The construction of a near-infrared fluorescent probe with dual advantages for imaging carbon monoxide in cells and in vivo

Abstract

As a kind of toxic gas, carbon monoxide (CO) can hinder uptake of oxygen in humans. However, more and more studies have shown that CO is an important gaseous messenger in the body and playing an indispensable role in intracellular signaling pathways. So, it is necessary to develop an analytical method to study CO in living organisms. Although there are many CO-responsive probes, most of them have the disadvantages of a small Stokes shift or short emission wavelength. In order to address the above issue, a novel probe (FDX-CO) with a large Stokes shift (190 nm) and long emission wavelength (770 nm) was firstly synthesized to detect CO. The probe shows high sensitivity and superior selectivity toward CO. Moreover, the probe was successfully used for visualizing exogenous and endogenous CO in cells by fluorescence imaging, 3D quantification analysis and flow cytometric analysis. More importantly, FDX-CO could excellently detect CO in mice, which suggests that this probe has the potential ability to image CO in vivo. This probe can be viewed as a useful tool in the research of CO.

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Introduction

Carbon monoxide (CO) is a kind of colorless and odorless gas molecule and is named an “invisible killer” due to its high toxicity. This could be explained because CO is very easy to combine with hemoglobin, leading to inadequate oxygen supply.¹ However, scientific studies in recent years have shown that endogenous CO can be produced by heme metabolism and it is an important gaseous messenger in the human body.² For example, CO plays an indispensable role in various signaling pathways including vasodilation, antiapoptosis and neurotransmission.^3,4 It was reported that CO is also a potential drug and has positive effects on many diseases, acting, in particular, as an anti-inflammatory.^5–7 Moreover, the abnormal up-regulation or down-regulation of CO was closely connected with heart disease, tumors, Alzheimer's disease, hypertension, inflammation, etc.^8–11 Therefore, it is needed to develop an analytical method to visualize CO in living organisms.

Fluorescence imaging, as a real-time and in situ observation technique, has been widely used for marking and detecting functional molecules in bio-samples.^12–17 Among them, molecular fluorescent probes based on organic fluorescent dyes have the advantages of simple operation and good selectivity. To date, although there are many CO-responsive fluorescent probes, their emission wavelengths are mainly located in the visible region.^18–27 For example, the Tang group has reported a new probe for the detection of CO based on the fluorine boron pyrrole dye, which successfully visualizes intracellular CO up-regulation under hypoxia by this probe.²⁸ Moreover, the Feng group has developed a probe based on naphthalimide derivatives. This probe was successfully used to rapidly detect CO in air, aqueous solution, and living cells providing the first promising ratiometric fluorescent probe system.²⁹ But, none of the above work is suitable for imaging CO in vivo.

Near-infrared (NIR) fluorescence imaging technology is expected to solve the above issue because it can effectively reduce the absorption and scattering of light from tissues, improve the tissue penetration depth, and enhance the spatial resolution of fluorescence imaging.^30–37 Recently, some NIR fluorescent probes have been designed in order to image CO.^38–45 Our group reported a NIR fluorescent probe (CyAPC) and this probe was applied for imaging CO.³⁸ But, this probe has a small Stokes shift (30 nm), which would lead to severe crosstalk between the excitation spectrum and emission spectrum, a low signal-to-noise ratio and severe fluorescence self-quenching in imaging. This would limit its application in biological imaging. The Qi group has successfully designed a probe (DC-CO) which is applied to detect CO in living cells.³⁹ This probe possesses a short emission wavelength (685 nm) in spite of a large Stokes shift (120 nm) (Fig. 1A). Thus, it is urgent to develop an excellent probe with dual advantages (large Stokes shift and long emission wavelength).

Fig. 1 (A) The reported probe to detect CO. (B) The design of probe FDX-CO to CO with a large Stokes shift and long emission wavelength.

Herein, a NIR fluorescent probe (FDX-CO) was successfully designed (Fig. 1B). After reacting with CO and Pd²⁺, FDX-CO displays a large Stokes shift (190 nm) and long emission wavelength (770 nm) with a significant turn-on fluorescence response. More importantly, FDX-CO could visualize exogenous and endogenous CO in cells by fluorescence imaging, 3D quantification analysis and flow cytometric analysis. Furthermore, FDX-CO is successfully applied to detect CO in mice, which suggests that this probe has the ability to image CO in vivo. To the best of our knowledge, this structure was firstly used to design a CO-responsive NIR fluorescent probe with a large Stokes shift and long emission wavelength.

Results and discussion

Spectral response of FDX-CO to CO

Probe FDX-CO was synthesized according to the routes depicted in Scheme S1† and the ¹H NMR, ¹³C NMR and mass spectra of probe FDX-CO are available in the ESI (Fig. S1–3†). The response of probe FDX-CO to CO was also studied. For probe FDX-CO, its own absorption is centered at 580 nm. After the probe reacted with CO (CORM-3, CO-releasing agent) and Pd²⁺, a boosted absorption in 580 nm is found (Fig. 2A). With the increasing concentration of CO, the fluorescence of FDX-CO is gradually increasing (Fig. 2B). As displayed in Fig. 2C, there is a good linear correlation between fluorescence intensity and CO concentrations from 1 to 30 μM (R² = 0.9937). The detection limit is determined to be 0.33 μM (3σ/slope). These results suggest that FDX-CO could quantitatively analyse CO with high sensitivity.

Fig. 2 (A) The absorption spectra of FDX-CO (10 μM) and PdCl₂ (10 μM) in the absence or presence of CO (30 μM) in PBS buffer solution (0.1 M, pH 7.4). (B) Fluorescence spectra of FDX-CO (10 μM) and PdCl₂ (10 μM) upon the addition of CO (0, 1, 2, 6, 10, 14, 18, 22, 26, and 30 μM). (C) Linearity plots of the fluorescence intensity of probe FDX-CO and PdCl₂ against CO concentrations. (D) Fluorescence response of FDX-CO (10 μM) and PdCl₂ (10 μM) toward different analytes: 1, blank; 2, ClO⁻; 3, H₂O₂; 4, ONOO⁻; 5, NO₂⁻; 6, HS⁻; 7, SO₃²⁻; 8, Cys; 9, Hcy; 10, GSH; 11, Ala; 12, Trp; 13, Glu; 14, Na⁺; 15, Ca²⁺; 16, Fe²⁺; 17, Zn²⁺; 18, CO. The concentration of CO is 30 μM and that of other analytes is 300 μM. (E) Time-dependent fluorescence intensity of FDX-CO (10 μM) and PdCl₂ (10 μM) to CO (0, 6, and 30 μM). (F) Effect of pH on the fluorescence intensity of FDX-CO (10 μM) and PdCl₂ (10 μM) before and after the addition of CO (30 μM). λ_ex = 580 nm.

Moreover, the selectivity of probe FDX-CO was investigated by examining various potential interfering substances such as reactive species (ClO⁻, H₂O₂, ONOO⁻, NO₂⁻, HS⁻, SO₃²⁻, Cys, Hcy, GSH, Ala, Trp, Glu, Na⁺, Ca²⁺, Fe²⁺, and Zn²⁺) (Fig. 2D and Fig. S4†). It could be seen that the above analytes have no obvious interference on the detection of CO. As shown in Fig. 2E, the fluorescence of FDX-CO increases with time and reaches a plateau within 8 min at various concentrations of CO (0, 6, and 30 μM). A big change of fluorescence intensity of FDX-CO for CO is achieved at pH 7.4 (Fig. 2F). Finally, the stability of probe FDX-CO was studied (Fig. S5†). There is almost no change of fluorescence after 10 days, which suggests that the probe has good stability. These results suggest that probe FDX-CO could be applied for detecting CO with outstanding preference under a physiological environment.

The reaction mechanism of FDX-CO

A possible reaction mechanism of FDX-CO with CO is proposed in Fig. 3A. FDX-CO has weak fluorescence owing to the protection of the hydroxyl group by the allyl formate unit. Once CO and PdCl₂ were added, Pd²⁺ is firstly reduced into Pd⁰. Then, the allyl formate unit is cleaved from FDX-CO after Pd⁰ triggers the Tsuji–Trost reaction. This leads to the formation of FDX-OH and the recovery of a strong fluorescence signal.

Fig. 3 (A) The reaction mechanism of FDX-CO with CO. (B) Frontier molecular orbitals of FDX-CO and FDX-OH. (C) HPLC chromatograms of FDX-CO in the absence or presence of CO and Pd²⁺. The HPLC mobile phase was as follows: solvent A (CH₃OH), solvent B (distilled water), A/B = 15/85 (V/V).

Then, density functional theory (DFT) calculations by the B3LYP/6-311G method using the Gaussian 09 package were carried out. The optimized structures of FDX-CO and FDX-OH are shown in Fig. S6.† The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of FDX-CO and FDX-OH are presented in Fig. 3B. The electron density on the HOMO of FDX-CO is located on the allyl formate group and the LUMO is positioned at the FDX unit. This shows that there is a PET process. So, FDX-CO is non-fluorescent. The electron densities on the HOMO and LUMO of FDX-OH are both primarily located on the FDX unit. No PET occurs in this system and FDX-OH shows strong fluorescence. The theory calculation well supports the change of fluorescence.

In order to verify the proposed mechanism, the mass spectrometry analysis was performed. The peak of FDX-CO is at m/z = 481.3. After reacting with CO and Pd²⁺, a new peak at m/z = 396.3 corresponding to FDX-OH appears (Fig. S7†). Meanwhile, HPLC experiments were also performed to confirm the reaction mechanism and the results are shown in Fig. 3C. FDX-CO itself displays a signal peak at 11.0 min. Upon adding CO and Pd²⁺, a decrease in the FDX-CO signal is seen along with the appearance of new signals at 8.0 min, which has the same retention time as FDX-OH. All the results display the CO-induced transformation of FDX-CO into FDX-OH.

Fluorescence imaging of exogenous CO in living cells

Before cell imaging, the cytotoxicity of probe FDX-CO was evaluated in HepG2 cells by conventional MTT assay (Fig. S8†). The result shows that the probe with different concentrations is of low cytotoxicity, which makes the probe exhibit good biocompatibility.

The capability of FDX-CO for imaging exogenous CO in HepG2 cells was investigated. As shown in Fig. 4A and B, the cells display a very weak fluorescence signal in the presence of FDX-CO, which reflects that there is no potential interference fluorescence. When treated with Pd²⁺, the cells did not emit a fluorescence signal. After the cells are treated with CORM-3 (a CO donor) and Pd²⁺, there is a remarkable fluorescence enhancement. And the fluorescence intensity gradually increases with the increase of CORM-3′ concentrations. Then, the experiment of fluorescence intensity with time was studied and the fluorescence intensity increased with time (Fig. S9†). With the purpose of observing the change of fluorescence intensity in three dimensions, 3D quantification analysis was performed and the results are shown in Fig. 4C. These results are consistent with the change of fluorescence intensity in the cells (Fig. 4D) and reflect that probe FDX-CO has the ability to detect exogenous CO in cells.

Fig. 4 Fluorescence imaging of exogenous CO in cells. Control group: the cells were incubated with FDX-CO (10 μM) for 30 min. Pd²⁺ group: the cells were pretreated with Pd²⁺ (10 μM) for 30 min, and then incubated with FDX-CO (10 μM) for 30 min. 50 μM CORM-3 group: the cells were pretreated with 50 μM CORM-3 for 30 min and then treated with 10 μM FDX-CO and 10 μM PdCl₂ for 30 min. 100 μM CORM-3 group: the cells were pre-incubated with 100 μM CORM-3 for 30 min, and then incubated with 10 μM FDX-CO and 10 μM PdCl₂ for 30 min. λ_ex = 568 nm, λ_em = 750–850 nm. Scale bar: 10 μm. (A) Bright field imaging of CO. (B) Fluorescence field imaging of CO. (C) 3D quantification analysis for the above groups. (D) Quantification analysis of (B).

Fluorescence imaging of endogenous CO in living cells

Probe FDX-CO was applied for detecting endogenous CO in HepG2 cells. As shown in Fig. 5A and B, the cells display a weak fluorescence signal after they were incubated with FDX-CO alone for 30 min. When the cells were treated with LPS or Heme, which would induce the generation of CO due to the over-expression of HO-1, a large increase in fluorescence is easily observed. To further confirm that the over-expression of CO was really caused by heme, zinc protoporphyrin (ZnPP) which was regarded as an inhibitor of HO-1 was chosen to incubate with cells. After the cells are incubated with ZnPP, the fluorescence intensity obviously decreases.

Fig. 5 (A) Fluorescence imaging of endogenous CO in HepG2 cells. Control group: the cells were incubated with FDX-CO (10 μM) for 30 min. LPS group: the cells were pre-incubated with 1.0 μg mL⁻¹ LPS for 12 h, and then incubated with 10 μM FDX-CO and 10 μM PdCl₂ for 30 min. Heme group: the cells were pretreated with Heme (100 μM) for 10 h, and then incubated with FDX-CO (10 μM) and 10 μM PdCl₂ for 30 min. ZnPP group: the cells were pretreated with 100 μM Heme for 2 h and 20 μM ZnPP for 2 h and then treated with 10 μM FDX-CO and 10 μM PdCl₂ for 30 min. λ_ex = 568 nm, λ_em = 750–850 nm. Scale bar: 10 μm. (B) 3D quantification analysis for the above groups. (C) The flow cytometric analysis for the above groups. (D) Quantification analysis of (A). (E) The time-dependent fluorescence response to CO in HepG2 cells. The cells were incubated with FDX-CO (10 μM) at different time points after pre-treatment in Heme (100 μM). (F) 3D quantification analysis of (E). (G) Quantification analysis of (E). (H) 3D fluorescence imaging of endogenous CO and the length of fluorescence imaging in HepG2 cells.

Similarly, 3D quantification analysis also was carried out to observe the change of fluorescence intensity in cells. In addition, flow cytometry experiments were carried out. As displayed in Fig. 5C, the movement of magnitude indicates the change of fluorescence intensity in the cells. This result was verified by quantification analysis (Fig. 5D). At the same time, the time-dependent fluorescence response to endogenous CO in cells was measured and the fluorescence was gradually enhanced (Fig. 5E), which was quantified by 3D quantification analysis (Fig. 5F) and quantification analysis (Fig. 5G). Finally, 3D fluorescence imaging of endogenous CO was firstly studied. As displayed in Fig. 5H, the cell was filled with fluorescence, which reveals that there is a certain amount of CO in the cells. More importantly, the length of fluorescence imaging was 126 μm. This result reflects that the probe has the potential ability for deep imaging. Taken together, probe FDX-CO is able to image the endogenous CO level in cells.

Fluorescence imaging of CO in mice

On the basis of NIR optical properties and excellent cell imaging, the fluorescence imaging of probe FDX-CO in vivo was performed (Fig. 6A). After the intravenous injection of FDX-CO, almost no fluorescence signal is found. When the mice were injected by CORM-3, a strong fluorescence signal is observed and increases with time. Then, 3D quantification analysis for the above groups was carried out and the results were consistent with the change of fluorescence intensity (Fig. 6B). This result was also verified by quantification analysis (Fig. 6C). The above results illustrate that probe FDX-CO is successfully applied for visualizing CO in vivo.

Fig. 6 (A) Time-dependent fluorescence imaging of CO in mice. Control group: the mice were injected intravenously with 10 μM FDX-CO. Other groups: the mice were injected intravenously with 10 μM FDX-CO and 10 μM PdCl₂, and then were injected intravenously with 10 μM CORM-3. λ_ex = 600 nm, λ_em = 750–850 nm. (B) 3D quantification analysis of the fluorescence in (A). (C) Quantification analysis of (A).

Conclusions

In conclusion, we have reported a novel near-infrared (NIR) fluorescent probe (FDX-CO) to detect CO in solution, cells and in vivo with a large Stokes shift and long emission wavelength. Probe FDX-CO provides a low detection limit for CO (0.33 μM), which is sensitive enough for determining the CO level. The probe also shows outstanding selectivity to CO over other potential analytes under a physiological environment. Thanks to the minimal cytotoxicity of probe FDX-CO, the probe could monitor exogenous and endogenous CO in HepG2 cells by fluorescence imaging, 3D quantification analysis, and flow cytometry. In addition, the probe is successfully applied for visualizing CO in vivo. All in all, the probe FDX-CO could provide a useful tool for the research of CO in the future.

Ethical statement

All experiments were performed in accordance with the Guidelines “Regulations of Hunan Province on the administration of experimental animals” and approved by the ethics committee at Xiangtan university.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21775133), Hunan Provincial Natural Science Foundation (2018JJ2385), Scientific Research Fund of Hunan Provincial Education Department (19A479), Degree & Postgraduate Education Reform Project of Hunan Province (2019JGYB113) and Degree & Postgraduate Education Reform Project of Hunan Province (CX20190483, XDCX2020B115).

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Footnote

† Electronic supplementary information (ESI) available: Experimental section, additional spectroscopic data, cell cytotoxicity and supplemental fluorescence images in biological samples. See DOI: 10.1039/d0an01719a

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