English

• Rheumatoid arthritis (RA) is a chronic inflammatory disease with multi-system involvement and autoimmune features [1-3]. The disease primarily affects peripheral joints in a symmetrical distribution and can damage articular cartilage and bone, which in turn can lead to dysfunction and disability [4-7]. Despite significant advances in understanding its pathophysiology, the exact etiology of RA remains an open question. During the inflammatory process of RA, activated immune cells release large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), which in turn exacerbate oxidative stress [8-11]. Oxidative stress not only damages cellular components but also participates in the amplification of the inflammatory response [12,13]. As a result, inflammation resulted in cellular protein condensation and cell debris in the joint fluid increasing intracellular viscosity [14,15]. At the same time, peroxynitrite (ONOO⁻) is an RNS with high oxidizing and nitrating activity, which is considered a potential biomarker of RA and is closely associated with key pathological activities of RA. The molecule has a relatively short half-life and is generated under physiological conditions mainly by the free diffusion reaction of nitric oxide and superoxide anion. Considering the crucial roles of ONOO⁻ and viscosity in RA, the development of imaging tools that are capable of monitoring changes in ONOO⁻ and cellular viscosity in real-time is of great scientific and clinical significance for unraveling the mechanisms of RA pathology, assessing disease activity, and guiding clinical interventions.

  Small molecule fluorescent probes are widely recognized as powerful tools in sensing and optical imaging due to their high sensitivity, specificity, rapid response, non-invasiveness, and visualization capabilities [16-19]. In the early stage of RA, in-vitro assays failed to analyze ONOO⁻ due to its short lifetime. On this occasion, the in-situ detection feature of fluorescent probes may provide a superior way [20]. Similar to the ONOO⁻ case, the subtle cellular viscosity also highlighted the necessity of fluorescent probes for in-situ analysis [21]. However, because of the fluorescence wavelengths and specificity requirement of probes for in vivo imaging, simultaneous analysis of two analytes via efficient spectra split and red-to-near-infrared fluorescence signals is quite challenging [22-29].

  Merocyanines, which fuse the modifiability and red-to-near-infrared fluorescence signals, are a good choice for constructing fluorescent probes for in vivo analysis. As shown in Scheme 1A, to enhance the fluorescence sensitivity toward viscosity, a rotatable moiety dimethylaminophenyl was introduced into the fluorophore via a C—C bond. For ONOO⁻ responses, diphenylphosphine was elected as the reaction site because of its relatively higher specificity compared with the borinic acids [30-32]. We named the new probe YLS. YLS responds to viscosity with turn-on fluorescence emission at 760 nm, and the reaction between the probe and ONOO⁻ resulted in turn-on fluorescence at 625 nm. The ~140 nm emission wavelength difference provided an efficient imaging channel split. In our cellular imaging experiments, we confirmed the utility of YLS for ONOO⁻ and viscosity detection simultaneously. Supported by this dual-response fluorescent probe, we evaluated the ONOO⁻ and viscosity conditions in the RA model.

  Scheme 1

  

  Scheme 1.  Mechanisms of YLS for ONOO⁻ and viscosity response.

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  The spectral properties of YLS and the corresponding responses toward ONOO⁻ and viscosity were first evaluated. As shown in Fig. 1A, in the ultraviolet-visible (UV–vis) absorption spectrum, YLS shows two significant absorption peaks at 560 and 360 nm, and as the concentration of ONOO⁻ increases, a decrease in the absorption peak at 560 nm and an increase at 360 nm were observed. Furthermore, through fluorescence titration experiments, we observed that the fluorescence emission peak at 625 nm gradually intensified with the addition of ONOO⁻ (Fig. 1B). The fluorescence enhancement is attributed to the reaction between ONOO⁻ and the diphenylphosphine moiety in YLS. The specificity of YLS was assessed in the presence of various anions, cations, metal ions, amino acids, and ROS. As shown in Fig. S6 (Supporting information), the fluorescence intensity of YLS at 625 nm increased significantly only after reacting with ONOO⁻, indicating the probe's high selectivity. We further determined the kinetics of the reaction between YLS and ONOO⁻. As shown in Figs. 1C and D, the reaction reached equilibrium within 168 s. The detection limit was 3.5 µmol/L, demonstrating the probe's high sensitivity. Next, the viscosity-dependent spectral characteristics of YLS in a methanol and glycerol mixture were analyzed. As shown in Fig. S7 (Supporting information), the UV–vis absorption peak of YLS at 590 nm showed a gradual enhancement with increasing glycerol volume ratio. Correspondingly, a synchronous increase in the intensity of the fluorescence emission at 760 nm was observed in the fluorescence spectrum (Fig. 1E). The spectra changes were attributed to the restriction of the C—C bond rotation within YLS caused by increased viscosity, which inhibited the twisted intramolecular charge transfer between the electron donor (dimethylaminophenyl) and the acceptor (merocyanine) and lightened fluorescence signal. Furthermore, a significant linear relationship is observed between the fluorescence intensity (LogI₇₆₀) and viscosity (Logη), with an R-squared (R²) value of 0.9901 (Fig. 1F), indicating that YLS can serve as an effective optical probe for viscosity analyze.

  Figure 1

  

  Figure 1.  Spectral properties of YLS toward ONOO⁻ and viscosity. (A) UV–vis titration of 10 µmol/L YLS with 0–63 µmol/L ONOO⁻. (B) Fluorescence titration of 10 µmol/L YLS with 0–220 µmol/L ONOO⁻. (C) Time-dependent fluorescence responses of YLS toward 150 µmol/L ONOO⁻. (D) Detection limit of YLS for ONOO⁻ (λ_ex = 440 nm. λ_em = 625 nm, slit width: 5 nm/5 nm). (E) Fluorescence responses of 10 µmol/L YLS toward viscosity. The different viscosity was mimicked by different methanol/glycerol volume ratios mimicked the different viscosity. (F) Linear relationship between logI_{760 nm} and logη.

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  The recognition mechanism of diphenylphosphine structure for ONOO⁻ has been reported in the literature. In this study, we used mass spectrometry (MS) to verify the reaction between YLS and ONOO⁻. The MS result showed that the mass-to-charge ratio (m/z) of YLS was 663.2226. Upon the addition of the ONOO⁻, a new signal, m/z 357.1415, was detected, which coincided with the theoretically calculated (m/z + H)⁺ value of 357.1425 (Fig. S5 in Supporting information). This result is consistent with our hypothesis that YLS reacted with ONOO⁻ to release the quinoline derivative via an oxidation-induced self-immolation mechanism (Scheme 1B). These findings provide a solid scientific basis for fluorescence analysis of ONOO⁻ using YLS.

  Sulforhodamine B (SRB) assay was then used to evaluate the cytotoxicity of YLS in HeLa cells before cell imaging experiments. As shown in Fig. S8 (Supporting information), the measured cell viability reached 80.96% with a concentration of 10 µmol/L and an incubation time of 10 h, indicating satisfactory biocompatibility of YLS. Subsequently, the mitochondria labeling capacity of YLS supported by the natural cation feature was evaluated by co-staining HeLa cells with Mito-Tracker Green, a commercially available mitochondrial dye. The fluorescence signal of YLS in the red channel was activated upon reaction with endogenous ONOO⁻, which was mainly formed in mitochondria during respiration. As shown in Fig. 2, the red fluorescent signal exhibited good co-relation with the green fluorescent signal of Mito-Tracker Green in the mitochondrial region of the cells with a Pearson's coefficient of 0.89. These results fully confirmed the ability of YLS to specifically target mitochondria.

  Figure 2

  

  Figure 2.  Colocalization of YLS in HeLa cells. (A) Fluorescence imaging of cells incubated with 10 µmol/L YLS. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm. (B) Fluorescence imaging of 0.2 µmol/L Mito-Tracker Green stained cells. Green channel: λ_ex = 488 nm, λ_em = 510–540 nm. (C) Merged image of the two channels. (D) Correlation analysis.

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  The cellular ONOO⁻ responsive specificity of YLS was further confirmed by stimulants such as lipopolysaccharide (LPS) and 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1). LPS is an endotoxin from Gram-negative bacteria that strongly activates the immune system and mimics the inflammatory response to infection or injury [33]. SIN-1 releases nitric oxide (NO) and O₂^•− under physiological conditions, and these two substances react rapidly to generate ONOO⁻ [34]. During the experiment, HL-7702 cells were treated with 1 µg/mL of LPS and 100 µmol/L of SIN-1 for 20 min, respectively, followed by a 15 min incubation of 10 µmol/L YLS for laser confocal microscopy imaging analysis. Indeed, both LPS and SIN-1 induced significant fluorescence enhancement in the red channel and confirmed the specificity of YLS for ONOO⁻ (Fig. 3A).

  Figure 3

  

  Figure 3.  (A) Fluorescence imaging of 10 µmol/L YLS in cellular inflammation models. (B) Fluorescence imaging of intracellular viscosity by 10 µmol/L YLS. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm; NIR channel: λ_ex = 633 nm, λ_em = 700–754 nm.

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  For the specificity evaluation toward viscosity, nystatin (Nys) and rapamycin (RAPA), compounds that can cause mitochondrial dysfunction and increase viscosity, were used in further cell imaging experiments [35-37]. Upon Nys and RAPA treatment, the cellular fluorescence intensities presented systematic enhancement in the near infrared (NIR) channel (Fig. 3B). Thus, the fluorescence signal in the NIR channel can indicate specific viscosity in the cell.

  Since most cancer cells exhibit higher levels of ROS, RNS, and viscosity than normal cells [38-40], specific fluorescent probes can help understand the biological mechanisms in cancer cells and provide new targets and strategies for cancer treatment and prevention. We used YLS to assess basal levels of ONOO⁻ and viscosity in a normal cell line (HL-7702) and cancer cell (HepG2). As shown in Fig. 4, after incubation of 10 µmol/L YLS, HepG2 cells exhibited higher fluorescence signals in both the red and NIR channels compared with HL-7702 cells, which suggests the potential capability of YLS for cancerization visualization. Besides, these results also demonstrated the efficiency of YLS for cellular ONOO⁻ and viscosity analysis simultaneously via dual-channel fluorescence imaging.

  Figure 4

  

  Figure 4.  Two-channel fluorescence imaging of 10 µmol/L YLS in HL-7702 cells. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm; NIR channel: λ_ex = 633 nm, λ_em = 700–754 nm.

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  Tissue injury during RA is mainly caused by inflammation-mediated cell damage and death via multiple mechanisms, such as ferroptosis and apoptosis [41-45]. The corresponding molecular and viscous conditions were evaluated by YLS during ferroptosis or apoptosis. For ferroptosis entry, erastin was used to construct a cell model. As shown in Fig. 5, the fluorescence intensities of both the ONOO⁻ and viscosity channels in the erastin group were significantly enhanced compared with the control group incubated with YLS. Notably, the fluorescence signal of both channels decreased when ferroptosis inhibitor Fer-1 was added. Thus, the fluorescence signal changes in both the ONOO⁻ and viscosity channels can indicate cell injury during RA.

  Figure 5

  

  Figure 5.  Two-channel fluorescence imaging of 10 µmol/L YLS in a ferroptosis model of HeLa cells. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm; NIR channel: λ_ex = 633 nm, λ_em = 700–754 nm.

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  Supported by the dual-channel red-to-NIR fluorescence responses of YLS toward ONOO⁻ and viscosity, we then performed in vivo fluorescence imaging experiments to detect ONOO⁻ and viscosity in RA mice. All the animal experiments were performed by following the protocols approved by the Radiation Protection Institute of Drug Safety Evaluation Center in China (Production license: SYXK (Jin) 2018–0005). All animal experiments were performed according to the protocols approved by the Animal Ethics and Use Committee. λ-Carrageenan was used to construct the RA mouse model. The corresponding inflammatory lesion was confirmed by histopathological analysis (Fig. S9 in Supporting information). In the following fluorescence imaging experiments, YLS was injected in the left (control) and right (RA) hind leg joints of mice, respectively. The real-time fluorescence imaging results (Fig. 6) confirmed that both ONOO⁻ and viscosity increased in RA, which was consistent with the cellular inflammation models. Based on YLS, we confirmed that the activation of lymphocytes during RA will lead to an increase in the production of ONOO⁻, while the increase in the metabolic activity of inflammatory cells may lead to an increase in the viscosity of the intracellular environment, which further affects the normal function of cells.

  Figure 6

  

  Figure 6.  Dual-channel in vivo fluorescence imaging of ONOO⁻ and viscosity in RA mice. Red channel: λ_ex = 465 nm, DsRed; NIR channel: λ_ex = 570 nm, Cy5.5. Data are presented as mean ± standard deviation (SD) (n = 3).

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  In this study, a novel fluorescent probe for simultaneous ONOO⁻ and viscosity detection via dual-channel fluorescence imaging was designed to illustrate their potential pathological roles in RA. Featuring 625 and 760 nm turn-on fluorescence responses toward ONOO⁻ and viscosity, respectively. The probe provided efficient in situ discriminative detection of the two indexes in both cellular level and mouse models. We demonstrated the corresponding sensitivity and specificity by a series of inducers, and we found that ONOO⁻ and viscosity levels changed concomitantly in both cell and mouse models during inflammation. The probe not only promotes the understanding of the pathomechanisms of RA but also provides a potential molecular imaging tool for clinical diagnosis and therapeutic monitoring.

  Declaration of interest statement

  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

  Qian Pang: Writing – original draft, Investigation. Fangjun Huo: Validation, Data curation. Yongkang Yue: Writing – review & editing, Data curation. Caixia Yin: Supervision, Project administration.

  Acknowledgments

  We thank the National Natural Science Foundation of China (Nos. 22325703, 22377071, U23A6009), Research Project Supported by Shanxi Scholarship Council of China (No. 2022-002), the Shanxi Province Science Foundation (No. 202203021221009), Shanxi Province Science and Technology activities for overseas people selected funding project (No. 2024001).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110713.

  
  
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  Scheme 1  Mechanisms of YLS for ONOO⁻ and viscosity response.

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  Figure 1  Spectral properties of YLS toward ONOO⁻ and viscosity. (A) UV–vis titration of 10 µmol/L YLS with 0–63 µmol/L ONOO⁻. (B) Fluorescence titration of 10 µmol/L YLS with 0–220 µmol/L ONOO⁻. (C) Time-dependent fluorescence responses of YLS toward 150 µmol/L ONOO⁻. (D) Detection limit of YLS for ONOO⁻ (λ_ex = 440 nm. λ_em = 625 nm, slit width: 5 nm/5 nm). (E) Fluorescence responses of 10 µmol/L YLS toward viscosity. The different viscosity was mimicked by different methanol/glycerol volume ratios mimicked the different viscosity. (F) Linear relationship between logI_{760 nm} and logη.

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  Figure 2  Colocalization of YLS in HeLa cells. (A) Fluorescence imaging of cells incubated with 10 µmol/L YLS. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm. (B) Fluorescence imaging of 0.2 µmol/L Mito-Tracker Green stained cells. Green channel: λ_ex = 488 nm, λ_em = 510–540 nm. (C) Merged image of the two channels. (D) Correlation analysis.

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  Figure 3  (A) Fluorescence imaging of 10 µmol/L YLS in cellular inflammation models. (B) Fluorescence imaging of intracellular viscosity by 10 µmol/L YLS. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm; NIR channel: λ_ex = 633 nm, λ_em = 700–754 nm.

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  Figure 4  Two-channel fluorescence imaging of 10 µmol/L YLS in HL-7702 cells. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm; NIR channel: λ_ex = 633 nm, λ_em = 700–754 nm.

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  Figure 5  Two-channel fluorescence imaging of 10 µmol/L YLS in a ferroptosis model of HeLa cells. Red channel: λ_ex = 561 nm, λ_em = 630–670 nm; NIR channel: λ_ex = 633 nm, λ_em = 700–754 nm.

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  Figure 6  Dual-channel in vivo fluorescence imaging of ONOO⁻ and viscosity in RA mice. Red channel: λ_ex = 465 nm, DsRed; NIR channel: λ_ex = 570 nm, Cy5.5. Data are presented as mean ± standard deviation (SD) (n = 3).

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