Fluolab【Angew.Chem.】复旦大学朱亮亮、柴先志联手安徽中医药大学韩海浩等|5秒显著下调！persulfurated arene探针实现衰老-正常细胞边界的“双向”精准辨别
通讯作者： Xianzhi Chai, Glib V. Baryshnikov, Hai-Hao Han, Liangliang Zhu
文章概要
引言
细胞衰老是机体衰老和多种疾病的核心病理过程，能够识别并清除衰老细胞对于治疗老年相关疾病至关重要。然而，与边界清晰的肿瘤不同，衰老组织在细胞水平上缺乏明确的界限，衰老细胞与正常细胞往往呈现空间交错状态。目前的荧光探针大多采用“开启”或单向信号变化模式，这导致信号缺失区域既可能是正常细胞，也可能是未被标记的衰老细胞，给手术切除或病理诊断带来了巨大的模糊性。为了解决这一生物学难题，朱亮亮教授团队及其合作者开发了一种基于硫醚取代苯（persulfurated arene）骨架的双向光调制荧光探针，通过环境依赖的不同光响应逻辑，第一次实现了对衰老与正常细胞边界的高动态对比度成像。
(A) Chemical structure of compounds HTB-OH, HTB-βGal, HTB-MOH, and HTB-MβGal. (B) Illustration of the lysosome-targeting probe HTB-MβGal enabling bidirectional photomodulation for imaging normal cells (Direction 1: Up regulation of emission) and senescent cells (Direction 2: Down regulation of emission), toward precise delineation of senescent–normal cell boundaries at the cellular scale. AIE: Aggregation-induced emission, β-Gal: β-galactosidase.
主要实验及结论
研究人员通过在疏水的六硫苯（HTB-OH） 核心上修饰亲水的β-半乳糖基，成功合成了探针HTB-βGal及其具有溶酶体靶向功能的变体HTB-MβGal。在光物理性质研究中，研究团队发现这套分子体系具有极其独特的“双向驱动”能力。在正常细胞环境中，由于探针具有良好的水溶性，分子最初处于不发光状态，但在连续的405 nm光照射下，会诱发光激发诱导聚集（PEIA） 效应，使荧光信号显著上调。而在衰老细胞中，高表达的衰老相关β-半乳糖苷酶（β-Gal） 会迅速剪切掉糖苷键，生成强疏水性的水解产物。这些产物因聚集诱导发光（AIE） 效应立即产生强烈的荧光。令人惊叹的是，在随后的光照射下，这些水解产物会经历光激发诱导的分子重排，导致其发光在短短5秒内迅速下调。 
Illustration of aggregation-induced phosphorescence and photo-responsive processes of (A) HTB-OH and (E) HTB-βGal. The luminescence intensity variation of (B) HTB-OH (20 µM) and (F) HTB-βGal (20 µM) in CH3CN/H2O mixed solvents with different water contents. Photoluminescence spectral change of (C) HTB-OH (20 µM) and (G) HTB-βGal (20 µM) in H2O (containing 1% DMSO) upon irradiation with 405 nm light. Photoluminescence lifetime of (D) HTB-OH in water collected at 571 nm and (H) HTB-βGal in water upon irradiation with 405 nm light collected at 503 nm. The power density of 405 nm light was 0.45 mW cm−2 for all measurements, and all emission events were recorded under 405 nm excitation. Data are presented as mean ± SD from three independent experiments.
Photoluminescence spectral change of HTB-OH (20 µM) in H2O (containing 1% DMSO) upon irradiation with 405 nm light at (A) 298 K and (B) 77 K. (C) Normalized photoluminescence spectral change of HTB-OH (20 µM) in H2O (containing 1% DMSO) from 77 to 298 K. Theoretical results of S1→S0 transition and S1→T1/T1→S0 conversions for HTB-OH and HTB-βGal. (D) HTB-OH symmetric geometry. (E) HTB-OH asymmetric geometry. (F) HTB-βGal asymmetric geometry. (G) HTB-βGal symmetric geometry. The power density of 405 nm light was 20 mW cm−2 for all measurements, and all the emission events were recorded under 405 nm excitation.
(A) Photoluminescence spectral change of HTB-MβGal (20 µM) in the presence of β-Gal (10 U/mL) for 12 min in PBS buffer (10 mM, pH 7.4, 0.5% DMSO). (B) Photoluminescence lifetime of HTB-MβGal (20 µM) at 567 nm after reaction with β-Gal. (C) Photoluminescence spectra of HTB-MβGal (in the absence of β-Gal, in the presence of β-Gal, and upon irradiation with 405 nm light after response to β-Gal). (D) Photoluminescence spectral change of HTB-MβGal (20 µM) upon reaction with different concentrations of β-Gal for 6 min in PBS buffer (10 mM, pH 7.4, 0.5% DMSO). (E) Plotting the emission intensity of HTB-MβGal (20 µM) at 567 nm as a function of β-Gal concentration (0−10 U/mL) after 6 min of incubation. (F) Photoluminescence intensity of HTB-MβGal (20 µM) at 567 nm in the presence of β-Gal (10 U/mL) or other competing species (100 µM or 10 U/mL; 1: Blank; 2: β-Gal; 3: Cellulase; 4: Lysozyme; 5: Esterase; 6: ATP; 7: NADH; 8: GSH; 9: Cys; 10: HCy; 11: L-Tryptophan; 12: L-Lyrosine; 13: H2O2; 14: NaClO; 15: •OH; 16: ONOO−) after incubation for 10 min in PBS buffer (10 mM, pH 7.4, 0.5% DMSO). All the power density of 405 nm light was 0.45 mW cm−2 and all emission events were produced upon excitation at 405 nm. Data are presented as mean ± SD from three independent experiments.
通过密度泛函理论（DFT）计算，研究进一步揭示了这种双向调控的底层机制。研究发现，分子在光激发下会从对称构型转变为非对称构型。对于水解后的产物，构型改变抑制了系间窜越过程，从而使原本的磷光信号淬灭；而对于原始探针分子，构型改变则增强了振子强度并促进了荧光发射。在细胞层面的验证实验中，HTB-MβGal表现出极佳的溶酶体靶向性（皮尔逊相关系数达0.89）和极低的细胞毒性。当研究者将正常细胞与诱导衰老的A549细胞进行共培养时，探针展现了强大的空间分辨力：通过光调制，正常细胞区域信号由弱变强，而衰老细胞区域信号则由强变弱，这种截然相反的动态对比使得两者的功能界面边界清晰可见，有效排除了传统探针因扩散或负载不均带来的假阳性干扰。
Cellular imaging (A) and quantification (B) of normal and senescent A549 cells incubated with HTB-βGal or HTB-MβGal (10 µM). Colocalization imaging (C) and intensity profile for the region of interest lines (D–G) of HTB-βGal or HTB-MβGal (10 µM) in senescent A549 cells co-stained with MitoTracker (100 nM) and LysoTracker (100 nM), respectively. HTB-βGal/HTB-MβGal channel: Ex/Em = 405/500–600 nm. Tracker channel: Ex/Em = 561/570–650 nm. Error bars represent S. D. (n = 3). Statistical significance was determined using two-tailed Student's t-test (****p < 0.0001).
Photomodulation imaging (A, B) and quantification (C, D) of normal and senescent A549 cells treated with HTB-MβGal (10 µM, 24 h) or HTB-MOH (5 µM, 4 h). Normal A549 channel: Ex/Em = 405/570–630 nm; senescent A549 channel: Ex/Em = 405/500–600 nm. Error bars represent S. D. (n = 3). PEIA imaging (E) and quantification (F) of cocultured normal and senescent A549 cells incubated with HTB-MβGal. Imaging channel: Ex/Em = 405/570–630 nm. The PEIA images were taken every 2 min of irradiation with 405 nm light.
总结及展望
该研究不仅成功构建了一种高度灵敏且具有环境自适应能力的分子工具，还为解决生物学领域的“边界模糊”问题提供了全新的设计范式。这种双向光调制策略通过同一个探针分子在不同细胞状态下表现出的相反动态响应，极大地提升了检测的准确性和空间分辨率。未来，这种基于硫醚取代苯体系的可编程动态响应探针，有望在衰老相关疾病的早期诊断、术中导航以及细胞疗法的质量监测等领域发挥重要作用，为精准医学提供更具逻辑性和判别力的成像方案。