【Angew.Chem.】具有NAD(P)H激活特性的荧光探针在肿瘤诊疗一体化中的潜力：评估癌症侵袭性并触发>50%癌细胞凋亡#

文章标题：Theranostic Potential of an NAD(P)H‐Activatable Fluorophore: Assessing Cancer Aggressiveness and Triggering Apoptosis 文章作者：Yujin Cha, Taehoon Oh, Hui Joo Kim, Minju Kang, Shin A Yoon, Yul Lee, Yeon-Ju Lee, Sun Young Park, Dong-Sik Shin, Sung-Kyun Ko, Jiyou Han, Min Hee Lee 文章链接：https://doi.org/10.1002/anie.7968058

1. 研究背景与核心痛点#

在现代精准医疗中，“诊疗一体化”（Theranostics）是一个备受关注的发展方向。简单来说，它就像给药物装上“雷达”与“定向炸药”，将精准的病灶诊断成像与定向的药物杀伤融合为一体，既能看清病灶在哪里、恶性程度如何，又能同时就地消灭癌细胞。

然而，传统的诊疗一体化系统大多依赖复杂的“多组件拼装”：通常需要把靶向配体、前药分子、荧光团以及可裂解连接链等好几个化学模块拼装在一块。这种多成分结构不仅合成路线极其繁琐、生产成本高，而且在复杂的体内环境中容易出现提前脱落或非特异性释放，带来难以预估的脱靶毒性。

为了化繁为简，科学家们将目光转向了癌细胞独特的代谢特征。恶性肿瘤细胞具有非常旺盛的糖酵解代谢（即“瓦伯格效应”，Warburg effect）。为了满足疯狂增殖的需求并抵抗氧化应激，高度恶性的癌细胞内部会蓄积高浓度的还原型烟酰胺腺嘌呤二核苷酸（磷酸），即 NAD(P)H。NAD(P)H 是细胞内至关重要的还原当量与代谢辅酶：癌细胞恶性程度越高、侵袭与转移能力越强，NAD(P)H 的生成与周转水平通常就越活跃。因此，NAD(P)H 既是判断癌症侵袭性的“代谢风向标”，也是开发新型特异性抗癌药物的绝佳靶标。

Scheme.1 (a) DE-CQ and CQ are sensitive to the reductive activity of NAD(P)H and act as fluorescence switches for NAD(P)H. (b) DE-CQ indicates cancer aggressiveness and induces cancer apoptosis and is proposed as a fascinating potential theranostic agent.#

2. 核心创新与探针设计原理#

针对传统探针结构复杂、功能单一的问题，研究团队成功设计并合成了新型小分子诊疗探针——DE-CQ（基于香豆素稠合喹啉鎓骨架构建）。

该分子在设计上实现了多重创新与化繁为简：

智能荧光“开关”：探针结构中的喹啉鎓部分充当 NAD(P)H 的特异性识别单元。在未遇到 NAD(P)H 前，探针分子内电荷转移（ICT）处于关闭状态，几乎不发荧光；一旦遇到细胞内的 NAD(P)H，喹啉鎓发生不可逆氢负离子还原反应，重新激活内部电荷转移通道，瞬间点亮强烈的红色荧光（发射波长在 640 nm 处，斯托克斯位移超过 110 nm）。
免洗实时成像能力：因为未激活的底物完全无背景荧光，在活细胞检测时无需繁琐的洗脱步骤，能直接实现对细胞内 NAD(P)H 动态变化的“即加即测”与高对比度成像。
自带药物药效活性：探针将具有潜在抗癌药理活性的香豆素骨架与响应单元融为一体，无需额外挂载化疗毒素，还原激活后可直接触发靶向细胞凋亡。

Fig.1 Synthesis of NAD(P)H-activatable fluorophores and their fluorescence switch properties to NADH. (a) Synthetic pathways of DE-CQ and CQ used in this study. (b, c) Photographs for their solution color and fluorescence before and after NADH addition and corresponding absorption and fluorescence spectra. (d, e) Kinetic curves of DE-CQ and CQ (each 5 µM) for NADH activity (50 µM).#

3. 体外光谱性能与超高选择性验证#

在溶液层面的基础理化测试中，DE-CQ 展现出优异的动力学与传感特性：

反应速度快与极高灵敏度：与结构相近的对照探针 CQ 相比，DE-CQ 对 NADH 的反应速率常数高达 $5.70 \times 10^{-4} \text{ s}^{-1}$ （约为 CQ 的 2 倍），检出限（LOD）低至 0.038 µM（38 nM），具备对微量辅酶变化的快速捕获能力。
抗干扰能力极强：面对细胞内常见的多种金属阳离子（如 $\text{K}^+、\text{Na}^+、\text{Cu}^{2+}、\text{Fe}^{3+}、\text{Zn}^{2+}$ 等）、阴离子、高浓度活性氧（ROS）、游离氨基酸/硫醇（如 GSH、半胱氨酸）、葡萄糖、ATP、ADP 以及氧化态 $\text{NAD}^+$ ，探针均完全不产生荧光响应，仅在遇到还原态的 NADH 和 NADPH 时被特异性点亮。
耐受生理 pH 波动：在 pH 4.0 到 8.0 的宽生理酸碱度范围内，探针均能保持稳定的荧光开启信噪比，这意味着无论是处于酸性的肿瘤微环境、溶酶体，还是中性的细胞质与线粒体中，均能准确工作。

Fig.2 Fluorescence activation of DE-CQ and CQ by NAD(P)H and analysis using liquid chromatography and mass spectrometry. (a, d) Fluorescence intensities of DE-CQ and CQ before and after NAD(P)H addition in different pH solutions. (b, e) Fluorescence response to various biocomponents (100 µM, respectively; 1 mM for GSH and glucose), (1) DE-CQ or CQ, (2) K+, (3) Na+, (4) Cu+, (5) Cu2+, (6) Ca2+, (7) Mg2+, (8) Zn2+, (9) Fe2+, (10) Fe3+, (11) Cl−, (12) CN−, (13) H2PO4−, (14) OAc−, (15) OH−, (16) H2O2, (17) HOOtBu, (18) ·O2−, (19) ·OH, (20) ·OtBu, (21) GSH, (22) Cys, (23) Hcy, (24) glucose, (25) ATP, (26) ADP, (27) NAD+, (28) NADPH, (29) NADH. All fluorescence data were recorded after 80 min incubation at 37°C in a PBS solution (pH 7.4, 10 mM) containing 1 mM CTAB and 1% (v/v) DMSO. Excitations were respectively applied at 490 nm for DE-CQ and 465 nm for CQ. (c, f) LC profiles before and after the NAD(P)H reaction. LC peaks were shown by detection of absorbance for DE-CQ and CQ.#

Fig.3 Cell permeability and fluorescence intensity of DE-CQ and CQ in live cells. (a) Time-dependent and dose-dependent fluorescence intensities of MDA-MB-231 cells treated with DE-CQ or CQ. (b) Fluorescence imaging of cells under glycolysis stimuli. Cells were pretreated with 20 mM glucose or 5 mM pyruvate, and then incubated with 10 µM DE-CQ or 10 µM CQ for 15 min. All fluorescence images were collected by the excitation and emission filter sets; λex: 540–552 nm and λem: 575–640 nm for DE-CQ or λex: 450–490 nm and λem: 500–550 nm for CQ. The bottom image is an overlay of the fluorescent and nonfluorescent phase contrast images. Scale bar: 50 µm. * p < 0.05, p < 0.01, and * p < 0.001.#

4. 癌症恶性程度的“可视化”代谢评估#

由于侵袭性越强的癌细胞其糖酵解代谢越活跃，研究人员巧妙地设计了“葡萄糖刺激对比法”：先用 20 mM 葡萄糖 刺激细胞加速糖酵解并短时间内大量产生 NAD(P)H，再加入 10 µM DE-CQ 孵育 15 分钟，通过荧光增强倍数来直观量化不同癌细胞的“恶性/侵袭程度”。

实验在多种不同来源的癌细胞株中进行了系统评估，结果呈现出高度吻合的病理规律：

乳腺癌细胞系梯度：荧光活化强度与恶性程度完全一致，呈现 MDA-MB-231（高转移性三阴性乳腺癌） > T47D > MCF-7（低恶性度） 的严格梯度。
前列腺癌细胞系对比：高侵袭性的 LNCaP-LN3 细胞在葡萄糖刺激后荧光急剧上升，而恶性度较低的 DU-145 细胞则响应微弱。
广泛谱系验证：在宫颈癌（HeLa）、胰腺癌（PANC-1）、胃癌（AGS）和结肠癌（HCT-116）等多种高代谢恶性细胞中均检测到了显著的荧光翻倍，而在代谢迟缓的正常细胞或低响应癌株中变化微弱。这表明 DE-CQ 可以作为快速评估肿瘤代谢活跃度与恶性侵袭性的有力工具。

Fig.4 Fluorescence responses of various cancer cell lines before and after glucose stimulation using DE-CQ. (a) Box plots of quantified fluorescence intensities of each cell line. *** p < 0.001, n.s.: not significant. (b) Heat map analysis of fluorescent difference (glucose-control) and fold change (glucose/control). Cells were treated with 20 mM glucose for 45 min, and then incubated with 10 µM DE-CQ for 15 min. All data were derived from fluorescence images collected with the excitation and emission filter set as λex: 540–552 nm and λem: 575–640 nm. All images were acquired and processed under identical instrumental settings for accurate quantitative comparison.#

5. 细胞杀伤机制：双重应激与凋亡信号通路#

除了诊断成像，DE-CQ 在治疗端展现出了对恶性癌细胞的高效选择性杀伤效果：

选择性毒性与高治疗窗：在 MTT 细胞毒性测试中，10 µM DE-CQ 作用于高恶性乳腺癌 MDA-MB-231 细胞 24 至 48 小时后，诱导了超过 50% 的癌细胞死亡；然而在相同浓度下，对正常人乳腺上皮细胞 MCF10A 表现出极低的毒性，证明其具有良好的肿瘤选择性。
线粒体与内质网双靶向定位：共聚焦荧光共定位实验证实，激活后的 DE-CQ 主要定位于线粒体（皮尔逊相关系数 PCC = 0.804）和内质网（PCC = 0.639），而在溶酶体分布较少（PCC = 0.560）。
触发线粒体与内质网应激凋亡级联：
转录层面：实时荧光定量 PCR 证实，线粒体凋亡关键基因（Fas、Bax、Caspase-9、Caspase-3）以及内质网应激凋亡基因（PERK、CHOP、JNK、Bim）均被显著上调。
蛋白层面：Western Blot 显示前体蛋白 Pro-caspase-9、Pro-caspase-3 和全长 PARP 水平显著下降，而活化的剪切体 Cleaved caspase-3 和 Cleaved PARP 大幅增加。
生理层面：探针引发细胞内活性氧（ROS）急剧累积，并导致线粒体膜电位（ $\Delta\Psi_m$ ）显著去极化丧失。
抑制肿瘤干性与侵袭转移：探针处理显著下调了肿瘤干细胞标志物（Nanog、CD133）和上皮-间充质转化（EMT）标志物（Snail1、E-cadherin、MMP9）的表达，在划痕愈合实验与 Matrigel 侵袭实验中有效限制了 MDA-MB-231 细胞的迁移与浸润能力。

Fig.5 Apoptosis mode and intracellular localization of active DE-CQ in live cells. (a) Apoptotic gene expression in MDA-MB-231 cells treated with DE-CQ (0, 10, or 20 µM) for 24 h determined by quantitative PCR analysis. * p < 0.05. (b) Schematic illustration of the mitochondrial and endoplasmic reticulum (ER) caspase-dependent apoptotic pathways. (c) Representative Western blot analysis and densitometric quantification of pro-caspase-9, pro-caspase-3, cleaved caspase-3, full-length PARP, and cleaved PARP in MDA-MB-231 cells treated with DE-CQ (0, 10, or 20 µM) for 48 h. (d) MDA-MB-231 cells were treated with DE-CQ (0, 10, 25, or 50 µM) for 24 h, stained with Annexin V-FITC, and analyzed by flow cytometry. (e) Intracellular fluorescence colocalization of DE-CQ (red) with organelle-specific trackers for mitochondria, ER, and lysosomes (green) evaluated by confocal fluorescence microscopy. Pearson correlation coefficient (PCC) indicates the degree of colocalization. All confocal fluorescence images were collected using a 488 nm excitation laser and 493–520 nm (green) and 644–700 nm (red) emission band-pass filters. Scale bar: 10 µm. Data in (a) and (c) are presented as the mean ± SD (n = 3). *p < 0.05.#

6. 3D 肿瘤微球与活体小鼠异种移植瘤验证#

为进一步评估该探针在更接近临床真实病灶组织中的实用价值，研究团队开展了 3D 仿生微球和活体动物实验：

3D 肿瘤微球深层穿透与杀伤：在由 MDA-MB-231 构建的 3D 肿瘤球中，DE-CQ 不仅能顺利渗透并呈现出鲜艳的葡萄糖响应荧光，而且在 12 小时后引发了肿瘤微球深部细胞的大面积坏死，伴随 ATP 水平和微球整体活性的大幅下调（Calcein AM/PI 活死细胞荧光染色验证）。
活体抗肿瘤疗效与低全身毒性：在 BALB/c 裸鼠皮下异种移植瘤模型中，研究人员采用 5 mg/kg 和 10 mg/kg 剂量的 DE-CQ 进行腹腔注射治疗。在给药的 10 天周期内，荷瘤小鼠的肿瘤体积与肿瘤重量呈现出剂量依赖性的显著抑制（ $p < 0.0001$ ）；与此同时，小鼠体重平稳无明显波动，展现出良好的生物相容性与较低的全身系统毒性。

Fig.6 The theranostic effect of DE-CQ in MDA-MB-231 tumor spheroids. (a) Confocal fluorescence imaging of DE-CQ in tumor spheroids. Cryosectioned tumor spheroids were treated with 20 mM glucose for 45 min, incubated with 10 µM DE-CQ for 15 min. Bright field indicates a corresponding nonfluorescent phase contrast image. Scale bar: 50 µm. (b) Therapeutic potential of DE-CQ. Tumor spheroids were incubated with 10 µM DE-CQ for 12 h and stained with PI for 2 h to visualize dead cells. Scale bar: 50 µm. (c) Quantitative analysis of the fluorescent intensities of DE-CQ-treated cells (green) and PI-stained dead cells (red) in cryosectioned tumor spheroids. (d) Live/dead cell imaging of tumor spheroids incubated with DE-CQ.#

Fig.7 DE-CQ exhibits antitumor activity in an MDA-MB-231 xenograft mouse model. (a) Scheme of the xenograft mouse experiment. (b) Body weight was measured daily during treatment with DE-CQ (5 and 10 mg/kg). (c) Tumor volume was measured daily during treatment with DE-CQ (5 and 10 mg/kg). Data were analyzed using two-way ANOVA followed by Dunnett’s multiple comparisons test for comparisons between treatment groups and the vehicle control at each time point. (d) Representative images of excised tumors. (e) Tumor weight was measured 10 days after the first treatment with DE-CQ (5 and 10 mg/kg). Data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test for comparisons between treatment groups and the vehicle control. Data are presented as mean ± SEM. Vehicle, n = 4; DE-CQ 5 mg/kg, n = 3; DE-CQ 10 mg/kg, n = 3. p < 0.01, ** p < 0.0001 compared to the vehicle control.#

7. 总结与未来展望#

本项研究成功构建了单分子一体化的 NAD(P)H 响应型诊疗探针 DE-CQ。它摆脱了传统多组件前药拼装的复杂桎梏，巧妙利用癌细胞糖酵解产物 NAD(P)H 作为启动“钥匙”，在诊断端以 640 nm 强荧光输出直观反映肿瘤恶性侵袭性，在治疗端则通过精准定位于线粒体与内质网引发细胞凋亡级联反应。该成果为开发下一代结构精简、靶向度高、兼备代谢评估与精准抗癌潜力的个性化分子诊疗平台提供了全新的设计思路与实验范例。

【Angew.Chem.】中山大学夏炜|细菌介导的四价铂前药靶向递送与肿瘤深层渗透实现协同化疗-免疫治疗：肿瘤体积缩小72.2%