Physicochemical-property guided design of a highly sensitive probe to image nitrosative stress in the pathology of stroke

Abstract

In vivo real-time imaging of nitrosative stress in the pathology of stroke has long been a formidable challenge due to both the presence of the blood-brain barrier (BBB) and the elusive nature of reactive nitrogen species, while this task is also informative to gain a molecular level understanding of neurovascular injury caused by nitrosative stress during the stroke episode. Herein, using a physicochemical property-guided probe design strategy in combination with the reaction-based probe design rationale, we have developed an ultrasensitive probe for imaging nitrosative stress evolved in the pathology of stroke. This probe demonstrates an almost zero background fluorescence signal but a maximum 1000-fold fluorescence enhancement in response to peroxynitrite, the nitrosative stress marker. Due to its good physicochemical properties, the probe readily penetrates the BBB after intravenous administration, and quickly accumulates in mice brain to sense local vascular injuries. After accomplishing its imaging mission, the probe is easily metabolized and therefore won't cause safety concerns. These desirable features make the probe competent for the straightforward visualization of nitrosative stress progression in stroke pathology.

Fig. 1. Physicochemical-property guided fluorescent probe design for non-invasive imaging of ONOO⁻ during stroke in live mice. (a) Partition coefficients (log P) of classical fluorophores were determined and benzo-BODIPY (F4) was predicted to have a potentially good BBB permeability. (b) Electron-rich phenols may be oxidized to quinine by ONOO⁻ and this reaction has been used as a trigger for ONOO⁻ probe development. (c) Probes B545a and B545b were designed based on the F4 fluorophore which showed desirable log P for BBB permeability. B545a and B545b were hypothesized to undergo a biocompatible reaction with ONOO⁻ to be transformed into highly fluorescent products.

Fig. 2. UV-Vis and fluorescence response of probe B545b to ONOO⁻. (a) Absorption spectra of B545b (10.0 μM) before and after being treated with various amounts of ONOO⁻. (b) Fluorescence spectra of probe B545b (5.0 μM) before and after treatment with ONOO⁻ at indicated final concentrations. (c) Time-lapse emission of B545b (5.0 μM) at 545 nm in response to ONOO⁻. (d) Fluorescence intensity of B545b (5.0 μM) at 545 nm after treatment with various biologically related species. (1) Probe blank, (2) NO, (3) NO₂⁻, (4) NO₃⁻ (5) H₂O₂, (6) THBP, (7) ˙OH, (8) ¹O₂ (9) O₂⁻, (10) ClO⁻, (11) GSH, (12) Cys, (13) Zn²⁺, and (14) Cu²⁺. All analytes were kept at a final concentration of 100 μM except ONOO⁻ which was kept at 10 μM. All measurements were carried out at ambient temperature with excitation at 475 nm and emission at 545 nm.

Fig. 3. Imaging SIN-1 induced ONOO⁻ in EA.hy926 endothelial cells. (a) Cells were incubated with various doses of SIN-1 for 1 h, and then stained with B545b (0.5 μM) for 30 min. (b) Quantified B545b fluorescence in (a) (mean ± S.E.M, n = 4). (c) Time-lapse series of EA.hy926 cells first loaded with B545b and then stimulated with SIN-1 (1 mM). (d) Quantified B545b fluorescence in (c). Data are presented as a densitometric ratio change compared with control. DAPI (4′,6-diamidino-2-phenylindole) counterstaining indicates nuclear localization (blue, λ_em 420–480, λ_ex 405 nm). B545b fluorescence was collected at 560–620 nm with λ_ex 543 nm. ***P < 0.001, **P < 0.01, and *P < 0.1 versus control.

Fig. 4. Characterization of B545b specificity for ONOO⁻ in endothelial cells. (a) Scavenging ONOO⁻ with uric acid (100 μM) or FeTPPS (1 μM) inhibited SIN-1 induced B545b fluorescence. (b) Pretreating cells with uric acid (100 μM), FeTPPS (1 μM) or TEMPO (300 μM) 1 h prior to OGD exposure (4 h) significantly suppressed B545b fluorescence. (c and d) Quantification of B545b mean fluorescence intensity (mean ± S.E.M, n = 4) in (a) and (b). Data are presented as a densitometric ratio change compared with control. DAPI counterstaining indicates nuclear localization (blue, λ_ex 405 nm and λ_em 420–480 nm). B545b fluorescence was collected at 560–620 nm with λ_ex 543 nm. ***P < 0.001, **P < 0.01 versus control, ###P < 0.001, and ##P < 0.01 versus SIN-1 or OGD.

Fig. 5. In vivo real-time imaging of the endogenous ONOO⁻ flux incurred by brain microvessel occlusion. (a) Schematic illustration of experimental methods for microvessel occlusion and imaging. First, probe B545b and FITC–dextran were intravenously injected into mice. Then, microvessel occlusion was induced by photothrombosis, and imaged using a two-photon laser scanning microscope (TPLSM). (b) The time-series images representing individual frames from a continuous time lapse movie which showed the dynamic B545b fluorescence change in response to vascular occlusion in live mice. (c) Individual fluorescence of B545b and FITC in the photothrombosis model.

Fig. 6. Imaging of ONOO⁻ accumulation after MCAO in the brain parenchyma. (a) Cerebral imaging and analysis. After B545b administration, the mice were treated with MCAO for 0.5 or 1 h. The whole brains were scanned using a Maestro in vivo imaging system with a 480 nm excitation wavelength and a 560 nm filter. (b) Immunofluorescence staining of FasL (green) and DAPI (blue) in the brain after B545b injection and MCAO treatment. Scale bar = 50 μm. (c) Confocal imaging of brain slices with the B545b signal (red) and DAPI (blue). B545b fluorescence was collected at 500–540 nm with λ_ex 488 nm. B545b fluorescence significantly increased 0.5 h after MCAO in the ischemic brain. The white dotted lines show the brain sections and the yellow dotted lines show the ischemic region of brain. Scale bar = 1 mm. (d) Nissl staining with the whole brain sections. Scale bar = 1 mm.