English

• The Parkinson’s disease (PD) is one of the most common neurodegenerative diseases that affects more than 1% population over 65 years [1,2]. The characteristic manifestations of PD included resting tremor, bradykinesia, rigidity, impaired coordination and often accompanied by comorbid symptoms such as anxiety, depression, and cognitive impairment [3-5]. These symptoms seriously affect the normal life of PD patients. Therefore, the research on the pathogenesis and diagnostic medical of PD has been at the forefront of neuroscientific research [6-8].

  Currently, the diagnosis of PD is mainly based on the detection of medical devices, including single-photon emission computed tomography (SPECT) [9], magnetic resonance imaging (MRI) [10], and positron emission tomography [11]. In addition, the detection of biomarkers (a-synuclein (a-syn)) in the plasma or cerebrospinal fluid was also an important means to estimate the progression of the disease [12-14]. Even so, none of these methods achieve satisfactory specificity, sensitivity, and non-invasiveness in the diagnosis of PD.

  Fluorescence imaging technology using light as the medium has recently captivated a broad spectrum of attention in the chemical biology field with respect to its multitudinous excellent features, including non-invasive, high sensitivity, and real-time response [15-18]. Against the backdrop, fluorescent probes with PD biomarkers as recognition sites have been developed for the diagnosis and pathological analysis of PD [19-21].

  Nitric oxide (NO) is an important neurotransmitter in the brain, and its homeostasis disorder indicates the occurrence of a variety of neurodegenerative disease [22-25]. Among them, the occurrence of PD is inextricably related to the change of nitric oxide content in the brain. Studies have shown that neuroinflammation caused by PD can cause a surge in NO levels in the brain [26]. Currently, masses of fluorescent probes have been reported to be developed to detect NO in living systems [27-30]. However, the probes used to reveal NO fluctuation in PD brain are rarely reported. A major reason may be the inadequacy of the probe’s blood-brain barrier (BBB) crossing ability [31,32]. The BBB is a natural barrier that protects the brain tissue from outside substances [33,34]. Therefore, developing a probe that can monitor NO in the brain remains pretty challenging task.

  Herein, we designed a NO-activated fluorescence probe PO-NH with near-infrared (NIR) emission. The aim is to study the complex relationship between NO and PD, and achieve in vivo diagnosis and evaluation of PD progression. The probe showed enhanced red emission by blocking the photo-induced electron transfer (PET) process through NO-mediated conversion of diamine into a triazole, and showed an excellent selectivity for NO (Scheme 1). Due to the small molecular structure and favorable lipophilicity of the PO-NH scaffold, the probe exhibits superior BBB crossing capabilities, enabling it for in vivo monitoring NO fluctuations in the brain. Notably, at the different pathological stages of NO was vividly revealed with PO-NH in PD mouse brains, which showed great potential in studying the fluctuations of NO during the pathogenesis of PD.

  Scheme 1

  

  Scheme 1.  The proposed sensing mechanism for PO-NH and NO.

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  The synthesis of probe PO-NH refers to previous work [35] and the specific synthesis route is shown in Fig. S1 (Supporting information). ¹H and ¹³C NMR spectra of all compounds are shown in Figs. S6–S10 (Supporting information).

  Phenoxazine dyes have conjugate rigid plane structure and excellent electron donating properties and most of them have NIR imaging capability and good biocompatibility [36,37]. We designed and synthesized an activated fluorescent probe for specific recognition of NO based on the parent structure of phenoxazine dyes. It was mainly composed of NO recognition site (O-phenylenediamine structure) and parent phenoxazine dye. As shown in Fig. 1b, PO-NH is nonfluorescent due to PET. In the presence of NO, it emits a strong fluorescence at 660 nm, which is due to the great attenuation of its electron donating ability after the conversion of O-phenylenediamine into triazole, resulting in the blocking of the PET process (Scheme 1). In addition, the density functional theory (DFT) calculations (Fig. S4 in Supporting information) are also confirmed the molecular sensing mechanism. The intensity values and geometry of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) for PO-NH and PO-NO fully confirm that the probe undergoing PET process to quench the fluorescence.

  Figure 1

  

  Figure 1.  (a) Absorption spectra of PO-NO (5 µmol/L) and PO-NH (5 µmol/L) after reaction with NO (100 µmol/L) for 30 min. (b) Fluorescence emission of PO-NH (5 µmol/L) after reaction with NO (100 µmol/L) for 30 min. (c) Kinetic study of PO-NH (5 µmol/L) upon addition of NO (0, 50, 100 µmol/L). (d) Fluorescence spectra of PO-NH (5 µmol/L) treated with NO (0–100 µmol/L) for 30 min. (e) Linear relationship between fluorescence intensity and NO (0–80 µmol/L) concentration at 660 nm. (f) Fluorescence intensity of PO-NH (5 µmol/L) at 660 nm after 30 min response with different substances (200 µmol/L). Black: PO-NH, 2: Ca²⁺, 3: Zn²⁺, 4: Mg²⁺, 5: CO₃²⁻, 6: SO₃²⁻, 7: NO₂⁻, 8: S₂O₃²⁻, 9: H₂O₂, 10: ClO₄⁻, 11: Cys, 12: GSH, 13: Hcy, 14: Glyoxal, 15: l-Trp, 16: l-Tyr, 17: l-Arg, 18: l-Ser. Data are presented as the mean value, and the error bars represented the standard deviation (SD) from the mean value (n = 3).

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  The fluorescent properties of the probe PO-NH, were first evaluated. To our delight, it exhibits very low fluorescence quantum yield (φ = 0.003) and brightness (ε × φ = 111.6) in phosphate buffered saline (PBS), which indicates that the fluorescence signal of PO-NH has the potential to be activated (Table S1 in Supporting information). In order to confirm the responsiveness of PO-NH to NO, we investigated its spectral properties for the detection of NO in PBS solutions. As shown in Fig. 1a, PO-NH essentially possessed a maximum absorption peak centered at 600 nm. After NO mediation, the absorption peak at 600 nm gradually moves to the pre-synthesized PO-NO, indicating that the O-phenylenediamine in the PO-NH structure was gradually transformed into triazole. Attendant to, with the increasing concentration of NO (0–100 µmol/L), there was a gradual increase about 15-fold in the emission peak at 660 nm (Fig. 1d), which indicated its favorable “off-on” ability as a NO probe. Further study on the linear fluorescence response of PO-NH to NO (0–80 µmol/L) show that it has a good linear relationship for the detection of NO (R² = 0.98, Fig. 1e). Moreover, the reaction kinetics of PO-NH at different concentrations of NO (0, 50, 100 µmol/L) was studied. As shown in Fig. 1c, after the addition of NO, the fluorescent intensity of PO-NH at 660 nm was nearly stable within 8 min (Fig. 1c, Fig. S2 in Supporting information), which provides a guarantee for real-time detection. In addition, the corresponding detection limits is calculated (k = 3) to be 0.651 µmol/L for NO. The complexity of the biological environment always causes difficulties for the probe application, so the specificity of the probe is the basis for in vivo analysis. Therefore, we evaluated the selectivity of PO-NH. When these bio-related substances co-existed with PO-NH in PBS buffer, there was no significant change in fluorescent intensity. However, when NO was added, the emission peak at 660 nm was significantly enhanced, which showed excellent selectivity for NO (Fig. 1f).

  Encouraged by the results in vitro testing, we further investigated its ability to monitor the change of NO content in cellular level. Prior to bioimaging, the cytotoxicity of PO-NH was assessed by cell counting kit-8 (CCK-8). We evaluated the biotoxicity of the probe in HeLa and PC12 cells, respectively. Until the probe concentration is increased to 20 µmol/L, the survival rate of both cells could reach above 80% (Fig. S5 in Supporting information). These results show that the probe itself has excellent biocompatibility. Therefore, we further explored the ability of PO-NH to monitor exogenous and endogenous NO in cells. HeLa cells stained by the probe showed a very weak fluorescent signal. But, when the cells were pretreated with sodium nitroprusside dihydrate (SNP), a widely used NO donor [38], the intracellular fluorescent signal was significantly enhanced (Fig. 2). This suggests that PO-NH can effectively detect exogenous NO in living cells.

  Figure 2

  

  Figure 2.  Confocal imaging of extrinsic to endogenous NO level in living HeLa cells. (a) Control group: Cells and probe PO-NH (5 µmol/L) were incubated for 5 min. (b) SNP group: The cells were first incubated with SNP (300 µmol/L) for 1 h, followed by probe staining. (c) LPS group: The cells were incubated with LPS (20 µg/mL) for 12 h, followed by probe staining. (d) The cells were incubated with LPS (20 µg/mL) and l-Arg (5 mg/mL) for 12 h, followed by probe staining. (e) The cells were incubated with LPS (20 µg/mL) and l-NAME (0.5 mmol/L) for 12 h, followed by probe staining. Red channel: (λ_em = 650–720 nm. λ_ex = 633 nm). Scale bars: 10 µm.

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  Lipopolysaccharide (LPS) and l-arginine (L-Arg) are well-known NO stimulants that induce endogenous NO level to rise [39]. When the cells were pretreated with LPS or LPS + l-Arg, the cells showed significantly enhanced fluorescent signal compared with the control group (Fig. 2). At the same time, NG-nitroarginine methyl ester hydrochloride (L-NAME) as an NOS inhibitor to eliminate endogenous NO in cells [40]. Herein, we used it as a negative control group. The fluorescent intensity of cells pretreated with L-NAME decreased significantly, indicating that it effectively inhibited the production of endogenous NO (Fig. 2). Therefore, PO-NH can effectively detect endogenous and exogenous NO.

  Rotenone is often used to establish PD model, which induces oxidative stress and leads to the loss of dopaminergic neurons [41]. As shown in Fig. 3, PO-NH was incubated with the cells, imaging results showed that the fluorescent intensity of PD model was significantly stronger than that of control group. This showed that NO concentration was increased by the rotenone-induced PD model group, in other words, NO in the cellular PD model was monitored by PO-NH.

  Figure 3

  

  Figure 3.  Imaging of PC12 cells PD model with PO-NH. (a) Control group: Untreated cells were incubated with PO-NH (5 µmol/L, 5 min). (b) PD model: Cells treated with rotenone (1 µmol/L) for 1 h were then incubated with PO-NH (5 µmol/L, 5 min). (c) Average fluorescence intensity ratio between control (a) and PD model (b) groups. Data are presented as mean ± SD (n = 3). Scale bars: 20 µm.

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  As an optimal intracranial NO imaging probe, several requirements must be met including excellent sensitivity and selectivity for NO, NIR emission, and excellent BBB penetration capacity. Previous experiments have demonstrated the superior reactivity of PO-NH to NO. Therefore, to evaluate whether PO-NH could effectively cross the BBB, lipophilicity (logP, one key consideration for crossing BBB for small molecules) was estimated [42,43]. The logP value of PO-NH was 1.08, indicating that PO-NH had the potential to penetrate the BBB.

  In order to study the changes of NO levels in the brain of mice at different PD stages, BALB/c-nu mice induced by methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for different days (7, 14 days) were selected as the middle and late PD models. All the animal experiments were performed by following the protocols approved by Radiation Protection Institute of Drug Safety Evaluation Center in China (Production license: SYXK (Jin) 2023–0007). BALB/c-nu mice injected with the same volume of saline were selected as the control group. As shown in Fig. 4a, after PO-NH (100 µmol/L 150 µL) was injected through the tail vein, the time-dependent fluorescence imaging showed different concentrations of NO in each group’s mouse brains. In PD mice, the fluorescent intensity increased significantly during 0–40 min and remained stable within 40–120 min. By contrast, without obvious fluorescent signal was observed in the control group. To one’s excitement, the fluorescent intensity of PD model group after 14 days of drug induction was higher than that control group and after 7 days of drug induction (Fig. 4b). After quantifying the data, we can observe this phenomenon more directly (Fig. 4d). To confirm that the probe was concentrated at the mouse brains after the tail vein was injected, we performed in vitro NIR imaging of the brains of anatomic PD and control group (Fig. 4c). In vitro imaging results showed that the level of NO in the brain increased significantly with the aggravation of PD. This further confirmed the BBB crossing ability and NO detection performance of PO-NH. Therefore, it is reasonable to believe that PO-NH can be used as a potential tool to study the involvement of NO in different stages of PD.

  Figure 4

  

  Figure 4.  In vivo imaging of NO in PD mice with different stages. (a) In vivo fluorescence imaging within 2 h in the brains of control and PD mice by injection of PO-NH (100 µmol/L, 150 µL) through a tail vein. (b) After injecting PO-NH (100 µmol/L, 150 µL) into the tail vein for 1 h, fluorescence imaging was performed on PD mice at different periods. (c) Fluorescence intensity of PD mice brain in different stages was observed by anatomical experiment. (d) Quantitative data of fluorescence intensity in brain of PD mice at different stages. Data are presented as mean ± SD (n = 3). *P < 0.05. Red channel: λ_em = 700 ± 20 nm (λ_ex = 640 nm).

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  In summary, we have designed and synthesized a NIR probe (PO-NH) that can be activated by NO. PO-NH showed remarkable responsiveness, selectivity and high sensitivity for NO. At the cellular level, PO-NH is able to monitor the dynamic process of NO levels in living cells. Moreover, due to its ideal structure characteristics including its small molecular weight, logP value and moderate water solubility, PO-NH can effectively cross the BBB. This advantage enables real-time in vivo imaging of fluctuations in NO concentrations in the brain. Notably, facilitated by PO-NH, we found that with the deterioration of PD, the level of NO gradually increased in the brain of PD mice. The current study results confirmed that the variation of NO concentration in the brain is closely related to the development of PD. We envision that this work will further improve our knowledge of the diagnosis and therapy for PD.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Tao Liu: Writing – original draft. Xuwei Han: Formal analysis. Xueyi Sun: Validation. Weijie Zhang: Validation. Ke Gao: Investigation. Runan Min: Validation. Yuting Tian: Formal analysis. Caixia Yin: Project administration.

  Acknowledgments

  We thank the Program of Graduate Education and Teaching Reform of Shanxi (No. 2022YJJG302), the Applied Basic Research Programs of Shanxi (Nos. 201801D221106, 202203021221228), the Key R&D Project of Lvliang (No. 2023GXYF04), and Scientific Instrument Center of Shanxi University (No. 201512). The authors sincerely thank Suzhou Deyo Bot Advanced Materials Co., Ltd. and Professor Wentai Wang for providing support on material characterization.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110170.

  
  
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  Scheme 1  The proposed sensing mechanism for PO-NH and NO.

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  Figure 1  (a) Absorption spectra of PO-NO (5 µmol/L) and PO-NH (5 µmol/L) after reaction with NO (100 µmol/L) for 30 min. (b) Fluorescence emission of PO-NH (5 µmol/L) after reaction with NO (100 µmol/L) for 30 min. (c) Kinetic study of PO-NH (5 µmol/L) upon addition of NO (0, 50, 100 µmol/L). (d) Fluorescence spectra of PO-NH (5 µmol/L) treated with NO (0–100 µmol/L) for 30 min. (e) Linear relationship between fluorescence intensity and NO (0–80 µmol/L) concentration at 660 nm. (f) Fluorescence intensity of PO-NH (5 µmol/L) at 660 nm after 30 min response with different substances (200 µmol/L). Black: PO-NH, 2: Ca²⁺, 3: Zn²⁺, 4: Mg²⁺, 5: CO₃²⁻, 6: SO₃²⁻, 7: NO₂⁻, 8: S₂O₃²⁻, 9: H₂O₂, 10: ClO₄⁻, 11: Cys, 12: GSH, 13: Hcy, 14: Glyoxal, 15: l-Trp, 16: l-Tyr, 17: l-Arg, 18: l-Ser. Data are presented as the mean value, and the error bars represented the standard deviation (SD) from the mean value (n = 3).

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  Figure 2  Confocal imaging of extrinsic to endogenous NO level in living HeLa cells. (a) Control group: Cells and probe PO-NH (5 µmol/L) were incubated for 5 min. (b) SNP group: The cells were first incubated with SNP (300 µmol/L) for 1 h, followed by probe staining. (c) LPS group: The cells were incubated with LPS (20 µg/mL) for 12 h, followed by probe staining. (d) The cells were incubated with LPS (20 µg/mL) and l-Arg (5 mg/mL) for 12 h, followed by probe staining. (e) The cells were incubated with LPS (20 µg/mL) and l-NAME (0.5 mmol/L) for 12 h, followed by probe staining. Red channel: (λ_em = 650–720 nm. λ_ex = 633 nm). Scale bars: 10 µm.

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  Figure 3  Imaging of PC12 cells PD model with PO-NH. (a) Control group: Untreated cells were incubated with PO-NH (5 µmol/L, 5 min). (b) PD model: Cells treated with rotenone (1 µmol/L) for 1 h were then incubated with PO-NH (5 µmol/L, 5 min). (c) Average fluorescence intensity ratio between control (a) and PD model (b) groups. Data are presented as mean ± SD (n = 3). Scale bars: 20 µm.

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  Figure 4  In vivo imaging of NO in PD mice with different stages. (a) In vivo fluorescence imaging within 2 h in the brains of control and PD mice by injection of PO-NH (100 µmol/L, 150 µL) through a tail vein. (b) After injecting PO-NH (100 µmol/L, 150 µL) into the tail vein for 1 h, fluorescence imaging was performed on PD mice at different periods. (c) Fluorescence intensity of PD mice brain in different stages was observed by anatomical experiment. (d) Quantitative data of fluorescence intensity in brain of PD mice at different stages. Data are presented as mean ± SD (n = 3). *P < 0.05. Red channel: λ_em = 700 ± 20 nm (λ_ex = 640 nm).

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