(a) Schematic Illustration of the Photophysical and Photodynamic Mechanisms of Different Photosensitizers (PSs); (b) Molecular Structures and Photophysical/Photodynamic Properties of the HLCT-Type Cyanine Dyes (CyTY-1, CyTY-2, CyTY-3); (c) The Unique Charge-Separated Structure of CyTY-3 and Its Long-Term Tumor Retention Performance In Vivo#

Fig.1 Evaluation of the synthesis and photophysical properties of three CyTY dyes: (a) Chemical structures; (b) UV–vis absorption spectra in dimethyl sulfoxide (DMSO); (c) PL spectra in DMSO; (d) transient PL decay spectra; (e) linear fitting of the Lippert–Mataga model; (f) transient absorption (TA) spectra and corresponding kinetic decay curves of CyTY-3 in DCM.#

为了深入探索分子的激发态演化机制，研究团队进行了理论计算与光谱验证。如图2所示，时间依赖密度泛函理论计算揭示了分子最高占据分子轨道与最低未占分子轨道的空间有效分离，证实了典型的HLCT特征。能级图分析显示，CyTY-3具有极小的S₁-T₂能级差（0.08 eV）和较大的T₂-T₁能级差（0.76 eV），大幅提升了单线态到高阶三重态的系间跨越速率（3.8 × 10⁷ s⁻¹）。在此基础上，局部激发态特性保障了高荧光发光效率并抑制了三线态湮灭，而电荷转移态特性则驱动了电子转移过程，从而同时激活了产生单线态氧的Ⅱ型途径以及产生超氧阴离子和羟基自由基的Ⅰ型途径。

Fig.2 (a) Frontier molecular orbitals from TDDFT calculations of CyTY in the ground state. (b) Singlet–triplet energy-level diagrams and spin–orbit coupling matrix element (SOCMe) values of CyTY and ICG. (c) Photoinduced ROS generation and photosensitization mechanism of CyTY-3.#

活性氧检测实验全面验证了分子的光动力性能。如图3所示，利用DPBF、DHR123和HPF等探针评估发现，CyTY-2和CyTY-3的单线态氧生成能力显著优于对照组。电子自旋共振光谱配合猝灭剂实验明确捕获到了单线态氧、超氧阴离子和羟基自由基的特征信号，证实了CyTY-3具备出色的Ⅰ/Ⅱ型协同光动力活性。如图4所示，光热性能与能量跃迁测试表明，CyTY-3较大的构象柔性促进了非辐射跃迁，使其兼具19.0%的光热转换效率。

Fig.3 (a) Kinetic curve of 1O2 generation derived from the decomposition rate of DPBF. (b) Graph of relative fluorescence intensity (I/_I_0) of DHR123 solution containing CyTY dyes as a function of irradiation time. (c) Graph of relative fluorescence intensity (I/_I_0) of HPF solution containing CyTY dyes as a function of irradiation time. (d) Fluorescence kinetic curves of DCFH for verifying the total ROS production of CyTY-3, together with the control groups pretreated with FFA (1O2) and TBA + SOD (O2–• and •OH). (e) EPR spectra of TEMP and DMPO spin-trapping adducts, confirming the production of 1O2, O2–•, and •OH upon white light irradiation for 5 min in the presence of 5 μM CyTY-3. (f) Photostability comparison of CyTY-3 and commercial ICG monitored by the absorbance retention ratio (A/_A_0) within 10 min of continuous light irradiation.#

Fig.4 (a) Temperature elevation profiles of CyTY-2 and (b) CyTY-3 aqueous solutions with different concentrations under 808 nm laser irradiation (1.5 W·cm–2, 600 s). (c) Photothermal stability tests of CyTY-2, CyTY-3, and ICG over three heating–cooling cycles. (d) Decomposition of reorganization energy into contributions from bond angles, dihedral angles, and bond lengths for CyTY-2 and (e) CyTY-3. (f) RMSD comparison between the optimized ground- and excited-state conformations of CyTY-2 and CyTY-3.#

在细胞与动物水平的生物学效应评价中，HLCT光敏剂展现出了突出的诊疗潜能。如图5所示，激光共聚焦显微镜观察到CyTY-3能在60分钟内被4T1乳腺癌细胞高效摄取。在常氧及1%氧气的缺氧条件下，结合808 nm激光照射，CyTY-3处理组的细胞存活率均显著下降至18%左右，证实其克服缺氧微环境的能力。如图6所示，将CyTY-3封装为纳米颗粒后，其平均粒径约为60 nm。借助于电荷分布带来的自靶向效应以及高局部正电荷与肿瘤细胞膜负电荷的静电作用，CyTY-3纳米颗粒在小鼠肿瘤部位的积累荧光强度达到ICG的15倍，且肿瘤滞留时间长达12天。如图7所示，在体内抑瘤实验中，经808 nm激光照射治疗14天后，CyTY-3纳米颗粒组的肿瘤完全消退，抑瘤率达到99.01%，且小鼠血生化指标与主要器官组织切片均未见明显毒副作用，表现出极佳的体内生物安全性。

Fig.5 (a) Fluorescence staining images of incubated 4T1 cells with CyTY dyes (λex: 580/640/640 nm, λem: 650–900 nm, scale bars: 50 μm). (b) ROS generation image of CyTY-3 dye after light irradiation (scale bars: 100 μm). (c) Survival rate of 4T1 cells treated with various concentrations of CyTY dyes under normoxia, hypoxia, light-irradiated, and nonirradiated conditions. Data are representative of n = 5 independent experiments. (d) Live/Dead cell costaining images of CyTY-3 treated groups under normoxic and hypoxic conditions with Calcein-AM and Propidium Iodide (PI) as markers (scale bars: 100 μm). (e) DHE and HPF staining images of ROS for CyTY dyes under hypoxic conditions (scale bars: 50 μm).#

Fig.6 (a) Schematic illustration of the preparation process of CyTY-3 NPs. (b) Particle size distribution of CyTY-3 NPs detected by DLS (inset: typical TEM images of CyTY-3 NPs. Scale bars: 100 nm). (c) NIR-II emission spectrum and NIR-II fluorescence images of CyTY-3 NPs. (d) NIR-II fluorescence images of heart, liver, spleen, lung, kidney, and tumor tissues from tumor-bearing mice at 72 h postintravenous injection of CyTY-3 NPs (after dissection). (e) Comparison of NIR-II fluorescence imaging between ICG and CyTY-3 NPs. The illustration includes: After intravenous injection of ICG and CyTY-3 NPs into tumor-bearing mice, respectively, tumor photographs and NIR-II fluorescence images were acquired at different time points (excitation wavelength λex = 808 nm, long-pass filter 880 nm, exposure time 100 ms). (f) Comparison of intratumoral fluorescence intensity between ICG NPs and CyTY-3 NPs. (g) CyTY-3 NPs zeta potential plot and surface electrostatic potential distribution of CyTY-3 and ICG within different electrostatic potential intervals.#

Fig.7 (a) Schematic diagram of the photodynamic antitumor therapy process. (b) Images of mice and tumors with different treatments 14 days post-treatment. (c) Relative tumor volume of mice after treatments. (d) Body weights of the mice after different treatments. (e) Photographs of tumors dissected from 4T1 tumor-bearing mice after 14 days of treatment. (f) H&E-stained tumor sections from different treatment groups. Scale bar = 100 μm. (g) Liver function indicators: detection levels of Alanine Aminotransferase (ALT). (h) Liver function indicators: detection levels of Aspartate Aminotransferase (AST). (i) Renal function indicators: detection levels of Creatinine (CREA). The results are representative of n = 3 independent experiments.#