【Angew.Chem.】大连理工彭孝军院士等|450 nm可见光激活、700 nm近红外发射与268 ms寿命：激发态构象动态学赋能高对比度活体肿瘤成像#

Fig.1 (a) Molecular design logic of target compounds and photophysical process of visible light-excited NIR-UOP. (b) Nano-assembly approach for constructing NIR-UOP nanoparticles.#

如图2所示，研究团队在PMMA掺杂薄膜中对系列分子（py-2OCH3、py-2pNO2和py-2mNO2）的光物理性质进行了详细对比。相比于甲氧基取代的py-2OCH3，硝基取代分子的吸收与激发光谱显著展宽并延伸至可见光区域，其稳态荧光发射分别红移至535 nm和550 nm。当切断365 nm激发光源后，py-2mNO2表现出最佳的近红外超长磷光性能，其主发射峰位于635 nm和700 nm，磷光寿命长达268 ms，发光强度高达12 cd/m²，磷光量子效率达到4.6%，在撤去光源后余辉肉眼可见持续长达2秒。更为关键的是，监测700 nm发射的激发光谱显示，py-2mNO2的吸收响应带可延伸至500 nm。实验证明，该分子在450 nm可见光照射下依然能够被有效激活，并产生明显的700 nm近红外超长磷光。

Fig.2 (a) PL spectra of py-2OCH3, py-2pNO2, and py-2mNO2 films (_m_PMMA:_m_guest = 300:1) in steady state (_λ_ex = 330 nm) and delay 8 ms (_λ_ex = 365 nm) at room temperature. (b) UOP intensity for three target doped films (The response time interval of the photometer used is 0.3 s; the power of the UV flashlight used for measuring phosphorescence intensity is 5 W. The recorded phosphorescence intensity is the first response value detected by the photometer after the UV light is turned off). (c) Emission decay curves of three doped films peaked at 700 nm (_λ_ex = 360 nm). (d) The photograph of doped films under UV lamp (_λ_ex = 365 nm) and at a delay of 0.25 s. (e) Excitation spectra of py-2OCH3, py-2pNO2, and py-2mNO2 films peaked at 700 nm. (f) Delay emission of three films under visible light excitation (delay 8 ms; _λ_ex = 450 nm).#

如图3所示，为了揭示长波长激活与高发光效率的微观机制，团队开展了极性溶剂光谱实验与变温核磁共振测量。光谱结果表明，py-2mNO2在非极性环己烷中仅呈现415 nm的局域激发态（LE）发光，而在高极性溶剂四氢呋喃中则展现出415 nm与550 nm的双重发光特性。随着溶剂极性增加，550 nm处的长波长发射峰显著增强并发生红移，展现出典型的分子内电荷转移（CT）特征。变温核磁共振谱图显示，随着温度升高，硝基苯环与芘环上的质子信号发生明显的化学位移上场移动与峰形展宽，证实了分子内单轴旋转导致的构象快速互变。密度泛函理论计算进一步证实，py-2mNO2在单重态S1态的二面角从基态的122°动态旋转至140°，存在局域激发势洼与电荷转移势洼。在刚性PMMA基质中，聚合物网络限制了分子的自由扭转，将分子锁定在具有强电荷转移特性的构象中，从而大幅提升了长波长可见光的吸收与激发效率。

Fig.3 (a) Solvent polarity-dependent spectra of py-2mNO2 at room temperature (c = 1×10−5 mol/L, _λ_ex = 330 nm). (b) Excitation spectra of py-2mNO2 in THF (c = 1×10−5 mol/L, grey line: the excitation spectrum corresponding to the emission at 415 nm; red line: the excitation spectrum corresponding to the emission at 550 nm). (c) Emission decay curves of py-2mNO2 in THF peaked at 415 and 550 nm (c = 1×10−5 mol/L, _λ_ex = 375 nm). (d) Variable-temperature 1H NMR spectra of py-2mNO2 in DMSO-_d_6 from 25°C to 80°C. (e) The optimized geometric structure of py-2mNO2 in the S1 (_θ_1: the dihedral angle between the pyrene and the nitrobenzene on the right; _θ_2: the dihedral angle between the pyrene and the nitrobenzene on the left; red plane: the plane of the pyrene core; blue plane: the plane of the right benzene ring; grey plane: the plane of the left benzene ring). (f) Potential energy surface scans of py-2mNO2 in S0, S1, and T1 as a function of _θ_1 variation (_θ_2 = 140°). (g) NTOs analysis of py-2mNO2 under different conformations (red and grey isosurfaces correspond to hole and electron distribution, respectively).#

如图4所示，低温77 K光谱与自旋-轨道耦合（SOC）理论计算阐明了该系统实现高效近红外发光的本质原因。计算结果表明，电荷转移构象（D-140）的自旋-轨道耦合常数（0.59 cm⁻¹）显著高于局域激发构象（0.11 cm⁻¹），极大地促进了单重态到三重态的高效系间篡越（ISC）过程。同时，py-2mNO2在激发态呈现出极为平缓的势能面，使分子能够自然采取更加平面的几何结构。在该平面构象下，分子内存在显著的吸引相互作用，有效抑制了高频分子振动与转动，从而将非辐射衰减速率大幅降低至3.78 s⁻¹（明显低于对位异构体py-2pNO2的5.87 s⁻¹）。这种电荷转移促进系间篡越与平面构象抑制无辐射衰减的协同作用，最终实现了高效率的近红外超长磷光。

Fig.4 (a) Delay spectra of py-2mNO2 in CYH and THF at 77 K (delay 8 ms, _λ_ex = 365 nm). (b) Calculated energy level of excited-state and spin-orbit coupling (SOC) matrix elements (ξ) of target compounds based on different conformation structure (the initial conformation corresponds to the potential energy minimum point under gas-phase optimization). (c) Potential energy surface scans of py-2mNO2 in S0, S1, and T1 as a function of _θ_1 variation from minimum point to 165° (_θ_2 = 140°, step: 5°). (d) Interaction region indicator (IRI) analysis of py-2mNO2 under different conformation. (e) Schematic diagram of the photophysical process for visible-light-excited NIR-UOP in the py-2mNO2.#

如图5所示，基于上述优异性能，团队将py-2mNO2与PMMA按照1:100的质量比进行基质隔离组装，成功制备出平均水力学粒径为153 nm的均一球形纳米颗粒。细胞毒性实验表明，在高达100 μM的浓度下，4T1细胞存活率依然保持在90%以上，展现出良好的生物相容性。在细胞发光成像中，由于三线态激子易被氧气猝灭，纳米探针在1%的低氧环境下表现出显著增强的磷光信号，具有优异的缺氧响应特性。组织穿透模拟实验表明，即使在覆盖2 mm厚牛肉块的情况下，纳米探针在普通手机手电筒照射后仍能产生清晰的近红外余辉信号。在活体小鼠肿瘤成像实验中，经过局部注射纳米探针后，在撤去365 nm紫外灯和手机手电筒照射10秒后，肿瘤部位均能捕获到极高对比度的磷光信号，其信号与背景比（SBR）分别高达39.47和14.55，显著优于以往报道的短波长发光探针（如C2D探针SBR为14.46），彻底清除了活体组织的背景自发荧光。

Fig.5 (a) Emission spectra in THF/water mixtures with varying water fractions (_λ_ex = 330 nm). (b) Preparation process of matrix-isolated nanoparticles. (c) The hydrodynamic size and SEM of py-2mNO2 nanoparticles in water. (d) Cell viabilities of 4T1 cells after incubation with nanoparticles in different concentrations for 24 h. (e) Confocal microscopy images of 4T1 cells with different oxygen concentrations incubated with py-2mNO2 nanoparticles (10 µM). Cells were imaged in the red channel for py-2mNO2 nanoparticles (_λ_ex = 405 nm, _λ_em = 600–800 nm). (f) UOP images of py-2mNO2 nanoparticles (1 mg/mL, 8.13 × 1010 particles/mL) with and without beef tissue under different excitation wavelength. (g) In vivo UOP imaging followed by intratumorally injections of py-2mNO2 nanoparticles with PMMA (1 mg/mL, 8.13 × 1010 particles/mL, 30 µL) under different excitation source. The UOP images were captured 10 s after the removal of the light source. The model of the smartphone used as the flashlight is HUAWEI P50 Pro. (h) SBR in live mice for UOP from py-2mNO2 and C2D [7] in previous work. Error bars represent standard deviation of three separated measurements, n = 3.#