文章概要#

针对三阴性乳腺癌易复发和远端转移的临床痛点，研究团队开发了一种基于无荧光近红外电子共振拉曼探针的诊疗一体化纳米平台。该探针消除了荧光背景干扰，展现出低至95 fM的检测限、高达69.9的体外信噪比以及良好的光稳定性。结合特异性靶向与光热效应，探针在术中实现了信噪比为22.6的肿瘤边缘精确导航，并在术后通过高吸光转化为63.9%的光热转换效率。配合免疫检查点抑制剂，成功清除残留微小肿瘤并抑制远端肺转移。

Scheme.1 (a) Jablonski diagram of the designed quenchers as fluorescence-free Raman reporters and advantages of fluorescence-free probes over fluorescent counterparts. (b) Schematic diagram of the non‑fluorescent probe strategy for TNBC. (c) Postoperative synergistic therapy based on photothermal effect and immunotherapy.

引言#

三阴性乳腺癌缺少明确的靶点，临床上主要依赖手术切除配合术后化疗。然而此类肿瘤侵袭性极强，残留的微小病灶常导致术后复发和远端转移。近红外荧光成像虽常用于导航，但易受光漂白影响，且存在背景干扰大、光热效率低的问题。表面增强共振拉曼散射技术具有极高特异性，但传统拉曼报告分子大多依赖荧光染料，未完全淬灭的残留荧光会掩盖微弱的拉曼信号，显著降低检测灵敏度。因此，开发一种无荧光干扰、高稳定性且兼具高效光热性能的近红外拉曼探针具有重要意义。

Fig.1 (a) Molecular architectures of Q820 and Q920. (b) UV–vis absorption profiles for Q820, Q920, and ICG recorded in DMF. (c) The highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) distributions of Q820 and Q920. (d) Fluorescence emission of Q820, Q920, and ICG in DMF excited by 875, 940, and 788 nm, respectively. The concentration of each molecule is 10 µM. (e) The Φaf of Q820, Q920, and ICG in DMF under 800 nm excitation. (f) Time-resolved absorption profiles of a mixture comprising Q820 (5 µM) and DPBF (20 µM) subjected to 808 nm laser excitation for 3 min. Note: the acquired spectral traces are perfectly superimposed. (g) Corresponding spectral evolution for the Q920 (5 µM) and DPBF (20 µM) co-solution under identical irradiation parameters. Note: complete overlap of all curves is observed. (h) Full-profile fs-TA mapping of Q820 in DMF under 20°C air conditions. The GSB region is outlined with a black dashed box. (i) fs-TA spectral profiles of Q820 in DMF, acquired at representative pump-probe delay intervals. The trends in the plots are indicated by black arrows. (j) Single-wavelength kinetic fitting curve of Q820 in DMF at 885 nm.

主要实验及结论#

研究团队首先设计合成了两种具有近红外强电子吸收但无荧光发射的暗黑猝灭剂分子 Q820 和 Q920。如图1所示，光谱与电化学分析表明，分子极低的荧光量子效应和被抑制的系间跨越路径，使其激发态能量主要通过非辐射跃迁形式释放。飞秒瞬态吸收光谱进一步证实，Q820的非发光特性源于皮秒级的高效内部转换过程。

随后研究人员将报告分子锚定在金纳米棒表面并包裹聚 dopamine 保护层，构建出无荧光纳米探针。如图2所示，在785 nm激光激发下，AuNR@Q820 与 AuNR@Q920 展现出清晰的金属-硫键特征拉曼峰，且完全没有荧光背景干扰。其增强因子相比传统荧光染料ICG组提升了两个数量级，体外信噪比高达 69.9，极限检测浓度降至 95 fM。在持续30分钟的强激光照射下，探针拉曼信号强度保持稳定，克服了传统荧光探针光漂白的弊端。

Fig.2 (a) TEM image of AuNRs with PDA coatings. (b) Raman spectra of AuNR@Q820, AuNR@Q920, and AuNR@ICG NPs with a same concentration of 0.2 nM. (c) SERRS EF and SBR of three NPs. (d–f) The LOD of AuNR@Q820, AuNR@Q920, and AuNR@ICG NPs under 785 nm excitation. The Raman mappings of these three NPs were derived from the bands at 381, 366, and 1150 cm−1, respectively. (g-i) Photostability contour mappings of AuNR@Q820, AuNR@Q920, and AuNR@ICG NPs during 30 min of uninterrupted 785 nm laser excitation. Error bars, mean ± s.e.m. (n = 3).

量子化学计算揭示了探针优异光热性能的分子机制。如图3所示，分子重组能与黄昆-里斯因子的计算结果表明，二面角扭转振动有效促进了非辐射耗散。当无荧光染料与金纳米棒的局部表面等离子体共振发生近场耦合时，产生的协同效应使 AuNR@Q820 探针的光热转换效率达到了 63.9%，显著高于传统物理混合组与荧光探针组。连续5次光热循环实验验证了其优异的结构热稳定性。

Fig.3 (a) Calculated minimum-energy structures of Q820 and Q920 in the S0 (ground state, black) and S1 (first singlet excited state, red) electronic states. (b) Illustration of the adiabatic potential energy surface employed for the four-point calculations, where Q denotes the nuclear coordinate. Total reorganization energy as a function of normal-mode frequencies calculated for Q820 (c) and Q920 (d). (e) Breakdown of the overall reorganization energies for both compounds, detailing the specific structural contributions derived from alterations in bond lengths, bond angles, and dihedral torsions. (f) Temperature evolution profiles of various NPs upon exposure to the 808 nm laser (1.0 W/cm2) for 10 min. (g) Photothermal heating-cooling curve of AuNR@Q820, AuNR@Q920 and AuNR@ICG NPs with the same concentration of 1 nM and (h) the corresponding linear relationship between -Ln(θ) and time (s) under the 808 nm laser irradiation (1.0 W/cm2) for 10 min. It should be noted that the PCE of Q920 is lower than that of Q820, possibly because its absorption is farther away from the laser wavelength used. (i) Schematic depicting the synergistic mechanism responsible for the enhanced photothermal performance of the hybrid plasmonic NPs. (j) Heating/cooling curves recorded over five consecutive 808 nm laser-induced cycles for AuNR@Q820 and AuNR@ICG NPs at a power density of 1.0 W/cm2.

Fig.4 (a) Schematic illustration detailing the fabrication process of AuNR@Q820@AMD NPs. (b) Experimental setup for SERRS-guided surgery. (c) Representative photograph of a 4T1 tumor-bearing mouse (left), the corresponding tumor (middle), and the respective in vivo Raman image (right). (d) H&E staining image of the complete surgical bed. (e) Sequential tumor resection process under Raman imaging guidance and (f) corresponding representative Raman spectra collected from the cutting bed during different surgical stages. The Raman images are derived from the peak intensity of 381 cm−1. (g) (i) Photographs and (ii) H&E staining of the representative excised tumor tissues during sequential surgery.

在体内手术导航实验中，研究团队利用靶向分子修饰探针，使其精准富集于高表达CXCR4受体的肿瘤部位。拉曼成像清晰勾勒出原位肿瘤与正常组织的边界，以 22.6 的高信噪比实现了微小肿瘤切除导航。在术后辅助治疗阶段，局部近红外激光照射触发高效光热消融，原位清除了残留的微小病灶。最后，光热治疗引发的肿瘤抗原释放与抗PD-1抗体产生协同免疫效应，系统性激活了细胞免疫响应，有效遏制了远端肺部转移灶的生长。

Fig.5 (a) Schematic illustration of the in vivo antitumor treatment regimens. (b) (i) Infrared thermographic images and (ii) the corresponding photothermal heating profiles of the tumor sites following the intravenous administration of AuNR@Q820@AMD NPs. (c) Primary tumor recurrence rates across the respective treatment cohorts. (d) Kaplan-Meier survival analyses of 4T1 tumor-bearing mice subjected to the designated therapeutic interventions. (e) Time-dependent changes in mouse body weight. (f) Biodistribution in major organs and tumors of AuNR@Q820@AMD NPs following intravenous administration to 4T1 tumor-bearing mice. (g) Optical photograph of 4T1 tumor mice receiving various treatments. (h) Individual tumor growth curves of the primary tumor after various treatments. (i) Individual growth curves of the distant tumors following the respective therapeutic interventions. Error bars, mean ± s.e.m. (n = 6).

Fig.6 Relative expression levels of (a) Cd8a, (b) Gzmb, (c) Ifng, and (d) Foxp3 in distant metastatic tumors, measured by RT-qPCR. (e) Multiplex immunofluorescence evaluation of splenic tissues excised from 4T1 tumor-bearing mice following the respective therapeutic interventions, illustrating CD8+ T cells (red), CD4+ T cells (green), and cellular nuclei (blue, DAPI). Systemic concentrations of (f) TNF-α and (g) IFN-γ quantified from the sera of 4T1 tumor-bearing mice across the various treatment cohorts. (h) Representative photographs of lung tissue (top) and corresponding H&E-stained sections (bottom). The white circles indicate the visible metastatic foci. Error bars, mean ± s.e.m. (n = 6). Statistical significance was defined as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s. denotes no significant difference, p > 0.05.

总结及展望#

该研究成功构建了一种无荧光近红外电子共振拉曼纳米探针，完美解决了传统拉曼探针荧光背景干扰大、光稳定性差与光热效率低的难题。探针集成了超灵敏光学导航与高效光热-免疫联合治疗功能，不仅为高精度肿瘤外科手术提供了全新的导航工具，也为防止肿瘤术后复发与远端转移开辟了可行的诊疗路径。未来的工作将重点推动该探针材料的生物安全性评价，并探索其在大动物模型及临床转化中的可行性。