Near-infrared fluorescence probes to detect reactive oxygen species for keloid diagnosis

Abstract

Development of molecular probes for the detection of reactive oxygen and nitrogen species (RONS) is important for the pathology and diagnosis of diseases. Although an abnormally high RONS level has been identified in keloids - a benign dermal tumour developed after lesion, the ability of employing RONS probes for keloid detection has not yet been exploited. Herein, we report two near-infrared (NIR) fluorescent probes (CyTF and CyBA) that can specifically distinguish keloid fibroblasts from normal dermal fibroblasts. Both CyTF and CyBA show a 15-fold NIR fluorescence enhancement at 717 nm upon reaction with RONS. However, because CyTF has higher specificity towards ONOO^- than CyBA, CyTF can detect stimulated fibroblasts in a more sensitive way, showing 3.76 and 2.26-fold fluorescence increments in TGF-β1 stimulated dermal fibroblasts and keloid fibroblasts, respectively. Furthermore, CyTF permits specific detection of implanted keloid fibroblasts in a xenograft live mouse model. Our work thus developed a new optical imaging approach that has the potential for early diagnosis and drug screening of keloids.

Scheme 1. (a) Design and mechanism of CyTF and CyBA for RONS imaging. (b) Illustration of CyTF and CyBA activation in KF cells due to higher RONS levels.

Scheme 2. Synthetic route of CyTF and CyBA. (a) H₂SO₄ (con.), reflux, 4 h, EtOH, 97%; (b) TMSCF₃, CsF, THF, 18 h, 86%; (c) TBAF, THF, 5 h, then 4 M HCl, 3 h, 97%; (d) BBr₃, –78 °C, room temperature, 12 h, CH₂Cl₂, 74%; (e) K₂CO₃, resorcinol, CH₃CN, 2 h; (f) 5, BTC, Et₃N, 0 °C, 2 h, CH₂Cl₂, 28%; (g) 4-methylphenylboronic acid pinacol ester, K₂CO₃, CH₃CN, 75%.

Fig. 1. UV-vis absorption spectra (a) and fluorescence (b) of CyTF or CyBA (20 μM) in the absence or presence of ONOO^– (25 μM) at 25 °C in PBS (1×, pH = 7.4) containing 20% DMSO. Excitation: 640 nm. (c) High performance liquid chromatography (HPLC) traces of the incubation mixture of CyTF (c) and CyBA (d) in the absence (upper panel) or presence (middle panel) of ONOO^– (25 μM), and HPLC traces of CyOH in water (lower panel). Wavelength: 600 nm. (e) Fluorescence titration of CyTF or CyBA (20 μM) as a function of ONOO^– concentration. Each spectrum was recorded 5 min after addition of ONOO^–. Regression equation for CyTF: FL = 1.07 × 10⁶ + 1.16 × 10⁶ [ONOO^–] μM (R² = 0.99); regression equation for CyBA: FL = 1.14 × 10⁶ + 1.27 × 10⁶ [ONOO^–] μM (R² = 0.99). (f) Fluorescence intensity of CyTF and CyBA in the presence of various RONS for 5 min in PBS (1×, pH = 7.4) containing 20% DMSO. Control represents assay buffer only. ONOO^– was 25 μM, O₂˙^–, OCl^–, ˙OH, and H₂O₂ were 50 μM. The incubation time for H₂O₂ was 2 hours. Error bars represent standard deviations of three separate measurements.

Fig. 2. (a) Fluorescence microscopy of NDF cells incubated with CyTF or CyBA (10 μM, 30 min) before imaging; first panel: untreated cells; second panel: cells treated with TGF-β1 (36 h); third panel: cells pre-treated with NAC (2 h) before being treated with TGF-β1 (0.5 h), followed with NAC for 1 h; fourth panel: cells treated with NAC for 20 min. [TGF-β1] = 2.5 ng mL^–1, [NAC] = 5 mM. Scale bars: 50 μm. (b) Quantification of fluorescence intensities of NDF cells after incubation with CyTF or CyBA in Fig. 2a, the fluorescence intensities were normalized by a total cell nuclei signal, and the values presented were relative to the control groups. Error bars represent standard deviations of three separate measurements. (c) Schematic illustration of TGF-β1 signalling and RONS generation in NDF cells. *p < 0.05, **n.s.

Fig. 3. (a) Fluorescence microscopy of NDF, KF and KF cells treated with RepSox incubated with CyTF or CyBA (10 μM, 30 min) before imaging, [RepSox] = 25 μM. Scale bars: 50 μm. (b) Quantification of fluorescence intensities of NDF cells after incubation with CyTF or CyBA as shown in Fig. 3a, the fluorescence intensities were normalized by the total cell nuclei signal, and values presented were relative to the control groups. Error bars represent standard deviations of three separate measurements. (c) Schematic illustration of the RepSox inhibiting TGF-β type 1 receptor signalling pathway in KF cells. *p < 0.05.

Fig. 4. In vivo fluorescence imaging of keloid. (a) Schematic illustration of keloid fluorescence imaging in living nude mice. (b) Whole animal dorsal (upper panel) and ventral (lower panel) fluorescence imaging before intravenous injection (left panel) and 3.5 hours after intravenous injection (right panel). The upper left green circle indicates subcutaneously injected Matrigel as a control. The lower left blue circle indicates subcutaneous injected mixture of NDF cells and Matrigel. And the lower right red circle indicates a subcutaneous injected mixture of KF cells and Matrigel. (c) Fluorescence imaging of different organs and injected Matrigel. The organs were imaged after incubating in 200 μM ONOO^– solution for 30 minutes. (d) Quantification of fluorescence intensity as a function of time. (e) Fluorescence quantification of major organs of mice. The fluorescence images were acquired at 720 nm upon excitation at 675 nm.