核心研究主旨与总体成果#

分子余辉成像是一种能够在停止激发后持续发光的先进光学成像技术，在生物医学领域具有极高的应用价值。然而，传统声余辉技术高度依赖复杂的多组件纳米系统，存在制作工艺繁琐、体内代谢行为难以预测等瓶颈。本研究成功开发出全球首个自维持的单分子声余辉发光体，通过精细的分子结构设计与筛选，合成了具备强红光发射、长半衰期和优异可充能特性的单分子探针，并进一步将其改造为针对肿瘤衰老标志物的特异性激活探针，实现了在活体动物体内对化疗诱导的衰老肿瘤细胞进行实时、长期的无创精准动态监测。

研究背景与传统技术的困局#

在生物医学成像领域，传统的荧光成像往往面临组织背景干扰大、穿透深度有限等固有难题，这是由于生物组织自身在光照下会产生背景自发荧光。分子余辉成像技术通过在化学键或晶体缺陷中预先存储能量，在停止激发后缓慢释放光信号，从而彻底消除了背景干扰。为了进一步提高对人体深部组织器官的穿透能力，科学家们结合高频超声技术开发出了声余辉成像。超声波具有极强的高组织穿透力，能够深入体内诱导发光。

目前已有的声余辉成像系统大多依赖于纳米级的多组件复合物。这类系统需要将声感光剂、余辉基质和发光元件等多种化学成分包裹在一起，不仅合成步骤极为复杂，不同组件之间的比例难以精确调控，而且进入活体后各个组分很容易发生分离，导致成像效果不稳定、生物毒性和体内清除规律难以掌控。因此，开发一种将超声响应、能量存储与余辉发射完美融为一体的单分子声余辉探针，成为了光学分子成像领域亟待突破的核心难题。此外，在癌症治疗中，化疗等手段常会导致癌细胞进入细胞衰老状态，这类衰老癌细胞虽然停止分裂但依然存活，甚至会分泌有害因子促进肿瘤复发，因而亟需一种能够在深层组织中精准、实时监测衰老癌细胞的无创成像工具。

Fig.1 Design and characterization of optical properties of sonoafterglow luminophores. (a) Chemical structures of sonoafterglow luminophores SL1–SL12. (b) Normalized fluorescence spectra of SL1–SL12 (100 μM) in PBS (0.01 M, pH 7.4, containing 10% DMSO, V/V). (c) Sonoafterglow spectra and (d) corresponding half-lives of SL1–SL12 (100 μM) in PBS (0.01 M, pH 7.4, containing 10% DMSO, V/V) solution after ultrasound irradiation (1.5 W cm–2, 180 s). (e) Sonoafterglow intensities of SL6 and SL12 (100 μM) in PBS (0.01 M, pH 7.4, containing 10% DMSO, V/V) solution over 5 consecutive cycles of ultrasound irradiation (1.5 W cm–2, 180 s per cycle). (f) Representative sonoafterglow and fluorescence images of SL6 and SL12 (100 μM) in PBS (0.01 M, pH 7.4, containing 10% DMSO, V/V) after ultrasound irradiation (1.5 Wcm–2, 180 s), acquired through varying tissue depths (0, 0.5, 1.0, 1.5, and 2.0 cm). (g) SBRs of sonoafterglow and fluorescence signals at varying tissue depths (0, 0.5, 1.0, 1.5, and 2.0 cm).

创新性的单分子分子设计与筛选流程#

为了打破多组件纳米系统的局限，研究团队提出了基于受体-酚基-金刚烷核心骨架的单分子设计策略。研究人员合成了12种结构精密的单分子发光体，分别命名为SL1至SL12。这套分子库的设计巧妙在于，研究团队挑选了6种不同吸电子能力的化学基团，分别连接在酚基的邻位或对位，以此精确调控分子的发光波长、化学反应活性以及电子转移速率。

通过系统的光谱性能筛选，研究团队发现对位取代的分子整体呈现出更显著的红移现象，这是因为对位结构具有更平整的分子构象，能够形成更广泛的共轭体系。在经过超声波照射的诱导测试中，所有12种分子均展现出了3分钟至24分钟不等的余辉半衰期。其中，邻位取代的SL6和对位取代的SL12脱颖而出，其余辉发射峰均位于638纳米的近红外红光区域，表现出最高的余辉发光强度。更令人瞩目的是，这两款单分子发光体展现出了极佳的可重复充能特性，在经历5次连续的超声照射循环后，其余辉信号仅分别衰减了8%和8.5%，证明了其在长期反复成像中的卓越稳定性。

深度组织穿透性能与发光机制拆解#

在深层组织穿透能力评估中，研究团队利用不同厚度的鸡胸肉组织模拟生物体深层环境。实验结果表明，尽管余辉和荧光信号都会随着组织厚度的增加而衰减，但声余辉信号在各个深度均展现出远高于传统荧光的对比度。在0.5厘米的组织深度下，SL6和SL12的声余辉信噪比分别达到了154.2和124.4，相比于传统荧光成像（信噪比仅为13.4和13.1）实现了11.5倍和9.5倍的显著提升。即使当组织厚度增加到2.0厘米时，传统荧光信号已几乎无法被识别，而SL6和SL12的声余辉信号依然清晰可见，信噪比维持在4.3和3.5，展现出了极其优异的深层组织成像潜力。

为了揭示单分子声余辉的产生机制，研究团队结合理论化学计算与高分辨质谱、红外光谱等多种检测手段进行了深入剖析。研究表明，该过程包含一个高效的两步级联反应。在超声波照射下，分子首先通过声动力学效应高效产生单线态氧。生成的单线态氧随后与分子结构中的金刚烷双键发生自发的环加成反应，生成高能量的中间体，并进一步重排为高度张力的1,2-二氧杂环丁烷结构。随后，酚羟基发生去质子化形成富电子的酚负离子，进而引发化学引发电子交换发光级联过程。在此过程中，电子转移促使过氧化物化学键断裂并释放巨大的能量，使分子自身跨越能级进入激发态，最终在回到基态时释放出强烈的红光声余辉。理论计算进一步证实，邻位与对位的化学结构差异决定了其化学键断裂的速率决定步骤，从而影响了整体的发光动力学特征。

Fig.2 Mechanistic investigation of sonoafterglow generation. (a) Generation of 1O2 determined by changes of ABDA absorbance in the presence of SL1–SL12 (100 μM) in PBS (0.01 M, pH 7.4, containing 10% DMSO, V/V) after ultrasound irradiation (1.5 W cm–2, 3 min). (b) ESR spectra of SL6 and SL12 (100 μM) in PBS (0.01 M, pH 7.4, containing 10% DMSO, V/V) before and after ultrasound irradiation (1.5 W cm–2, 3 min), using TEMP as the trapping agent for 1O2. (c) DFT calculations illustrating the proposed reaction pathways of SL6 and SL12 with 1O2 and the generation of sonoafterglow emission. (d) High-resolution mass spectrometry (HRMS) spectra of SL6 and SL12 in an aqueous solution after ultrasound irradiation (1.5 W cm–2, 3 min). (e) Fourier transform infrared (FTIR) spectroscopy spectra of SL6 and SL12 (100 μM) in CHCl3 solution before and after ultrasound irradiation (1.5 W cm–2, 3 min). (f) Schematic diagram of the proposed sonoafterglow mechanism of SL6 and SL12.

肿瘤衰老标志物特异性激活与活体应用#

在成功明确了SL6和SL12的优异发光特性后，研究团队立足于肿瘤临床监测的现实需求，将研究进一步延伸至疾病生物标志物的响应性成像。衰老细胞通常会高表达一种名为β-半乳糖苷酶的生物标志物。研究人员分别在SL6和SL12的活性位点上偶联了β-半乳糖苷基团，构建出两款具备酶响应潜力的激活型探针，命名为GP6和GP12。由于半乳糖苷基团的封端屏蔽作用，探针在初始状态下处于“熄灭”状态，不会产生明显的余辉信号。

在体外酶解响应实验中，两款探针展现出了截然不同的化学命运。当与β-半乳糖苷酶共同孵育时，GP12能够被酶高效精准地切除封端基团，成功释放出活性发光体SL12。经过超声照射后，GP12表现出极其强烈的信号开启效应，其荧光强度提升了9.3倍，而声余辉信号提升更是达到了惊人的32.3倍，且对其他干扰酶及活性氧物质表现出高度的特异性选择。相比之下，GP6由于邻位结构的空间位阻影响，未能被酶识别，反而引发了苯并恶唑环的异常羟基化破坏，导致探针失效。这一现象深刻揭示了分子取代位置对酶底物亲和力与化学稳定性的关键作用。最终，研究团队将GP12成功应用于小鼠乳腺癌皮下肿瘤模型中，实现了对阿霉素（DOX）化疗诱导的衰老肿瘤的高对比度、实时、纵向无创跟踪监测。

Fig.3 In vitrocharacterization of β-gal-activatable sonoafterglow probes. (a) Fluorescence spectra of GP12 (100 μM) after incubation with or without β-gal (10 U mL–1) for 6 h, followed by ultrasound irradiation (1.5 W cm–2, 3 min). (b) Sonoafterglow spectra of GP12 (100 μM) after incubation with or without β-gal (10 U mL–1) for 6 h, followed by ultrasound irradiation (1.5 W cm–2, 3 min). (c) Fold enhancement of fluorescence and sonoafterglow signal enhancement in (a) and (b). (d) HPLC chromatography of SL6, SL12, GP6, and GP12 before and after incubation with β-gal (10 U mL–1) for 6 h. (e) Scheme of the proposed decomposition mechanism of GP6 and the β-gal-activatable sonoafterglow mechanism of GP12. (f) Substrate-dependent cleavage of GP12 by β-gal. (g) Sonoafterglow intensities of GP12 after incubation with the indicated enzymes or ROS for 6 h, followed by ultrasound irradiation (1.5 W cm–2, 180 s). (h) 1O2 generation efficiency of GP12 after incubation with or without β-gal (10 U mL–1) for 6 h, followed by ultrasound irradiation (1.5 W cm–2, 180 s). For inhibitor group, β-gal was treated with inhibitor (d-galactose, 100 μM) for 1 h. Data are presented as mean ± SD (n = 3). Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. The mean values and SD are presented. The values are relative to the control groups, where **** p < 0.0001.

Fig.4 In vitro evaluation of GP12 for sonoafterglow and fluorescence imaging in DOX-induced senescent cells. (a) Schematic illustration of the cell treatment layout for sonoafterglow and fluorescence imaging of senescent cancer cells. (b) Representative sonoafterglow images and (c) corresponding quantified intensities of 4T1 cancer cells and 3T3 fibroblasts. Cells were pretreated with either DOX or without DOX, followed by sequential incubation with GP12 (100 μM, 10 h). DOX-treated cells were also incubated with the β-gal inhibitor, d-galactose (d-gal, 1 mM). (d) Representative fluorescence images of 4T1 cancer cells and 3T3 fibroblasts under the same treatment conditions. (e) Corresponding mean fluorescence intensities quantified from (d). (f) Representative confocal images of 4T1 and 3T3 cells under various treatments. Green signal corresponds to oxidized H2DCFDA (25 μM), indicating ROS generation. (g) Quantification of mean fluorescence intensity of DCF (reflecting ROS levels) from the images in (g). (h) Western blot analysis of β-gal expression in 4T1 cancer cells and 3T3 fibroblast cells. Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. The mean values and SD are presented. The values are relative to the control groups, where *** p < 0.001, **** p < 0.0001.

Fig.5 In vivo real-time, longitudinal sonoafterglow and fluorescence imaging of a DOX-induced senescent tumor. (a) Experimental timeline of chemotherapy and subsequent sonoafterglow and fluorescence imaging. DOX (10 mg kg–1 body weight, 100 μL in PBS) was administrated intravenously as a single dose. The inhibitor-treated group received oral dasatinib (5 mg kg–1) and quercetin (50 mg kg–1) in olive oil containing 10% DMSO (100 μL) once daily for 5 days prior to imaging. GP12 (100 μM in PBS, containing 10% DMSO) was administered via intratumoral injection. (b) Representative longitudinal sonoafterglow and fluorescence images acquired under different treatments. Quantification of (c) sonoafterglow and (d) fluorescence intensities at indicated time points after GP12 injection. Tumor signal-to-background ratio of (e) sonoafterglow and (f) fluorescence signal intensities in tumors at indicated time points after GP12 injection. (g) Western blot and (h) immunofluorescence staining of β-gal expression in tumor sections from 4T1 tumor-bearing mice after different treatments. Statistical significance was calculated via one-way ANOVA with a Tukey post hoc test. The mean values and SD are presented. The values are relative to the control groups, where *** p < 0.001, **** p < 0.0001.

研究价值、现存局限与未来展望#

本项研究在学术理论与临床应用层面均具有里程碑意义。在理论层面，它打破了以往声余辉成像必须依赖复杂纳米系统的传统思维，开创性地证实了单分子结构同样可以完美兼具声感光、能量储存与级联化学发光的多重功能，为下一代深层组织光学探针的设计提供了全新的化学范式。在实际应用层面，该探针凭借高组织穿透力、超高信噪比以及对衰老标志物的高灵敏激活响应，为临床无创评估肿瘤化疗疗效、动态追踪化疗诱导的细胞衰老提供了强有力的工具，有助于避免因衰老肿瘤细胞积聚导致的肿瘤复发与副作用。

客观来看，该项研究仍存在一定的局限性与拓展空间。目前所有的活体验证均建立在小鼠皮下肿瘤模型之上，未来仍需在更贴近人类生理特征的原位肿瘤模型及大动物模型中进一步验证其成像深度与药代动力学特性。同时，单分子探针在体内的长远生物安全性、体内降解代谢产物的毒理学评估以及大规模合成的成本控制，也是推动其走向临床转化必须跨越的障碍。研究团队未来可以进一步丰富该单分子骨架，通过替换不同的酶响应基团或疾病靶向头基，将这一自维持声余辉平台扩展至心血管疾病、器官损伤及其他恶性肿瘤的早期诊断与精准分子成像领域。