Fluolab【Angew.Chem.】南开大学熊虎|灵敏度达1.37 nM！科学家开发新型氟化荧光素探针，首次揭示帕金森病脑部D-半胱氨酸显著下降
通讯作者： Hu Xiong (熊虎)
文章概要
引言
大脑作为代谢极其活跃的器官，其氧化还原平衡的维持对于神经功能至关重要。D-半胱氨酸（D-Cys） 作为一种在哺乳动物脑中产生的关键抗氧化剂，在调节神经祖细胞增殖和保护神经元免受氧化应激损伤方面发挥着不可替代的作用。然而，长期以来，精确监测活体脑部D-Cys的水平一直面临巨大挑战。一方面，传统的荧光成像存在组织穿透浅和背景干扰大的问题；另一方面，血脑屏障（BBB）严格限制了大多数诊断分子进入中枢神经系统。即便如天然的D-荧光素，也因为在生理pH下带电荷且易受外排转运体影响，难以进入大脑。因此，开发一种能够高效跨越血脑屏障并实现高灵敏度、原位监测的新型工具，对于理解帕金森病、胶质母细胞瘤等神经系统疾病的致病机理具有深远意义。
Synthesis of BBB-crossing fluorinated CBT for in situ detection of D-Cys in the brain. (a) Synthetic routes of fluorinated CBT derivatives (R-CBT). (b) Schematic illustration of 7'F-CBT traversing the BBB and producing fluorinated luciferase substrates in situ, enabling bioluminescence imaging of D-Cys in the brain.
主要实验及结论
研究团队基于腈基-氨基硫醇生物正交反应原理，巧妙地设计并合成了一系列氟化2-氰基苯并噻唑（R-CBT） 衍生物作为火放线虫荧光素酶的底物前体。通过在CBT分子上引入氟原子或三氟甲基，显著提升了分子的亲脂性，从而增强了其穿透血脑屏障的能力。在对六种候选化合物的系统筛选中，研究人员发现7'F-CBT表现最为出色。实验结果显示，7'F-CBT在体外对D-Cys表现出惊人的灵敏度，检测限低至1.37 nM，且对L-半胱氨酸及其他生物硫醇具有极高的选择性。在细胞实验中，7'F-CBT不仅能够原位检测内源性D-Cys，其生成的荧光素类似物还展现出了比天然底物更长、更稳定的生物发光信号，半衰期延长了约13倍。
7'F-CBT exhibited improved BBB penetration and enabled intracellular in situ generation of luciferase substrates for prolonged bioluminescence imaging. (a) Calculated log_P_ values of R-CBT measured by the shake-tube method. (b) Bioluminescence intensity of fLuc-transfected U87 cells incubated with R-CBT (50 µM) for 20 min (mean ± SD, n = 3). (c) Time-dependent bioluminescence intensity of fLuc-transfected U87 cells incubated with 50 µM R-CBT (mean ± SD, n = 3). (d) HPLC analysis of R-Luc (solid line), R-CBT (dashed line), and cell lysate (dotted line). Cells were incubated with 500 µM R-CBT for 40 min, followed by lysis with cell lysis buffer. Wavelength for detection: 320 nm. (e) Plasma membrane permeability of CBT, 4'F-CBT, and 7'F-CBT (mean ± SD, n = 3). U87 cells and HT22 cells were separately incubated with 500 µM R-CBT for 40 min. (f) Illustration of the transwell assay. bEnd.3 cells were incubated with 50 µM R-CBT for 12 h. (g) BBB permeability of CBT, 4'F-CBT, and 7'F-CBT (mean ± SD, n = 3). (h) Time-dependent bioluminescence imaging of fLuc-transfected MDA-MB-231 cells incubated with 50 µM R-Luc (exposure time = 15 s). (i) Time-course analysis of bioluminescence intensity for 50 µM D-Luc, 4'F-Luc, and 7'F-Luc in fLuc-transfected MDA-MB-231 cells (mean ± SD, n = 3). (j) Molecular docking analysis of 7'F-Luc with firefly luciferase (4G36). Statistical significance was analyzed by one-way ANOVA: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, P > 0.05.
7'F-CBT enabled the highly sensitive detection of D-Cys in vitro. (a) Linearity plots of the bioluminescence intensity of 7'F-CBT (50 µM) against D-Cys concentrations in the presence of luciferase (15 µg/mL), ATP (2 mM), and Mg2+ (10 mM) (mean ± SD, n = 3). (b) Bioluminescence intensity of 7'F-CBT (50 µM) toward reactive sulfur species (100 µM) in DPBS solution containing luciferase (15 µg mL−1), ATP (2 mM), and Mg2+ (10 mM) (mean ± SD, n = 3). (c) Bioluminescence imaging of D-Cys in fLuc-transfected U87 cells. Cells were incubated with D-Cys (0-1.0 mM) for 1 h and washed with fresh DMEM for three times, followed by incubation with 7'F-CBT (50 µM) (exposure time = 15 s). (d) Quantification of the total luminescence intensity of panel (c) (mean ± SD, n = 3). (e) Comparison of luminescence intensity of fLuc-transfected U87 cells pretreated with D/L-Cys for 1 h and then incubated with 7'F-CBT (50 µM) after 30 min (mean ± SD, n = 3). (f) Bioluminescence intensity of fLuc-transfected U87 cells in different groups (mean ± SD, n = 3). Group 1: cells were treated with 7'F-CBT (50 µM). Group 2: cells were pretreated with NEM (50 µM) for 1 h, followed by incubation with 7'F-CBT (50 µM). Group 3: cells were pretreated with NEM (50 µM) and D-Cys (1.0 mM) for 2 h, followed by incubation with 7'F-CBT (50 µM). (g) Bioluminescence imaging of D-Cys in fLuc-transfected U87 cells. Cells were incubated with D/L-NAC (1.0 mM) for 1 h and washed with fresh DMEM for three times, followed by incubation with 7'F-CBT (50 µM) (exposure time = 15 s). (h) Quantification of the average luminescence intensity of panel (g) (mean ± SD, n = 3). (i) Bioluminescence intensity of fLuc-transfected U87 cells in different groups (mean ± SD, n = 3). Group 1: cells were treated with 7'F-CBT (50 µM). Group 2: cells were pretreated with NEM (50 µM) for 1 h, followed by incubation with 7'F-CBT (50 µM). Group 3: cells were pretreated with NEM (50 µM) and D-NAC (1.0 mM) for 2 h, followed by incubation with 7'F-CBT (50 µM).
7'F-CBT enabled the highly sensitive detection of D-Cys in vivo. (a) Schematic illustration of brain BL imaging in transgenic mice (R26-CAGLuc-2A-EGFP) using 7'F-CBT. (b) Bioluminescence images of transgenic mice in different groups. Prior to imaging, mice were injected i.p. with 100 µL D/L-Cys (0-3.0 mM), and 1 h later, the mice were injected i.v. with 7'F-CBT (2.0 mM, 100 µL) (exposure time = 15 s). (c) In vivo quantification of the bioluminescence intensity in the brain area of mice from different groups (mean ± SD, n = 3). (d) Relative SNR of the brain (mean ± SD, n = 3). (e) Bioluminescence imaging of endogenous D-Cys in transgenic mice. Group 1: mice were injected i.v. with 7'F-CBT (2 mM, 100 µL). Group 2: mice were injected i.p. with NEM (0.5 mM, 100 µL), followed by i.v. administration of 7'F-CBT (2 mM, 100 µL) at 1 h post-injection. Group 3: mice were injected i.p. with NEM (0.5 mM, 100 µL) and D-Cys (2 mM, 100 µL), followed by i.v. administration of 7'F-CBT (2 mM, 100 µL) (exposure time = 15 s). (f) Bioluminescence imaging of excised brains 10 min after i.v. injection of 7'F-CBT into the mice of (e). (g) Quantification of the brain luminescence intensity in (f) (mean ± SD, n = 3). Statistical significance was analyzed by one-way ANOVA: ****P < 0.0001; ***P < 0.001.
在动物模型研究中，通过静脉注射给药，7'F-CBT成功穿越了转基因小鼠的血脑屏障，并在脑部与内源性D-Cys反应生成荧光素。成像结果显示，脑部发光强度比普通CBT提高了4.1倍，信噪比高达60:1。利用这一特性，研究团队在原位脑肿瘤模型中实现了视频级（video-rate）的实时监测，清晰地观察到自由移动小鼠脑内D-Cys的分布。最具突破性的发现是在帕金森病（PD）小鼠模型中，通过7'F-CBT介导的成像技术，研究人员首次观察到PD小鼠脑部D-Cys水平发生了剧烈下降。这一现象结合行为学测试中观察到的运动能力受损，有力地证明了D-Cys代谢异常与神经退行性疾病病理过程之间的紧密联系。
High-contrast bioluminescence imaging of D-Cys in an orthotopic brain tumor model. (a) Establishment of an orthotopic brain tumor model for imaging study. (b) In situ injection of U87-Luc cells (1*105 cells, 8 µL, 1 µL min−1) into the mouse brain using a stereotactic device. (c) Walking path chart of normal and tumor-bearing mice. Comparison of the walking distance (d), walking speed (e), and rest time (f) in normal and tumor-bearing mice (mean ± SD, n = 3). (g) Bioluminescence images of BALB/c mice, including normal and tumor-bearing mice, after i.v. injection of CBT or 7'F-CBT (10 mM, 50 µL). H&E staining analysis of harvested brain tissue sections, inset: brightfield image of the brain. (h) Quantification of bioluminescence intensity in the mouse brain of (g) at 30 min post-injection (mean ± SD, n = 3). (i) SNR of the mouse brain in different groups (mean ± SD, n = 3). (j) Real-time bioluminescence imaging of freely moving mice. 100 µL 7'F-CBT (10 mM) was subcutaneously injected into U87 brain tumor-bearing mice. Exposure time: 1 s. Statistical significance was analyzed by one-way ANOVA: ****P < 0.0001; ***P < 0.001; NS, P > 0.05.
Mapping D-Cys levels in PD cell and mouse models using 7'F-CBT. (a) Schematic diagram of imaging intracellular D-Cys levels in a PD cell model using 7'F-CBT. Firefly luciferase (15 µg mL−1) was added for the final bioluminescence assay. (b) Bioluminescence imaging of endogenous D-Cys in a MPTP-induced PD cell model. PC12 cells were pretreated with MPTP (0–0.5 mM) for 24 h and then incubated with 7'F-CBT (50 µM) for imaging (exposure time = 15 s). (c) Quantification of luminescence intensity of panel (b) (mean ± SD, n = 3). (d) Bioluminescence imaging of exogenous D-Cys fluctuations in PC12 cells. The cells were pretreated with MPTP (0.5 mM) for 24 h and then incubated with D-Cys (0–1.0 mM) for 1 h, followed by addition of 7'F-CBT (50 µM) for imaging. (e) Quantification of luminescence intensity of panel (d) (mean ± SD, n = 3). (f) Experimental timeline for establishing the MPTP-induced PD mouse model and workflow of the imaging study. (g) Illustration of the open-field test. (h) Walking path chart of the normal and PD model mice. (i) Schematic representation of the pole test. (j–m) Comparison of the walking distance (j), rest time (k), time in central area (l), and the time taken to crawl in the pole test (m) (mean ± SD, n = 4). (n) Bioluminescence images of the normal and PD model mice after i.v. injection of 7'F-CBT (2 mM, 100 µL) (exposure time = 15 s). (o) Quantification of the brain luminescence intensity of panel (n) (mean ± SD, n = 3). (p) Brightfield and bioluminescence images of the dissected brains. (q) Quantification of the average radiance of brains in (p) (mean ± SD, n = 3). Statistical significance was determined using a two-tailed Student's t test: ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; NS, P > 0.05.
总结及展望
本研究成功开发了一种新型氟化生物发光探针平台，突破了传统成像工具在血脑屏障渗透性和空间分辨率上的局限。7'F-CBT探针凭借其卓越的灵敏度和体内稳定性，不仅为神经科学研究提供了一种强大的非侵入式监测手段，也为脑部疾病的早期诊断开辟了新途径。未来，这种通过原位生成荧光素底物的策略有望扩展到更多脑部生物标志物的检测中。通过深入研究D-Cys在不同神经疾病中的动态变化，科学家们或许能够开发出更精准的治疗方案，为帕金森病及其他氧化应激相关脑病的临床诊断与药效评估提供关键的数据支持。