【ACS Nano】天津大学孟舒献|发光波长红移60nm且抑瘤率超90%！分子间非共价锁策略开启高效肿瘤光诊疗新途径
【ACS Nano】天津大学孟舒献|发光波长红移60nm且抑瘤率超90%！分子间非共价锁策略开启高效肿瘤光诊疗新途径
文章标题：Intermolecular Noncovalent Locks Enable Red-Shifted Emission, Modulated Reactive Oxygen Species Generation Pathway, and Shuttle-Like Nanoassembly for Phototheranostics
文章作者：Zhichao Gong, Guangbo Kang, Xuejiao Rong, Shenao Li, Lina Wang, He Huang*, Shuxian Meng*
文章概要
光诊疗制剂的传统研发往往依赖于繁琐多步的有机合成。天津大学研究团队提出了一种分子间非共价锁策略，将聚集诱导发光分子与聚集导致猝灭分子通过超分子作用力巧妙结合。该策略避免了复杂的化学修饰，使组装体系实现了荧光强度提高2倍、发射波长红移60 nm以及Ⅰ型活性氧生成路径的激活。此外，非共价锁诱导分子紧密排列，形成了独特的梭形纳米组装体。动物实验表明，该纳米制剂具备优异的肿瘤富集与长滞留特性，单次给药后配合光照即可实现超过90%的肿瘤抑制率，且生物安全性良好。
引言
光动力疗法与荧光成像相融合的光诊疗技术，因其高时空选择性和低毒副作用，在肿瘤精确诊疗领域展现出广阔的应用前景。理想的光敏剂通常需要同时具备近红外发射、高效活性氧产生以及良好的肿瘤组织穿透与滞留能力。然而，传统的分子设计思路主要依靠拓展共轭体系或引入大体积空间位阻基团等有机合成手段。这种方法步骤繁琐、合成成本高昂，且难以协同优化分子的光物理性质与纳米聚集体的形貌。
为突破这一瓶颈，研究团队将非共价锁的概念从单分子内部拓展至分子间体系。通过将包含特定杂原子的硼二吡咯衍生物与三苯胺衍生物进行组合，利用分子间多重非共价相互作用锁定组装构象。这一设计不仅大幅降低了合成复杂度，更实现了发光性能、活性氧产生机制与纳米形貌的三重协同调控。
Scheme 1 Schematic Representation of the Intermolecular Noncovalent Locking Strategy and Its Application in Phototheranostics
主要实验及结论
研究人员首先以一步克诺文盖尔缩合反应制备了两类基础分子，并构建了四种双分子组装体系。如图1所示，随着水相比例的增加，所有双分子体系均展现出渐进式的荧光增强与光谱红移特征。其中，BODNS与MeTPA构建的体系表现最为优异，其荧光强度相比单体提升了约2倍，发射峰显著红移60 nm并延伸至近红外810 nm区域。活性氧检测与电子自旋共振实验证实，该体系能高效产生超氧阴离子自由基与羟基自由基，成功激活了Ⅰ型光动力学机制。密度泛函理论计算揭示，双分子结合缩小了单重态与三重态之间的能隙，同时增大了自旋-轨道耦合常数，解释了Ⅰ型活性氧产率提升的原因。
Fig.1 Fluorescence emission of (A) BODNS, (B) BODNO, (C) MeTPA, (D) MeOTPA, (E) BODNS&MeTPA, (F) BODNS&MeOTPA, (G) BODNO&MeTPA, and (H) BODNO&MeOTPA in THF/water mixtures with varying fw. (I) Comparison of fluorescence emission between BODNS&MeTPA and MeTPA at fw = 70%–90%. (J) Comparison between BODNS&MeOTPA and MeOTPA at fw = 80%–90%. (K) Comparison between BODNO&MeTPA and MeTPA at fw = 70%–90%. (L) Comparison between BODNO&MeOTPA and MeOTPA at fw = 80%–90%. (M–O) ESR spectra of BODNS&MeTPA (fw = 90%). Comparison of (P) 1O2, (Q) O2•– and (R) •OH generated by BODNS, BODNS&MeTPA and MeTPA under laser irradiation (fw = 90%). (S) Distribution of HOMOs and LUMOs, and (T) calculated excited-state energy and SOC constants for BODNS, BODNS&MeTPA and MeTPA.
为进一步揭示激发态动力学过程，研究团队开展了超快瞬态吸收光谱分析。如图2所示，单体分子在激发后表现出较快的光致衰减，而BODNS&MeTPA双分子组装体在长波长区域的受激辐射信号衰减明显推迟，激发态寿命延长至697.56 ps。这一现象证实了电子供体与受体之间形成了稳定的分子间电荷转移态，该电荷分离态作为关键中间体促成了活性氧生成路径的转变。
Fig.2 fs-TA maps of (A) BODNS, (B) BODNS&MeTPA and (C) MeTPA in THF/H2O solution (fw = 90%). Extracted TA spectra of (D) BODNS, (E) BODNS&MeTPA and (F) MeTPA at selected delay times. Kinetic decay curves and their corresponding fitting lines for (G) BODNS, (H) BODNS&MeTPA and (I) MeTPA at representative wavelengths. Fitting parameters for (J) BODNS, (K) BODNS&MeTPA and (L) MeTPA.
在分子间相互作用的理论剖析方面，研究人员利用多种计算手段进行了深度探讨。如图3所示，静电势与独立梯度模型分析表明，BODNS与MeTPA之间存在丰富的C–H···N、C–H···S及S/O···N等非共价相互作用。这些分子间非共价锁不仅有效抑制了导致荧光猝灭的π–π堆积，还稳定了电荷转移构象。分子动力学模拟进一步证实，双分子在自组装过程中相互作用能持续增强，体系稳定性显著高于单组分聚集体。
Fig.3 (A) ESP distribution maps of BODNS, MeTPA and MeOTPA. (B) ESP penetration maps of BODNS&MeTPA and BODNS&MeOTPA. (C) mIGM and (D) Hirshfeld surface analysis of BODNS&MeTPA. (E) Percentage contributions of major intermolecular interactions to the Hirshfeld surface area for BODNS&MeTPA. (F) mIGM and (G) Hirshfeld surface analysis of BODNS&MeOTPA. (H) Percentage contributions of major intermolecular interactions to the Hirshfeld surface area for BODNS&MeOTPA. (I) Trajectories of the self-assembled system of BODNS&MeTPA. (J) Intermolecular interaction energy between different molecular pairs, (K) color-filled map of interaction angles and distances between different molecular pairs. (L) Time of first contact for different molecular pairs within the BODNS&MeTPA system.
随后，研究团队采用两亲性聚合物封装构建了纳米组装体。如图4所示，单组分对照组仅形成无定形的球形纳米颗粒，而引入分子间非共价锁的双分子体系则自发演变为形貌规整的梭形纳米组装体，其长宽比约为15<1>。体外细胞实验表明，梭形纳米组装体具有良好的生物相容性，且能被肿瘤细胞快速摄取。在激光照射下，梭形组装体展现出高达40以上的光毒性指数，引发了高效的细胞凋亡。
Fig.4 (A) Size distribution of BODNS&MeTPA NSs (means ± SD; n = 3). B) TEM images of BODNS&MeTPA NSs. (C) UV–vis absorption and fluorescence emission spectra of BODNS&MeTPA NSs. (D) Comparison of fluorescence emission between BODNS&MeTPA NSs and BODNS&MeTPA. Concentration-dependent generation curves of (E) total ROS, (F) 1O2, G) O2•– and (H) •OH. (I) UV–vis absorption spectra of BODNS&MeTPA NSs before and after laser irradiation. Concentration-dependent cell viability of (J) CT26 cells and (K) 3T3 cells treated with BODNS&MeTPA NSs without laser irradiation. (L) Concentration-dependent cell viability of CT26 cells treated with BODNS&MeTPA NSs with laser irradiation. (M) Time-dependent cellular uptake profiles. Intracellular generation of (N) ROS, (O) 1O2, (P) O2•– and (Q) •OH generation after various treatments. (R) Live/dead staining (calcein-AM/PI) after various treatments. (Statistical significance between groups was determined using Student’s t-test; means ± SD; n = 6; **p < 0.01, ***p < 0.001) (scale bar: 25 μm).
活体应用实验进一步验证了其诊疗潜能。如图5所示，小鼠全身荧光成像显示，梭形纳米组装体在静脉注射后能够高效靶向肿瘤，并在肿瘤组织中实现长达72小时的高效滞留。在活体治疗实验中，受试小鼠在接受单次给药与三次光照后，肿瘤体积持续缩小，治疗结束时肿瘤抑制率超过90%。同时，小鼠体检指标与主要器官组织切片均未见损伤，证实了该策略良好的生物安全性与应用潜能。
Fig.5 (A) In vivo whole-body fluorescence images acquired at designated intervals after intravenous injection. (B) Fluorescence images of isolated major organs and tumors collected at 3, 24, and 72 h postinjection (0 h shown as control). (C) Quantification of tumor fluorescence intensity over time (means ± SD; n = 3). (D) Quantified fluorescence signals in major organs and tumors at 3, 24, and 72 h postinjection (means ± SD; n = 3). (E) Schematic representation of the treatment regimen. (F) Body weight changes and (G) tumor growth profiles of mice across different groups during the 12 day observation period. (H) Tumor weights measured in the end of the treatment regimen. (means ± SD; n = 5; Student’s t-test, ***p < 0.001). (I and J) Representative photographs of mice and (K) corresponding excised tumors from each group. (L) Blood routine parameters and (M) blood biochemistry of mice from different groups after various treatments. (N) Representative histological and immunohistochemical staining (H&E, TUNEL, K i-67, and CD31) of CT26 tumor sections from each treatment group.
总结及展望
该研究成功开发了一种分子间非共价锁策略，摆脱了传统光敏剂对复杂有机合成的依赖。通过简单分子的自组装，不仅实现了荧光发射的红移与增强，还调控了活性氧产生路径并构建了具备肿瘤靶向特性的梭形纳米结构。这种将研究视角从单分子工程转向超分子组装的设计思路，为开发高性能、低成本的光诊疗药物提供了新途径。未来，随着代谢动力学与长期毒理学评价的深入，该策略有望在肿瘤精准诊疗领域展现更大的应用价值。
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