugation and lysed and then subjected to 19F NMR (Fig. 4a). A very broad peak was observed with the lysate of the cells stimulated with PBS, suggesting that the concentration of endogenous HClO is too low to activate Probe 1, which necessitates the introduction of exogenous HClO. As expected, a strong 19F NMR signal could be detected with the cells stimulated with HClO (Fig. 4b). In contrast, no evident change was observed with the lysate of the cells stimulated with H2O2 up to 15 mM. Addition of taurine successfully prevented the recovery of the 19F NMR signal, which is consistent with the aforementioned experiments. A quantitative analysis reveals the superior relative SNR of the HClO-stimulated group compared to those of the other groups, indicating the high selectivity of Probe 1 (Fig. 4c). Meanwhile, Probe 1 shows no appreciable cytotoxicity even at concentrations up to 3 mM (Fig. S5, ESI). These results reveal the good cell permeability of Probe 1 and evidence its intracellular reaction with exogenous HClO, indicating the feasibility and specificity of Probe 1 for the detection of HClO in cells.

image file: c9cc06622e-f4.tif
Fig. 4 (a) Schematic illustration of the protocol for the detection of HClO with Probe 1 by 19F NMR at the cellular level. (b) 19F NMR spectra of SMMC-7721 cells incubated with Probe 1 (1.5 mM) for 4 h followed by PBS, H2O2, HClO (1.5 mM) + taurine (1.8 mM), or HClO (1.5 mM) for 40 min. (c) Relative SNRs of 19F NMR spectra in (b). The SNR of the PBS-treated sample was normalized as 1.0. n.s., not significant.

Finally, the capability of Probe 1 for in vivo imaging of HClO was assessed with nude mice. A nude mouse was subjected to subcutaneous injection with Probe 1 into both the left and right flanks followed by injection of HClO into the right flank. Strong 1H MRI signals were observed for both the left and right flanks due to the contrast-enhanced effect of Gd3+ chelate molecules (Gd-DOTA) for 1H MRI. However, a strong 19F MRI signal was only observed for the right flank, indicating the outstanding specificity of Probe 1 for imaging of HClO. Quantitative analysis of the merged image reveals the excellent colocalization of 1H MRI and 19F MRI signals into the right flank (Fig. 5d). These results demonstrate that Probe 1 is feasible for in vivo imaging of HClO with 1H MRI providing anatomical details and 19F MRI offering specific distribution in deep tissues. Similar to most small molecular probes, challenges including rapid clearance and limited delivery to targets should be considered for design in the future to realize the targeted 19F MRI in living subjects.

image file: c9cc06622e-f5.tif
Fig. 5 (a) 1H and (b) 19F MRI of a nude mouse after subcutaneous injections of Probe 1 (10 mM, 150 μL) into both the right and left flanks, followed by an additional injection of HClO (1 mM, 25 μL) into the right flank. (c) The merged image of (a) and (b). (d) A plot showing the signal intensities of 1H MRI and 19F MRI along the yellow line as indicated in (c), which reveals the colocalization of 1H MRI and 19F MRI signals in the right flank of the mouse.

In summary, we have developed an activatable probe based on a fluorinated bihydrazide conjugate for sensing and imaging of hypochlorous acid via19F NMR/MRI. This small molecular conjugate could selectively respond to hypochlorous acid, which cleaves the bihydrazide, leading to the recovery of the 19F signals that are “extinguished” in the intact probe due to the PRE effect. The recovered signals could be promptly detected by 19F NMR/MRI. We believe that this small molecular conjugate holds great potential as a promising probe for selective detection and real-time imaging of hypochlorous acid in organisms and would be inspiring for the design and construction of probes for sensing and imaging of other chemical species in biological systems.

The authors acknowledge the research support from the National Natural Science Foundation of China (21771148, 21602186, 21521004, and 81430041), the Natural Science Foundation of Fujian Province of China (2018J01011), and the Fundamental Research Funds for the Central Universities (20720170020, 20720170088, and 20720180033).

Conflicts of interest

There are no conflicts of interest.

Notes and references

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Electronic supplementary information (ESI) available: Experimental details and Fig. S1–S3. See DOI: 10.1039/c9cc06622e
These authors contributed equally to this work.

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