【JACS】华西医院毛梧宇、詹梓炫|300倍荧光激活！新型TBCy探针攻克活细胞细胞器膜超分辨成像与精准破坏难题#

Scheme 1. (A) Representative Molecular Structures of Previously Reported Small-Molecular Organelle Membrane Probes; (B) Molecular Structures of Fluorogenic Tetrazine-Fused BF2-Bridged Cyanine (TBCy) and Schematic Overview of Its Photophysical Properties and Application Potentials; (C) Illustration of Organelle Membrane Imaging and Functional Disruption Enabled by TBCy Probes#

Figure 1. (A) Synthetic routes and molecular structures of fluorogenic TBCy probes. (B) Normalized absorption (Blue) and emission (purple) spectra of representative probes before (dotted line) and after (solid line) reacting with BCN in DMSO. (C) Photophysical properties of TBCy-BCN adducts, including the peak absorption (λabs)/emission wavelengths (λem), aStokes shift (Δλ), bmaximum molar extinction coefficients (εmax), fluorescence turn-on ratios, and cquantum yields (Φ). (D) Photophysical pathway of TBCy5–1. (E) Electronic energies of different frontier molecular orbitals in TBCy5–1 during the photoexcitation. (F) Tetrazine fragment extracted from the precursor for calculating the fragment contributions (top), representative example of calculating fragment contribution to the LUMO in TBCy5–3 (middle), and calculated fragment contributions of different molecules to LUMO (ηL), HOMO (ηH), and their difference (Δη; bottom).#

为了验证其在活细胞内的实际成像能力，团队针对性地设计并合成了带有十二烷基链和特定靶向基团的反应标签。实验中，细胞在预先孵育这些标签后，直接加入经过PEG修饰增强生物相容性的TBCy探针，无需任何繁琐的洗涤步骤即可清晰显现出精细的细胞器结构。定量共定位分析表明，激活后的荧光信号与商品化线粒体、溶酶体以及内质网绿色荧光染料的皮尔逊共定位系数高达0.84至0.95，且成像对比度较对照组提升了高达29至80倍。得益于BCy核心优异的光化学稳定性和极低的细胞毒性，该探针在面对持续高强度的激光照射时未表现出明显降解，不仅能长时间追踪细胞器形态，还彻底克服了商品化染料常导致的细胞受热变形与光毒性萎缩难题。

Figure 2. (A, B) Molecular Structures of biocompatible TBCy3P, TBCy5P, and TBCy7P (A) and organelle membrane-targeted dienophile probes MemMito, MemLyso, and MemER (B). (C–E) Bioorthogonal imaging of mitochondria (B, D) and lysosomes (C, E) labeled with the indicated TBCy probes with (+) or without (−) the preincubated membrane probes, together with the corresponding imaging contrast in labeled and control cells. (F–H) Photostability comparison of TBCy3P vs MTR and TBCy3P vs LTR under continuous laser irradiation. Scale bar = 20 μm.#

有了高光稳定性的基础，团队进一步引入吸电子能力更强的氰基取代基，制备出了专为超分辨受激发射损耗（STED）纳米成像量身定制的探针。在STED成像模式下，溶酶体、线粒体和内质网的超微膜结构被清晰剥离，空间分辨率跨越到了120至181纳米的纳米级尺度，完美捕捉到了共聚焦镜下无法分辨的环状、弯曲等异质性结构。通过长达数分钟的连续高速动态扫描，探针记录下了活细胞内溶酶体膜频繁发生的接触、融合、 round-up、收缩和扩张等极其复杂的“吻过即跑（kiss-and-run）”行为。更令人惊叹的是，利用该系统与其它生物正交体系的互不干扰性，研究人员成功在一根 depletion 激光线下实现了溶酶体膜与线粒体、溶酶体膜与脂滴的双色超分辨实时追踪，直观展现了亚细胞结构之间的动态异质性通讯过程。

Figure 3. TBCy probes for organelle membrane visualization using super-resolution STED microscopy. (A) Molecular structures of TBCy3-CN and TBCy5-CN. (B, C) STED imaging of lysosomal membranes labeled with TBCy3-CN and MemLyso, and corresponding intensity profiles along the dashed lines; enlarged views are shown in the boxed regions of interest (ROIs). (D) Dynamic STED imaging of the lysosomal membrane. Selected frames depict real-time lysosomal membrane changes. (E) Photostability of the TBCy3-CN adduct. (F) Time-dependent intensity profiles of arrow-indicated lysosomes along the lines in panel D. (G) STED imaging and intensity profiles of endoplasmic reticulum membranes labeled with TBCy3-CN and MemER. (H, I) STED imaging of mitochondria membranes labeled with TBCy3-CN and MemMito in A549 (H) and HUEVC (I) cells, and intensity profiles along the dashed lines. (J, K) Dual-color dynamic STED imaging of lysosomal membranes with mitochondria (J) and LDs (K) using TBCy3-CN together with SiR and LDBCy5, respectively. Selected frames show real-time organelle interactions. (L) Molecular structures of previously reported imaging reagents. Scale bars: 1 μm.#

Figure 4. Bioorthogonally activatable lysosomal membrane photodamage and the associated downstream biological responses. (A) Chemical structure of TBCy5-Br. (B) Schematic illustration of lysosomal membrane photodamage by the bioorthogonal activation of TBCy5-Br with MemLyso. (C) Phototoxicity and dark toxicity of TBCy5-Br at different concentrations in the presence of MemLyso. (D) Flow cytometric analysis of ROS generation in cells subjected to different treatments. (E) Fluorescence images of cells stained with Calcein-AM/PI, acridine orange (AO), and C11-BODIPY after various treatments. Scale bars: 20 μm. (F) Mean fluorescence intensity (MFI) of AO in the yellow channel. (G) ZRR-AMC assay results following different treatments. (H) Chemical structures of Pz5-Br (an adduct of TBCy5-Br and MemLyso, see also Figure S27) and BCy-Lyso. (I–L) Cellular phototoxicity (I), MFI of AO staining in the yellow channel (J), cytosolic cathepsin B activity (K), and merged fluorescence images of C11-BODIPY staining (L) upon indicated treatments. Scale bar: 10 μm. (M) Immunofluorescence staining of CRT, HMGB1, and LC3 in treated cells. Scale bars: 100 μm. (N) Western blot analysis of LC3, p62, GPX4, and caspase-3 expression levels under different treatment conditions.#

最后，研究团队展现了该平台在精准光动力治疗层面的巨大医疗潜力。他们通过将分子中的氯原子替换为溴原子，开发出了可被生物正交激活的具有高效单线态氧生成能力的探针。实验显示，只有在靶向标签和探针同时存在并接受光照的AND逻辑门控条件下，才会引发细胞器膜局部的强氧化损伤与脂质过氧化反应。这种局部高Permeabilization压力会导致溶酶体内的组织蛋白酶B等破坏性蛋白大量泄漏至细胞质中。这一上游级联反应不仅有效切断了细胞内部的自噬流，造成LC3-II和p62蛋白的显著堆积，还联合引发了铁死亡与细胞凋亡，并在体内肿瘤小鼠模型中实现了高达3.6倍的荧光富集和极其显著的肿瘤生长抑制效果。

Figure 5. (A, B) In vivo fluorescence images and corresponding intensity analyses of A549-tumor-bearing mice after intratumoral injection of TBCy5-Br or TBCy5-Br combined with MemLyso. (C) Photographs of excised tumors collected from A549-tumor-bearing nude mice subjected to different treatments. (D) Change of tumor volume in each group during the treatment period.#