【Angew.Chem.】港中深唐本忠院士课题组|基于近红外二区荧光半导体聚合物探针实现子宫内膜异位症的高精度手术导航与49天长期监测#

文章标题：Precise Surgical Navigation of Endometriosis via NIR‐II Fluorescence Imaging With a Semiconducting Polymeric Probe 文章作者：Zhihui Huang, Chao Li, Jinju Lin, Yucheng Wang, Pengfei Zhang, Dan Ding, Huilin Xie, Jianquan Zhang, Ben Zhong Tang 文章链接：https://doi.org/10.1002/anie.2407891

一、研究背景与临床痛点：肉眼难辨的隐匿病灶#

子宫内膜异位症（Endometriosis，简称 EMs）是一种影响全球约 10% 至 15% 育龄期女性的慢性炎症性疾病，其典型特征是本该生长在子宫腔内的内膜组织“跑”到了盆腔腹膜、卵巢等其他部位异常生长。这种异位生长不仅会导致慢性盆腔痛、痛经与不孕，还与卵巢癌等全身性并发症风险相关。

目前临床治疗高度依赖腹腔镜手术切除病灶，但手术面临着极大的现实困境：异位病灶通常体积微小、呈多发弥漫分布，且在常规白光视野下与正常盆腔组织边界模糊。传统影像学手段（如超声、CT）对早期或深部浸润病灶的分辨率有限，导致手术往往难以彻底切除病灶，术后复发率居高不下。

为了让手术医生拥有“透视眼”，波长在 1000 至 1700 纳米的近红外二区（NIR-II）荧光成像技术应运而生。相比可见光与近红外一区，近红外二区光在生物组织中具有穿透深、组织散射低以及几乎无背景自发荧光干扰的显著优势。然而，现有用于近红外二区的发光材料——半导体聚合物（SPs），由于其平面骨架极易在纳米颗粒聚集成团时发生紧密堆积，引发“聚集导致发光猝灭”（ACQ）效应，导致探针发光亮度骤降、光稳定性差，无法满足复杂腹腔手术导航与长期术后监测的苛刻需求。

Scheme.1 Schematic illustration of the fabrication of P-BPTI nanoparticles (NPs) and their application in NIR-II fluorescence–guided surgical resection of endometriosis.#

二、分子设计与机理突破：打破聚集猝灭的分子扭曲策略#

针对传统探针聚集后发光变暗的关键瓶颈，研究团队从有机光伏领域常用的非富勒烯受体分子中汲取灵感，设计并合成了新型半导体聚合物荧光探针 P-BPTI，并制备成水相分散的纳米颗粒（P-BPTI NPs）。

研究的核心创新在于“通过聚合引入几何扭曲，破坏分子的紧密堆叠”。对比常规小分子单体 M-BPTI 容易形成紧密平面的芳香环 $\pi-\pi$ 堆积，P-BPTI 聚合物在链生长过程中由于聚合位点具有特定夹角，形成了空间受阻的扭曲螺旋构象。

理论模拟证实松散构象：分子动力学模拟显示，单体在水溶液中聚集后溶剂可及表面积（SASA）骤降至 26%，表现出紧密聚集；而聚合物体系的 SASA 稳定在 45%，其分子间疏水相互作用数（77.54 对比单体的 156.26）与 $\pi-\pi$ 堆积数（24.69 对比单体的 33.21）均显著降低。
晶体与固态表征印证无定形优势：掠入射广角 X 射线衍射（GIWAXD）表明，聚合物的 $\pi-\pi$ 堆积距离增大至 3.65 Å，相干长度缩短至 15.8 Å；差示扫描量热法（DSC）显示单体具有高达 187.2 °C 的尖锐熔融峰，而聚合物呈现完全的无定形特征。这种结构特性从根源上减弱了非辐射能量损耗，成功抑制了聚集猝灭效应。

Fig.1 Chemical structures, molecular dynamics (MD) simulation and solid-state characterizations of M-BPTI and P-BPTI. (a) Chemical structures of M-BPTI and P-BPTI and schematic illustration of the transition from the ordered packing of M-BPTI to the disordered packing of P-BPTI. (b) Optimized molecular geometry of M-BPTI. (c) Optimized molecular geometry of P-BPTI (decamer with 10 repeating units). (d) Assembly process of 30 M-BPTI molecules in aqueous solution and snapshots of the M-BPTI system. (e) Assembly process of 3 P-BPTI decamers in aqueous solution and snapshots of the P-BPTI system. (f) Time-dependent solvent-accessible surface area (SASA) of M-BPTI and P-BPTI systems. (g) Time-dependent number of hydrophobic interactions in M-BPTI and P-BPTI systems. (h) Time-dependent number of π–π stacking in M-BPTI and P-BPTI systems. GIWAXD patterns of (i) M-BPTI and (j) P-BPTI showing distinct molecular packing parameters, dπ–π and CLπ–π, from Scherrer equation. (k) DSC thermograms of M-BPTI and P-BPTI.#

三、体外性能与血管成像：高亮度、高稳定性与深层组织穿透#

通过高分子脂质材料 DSPE-PEG2000 对荧光分子进行包裹，所制得的 P-BPTI 纳米颗粒流体动力学直径约为 89 nm，在 pH 2 至 12 的极端环境下及常温水溶液中放置 21 天 均保持均一稳定。

优异的光物理性能：P-BPTI NPs 的摩尔消光系数高达 $1.79 \times 10^5\text{ M}^{-1}\text{ cm}^{-1}$ ，荧光量子产率达到 0.58%，是单体纳米颗粒（0.18%）的 3 倍以上，更大幅超越了临床红外染料基准 IR-26（0.05%）。
光稳定性与组织穿透深度：在 808 nm 激光连续照射下，临床常用染料吲哚菁绿（ICG）发生快速光漂白，而 P-BPTI NPs 经历长时间照射后荧光强度几乎没有衰减；在 1% 脂肪乳组织模拟液与鸡胸肉组织实验中，P-BPTI NPs 在 3 mm 穿透深度 下依然能够清晰分辨毛细管轮廓，有效成像深度可达 4 mm。
活体全身与后肢血管高分辨率造影：在小鼠尾静脉注射造影实验中，利用 1300 nm 长通滤波片，P-BPTI NPs 获得的局部血管信噪比（SBR）达到 1.79，显著优于单体组（1.36）和 ICG 组（1.23）。快速傅里叶变换（FFT）分析证实其能完好保留高频微血管边界信息，成功在活体后肢中以极小的半高全宽（动脉 0.225、静脉 0.194）实现动静脉血管的精准空间区分。

Fig.2 Characterizations of NIR-II fluorophores and their NPs. (a) Illustration of NP preparation. UV–vis absorption and photoluminescence spectra of (b) M-BPTI NPs and (c) P-BPTI NPs dispersed in deionized water. (d) Plots of the integrated PL intensity of M-BPTI NPs and P-BPTI NPs in water and IR-26 in 1,2-dichloroethane (DCE), (Reference QY = 0.05%). (e and f) DLS analysis of M-BPTI NPs and P-BPTI NPs, insert: TEM image of M-BPTI NPs and P-BPTI NPs, Scale bar 50 nm. (g) The particle size changes of M-BPTI NPs and P-BPTI NPs recorded over 21 days. (h) NIR-II images of M-BPTI NPs, P-BPTI NPs and ICG in mouse serum with various concentrations (808 nm laser excitation, LP 1000 nm, 50 mW cm−2). (i) Quantitative analysis of NIR-II fluorescence intensity for M-BPTI NPs, P-BPTI NPs and ICG at different concentrations in (h). (j) NIR-II image of M-BPTI NPs, P-BPTI NPs and ICG in mouse serum after different time periods upon 808 nm laser irradiation (808 nm laser excitation, LP 1000 nm, 80 mW cm−2). (k) Quantitative analysis of NIR-II fluorescence intensity for M-BPTI NPs, P-BPTI NPs and ICG at different 808 nm laser irradiation time in (j). (l) NIR-II images of M-BPTI NPs, P-BPTI NPs and ICG in mouse serum with different LP filters (from LP 900 nm to LP 1300 nm, 808 nm laser excitation: 80 mW cm−2). (m) Quantitative analysis of NIR-II fluorescence intensity for M-BPTI NPs, P-BPTI NPs and ICG with different LP filters in (l). (n) NIR-II imaging of glass capillary tubes perfusing M-BPTI NPs, P-BPTI NPs and ICG, respectively, immersed in various depths of 1% Intralipid solution (808 nm laser excitation, LP 1000 nm, 80 mW cm−2). (o) Correlation of FWHMs versus depths of Intralipid solution in (n).#

四、手术导航与体内代谢：精准切除与低网状内皮系统截留#

在活体病灶靶向机制上，P-BPTI NPs 表现出近中性的表面电位（-5.8 mV），相比于单体 M-BPTI NPs 携带的强负电位（-25.3 mV），能有效规避单核巨噬细胞系统（MPS）在肝脾中的过度吞噬与清除。

长循环与高富集：药代动力学分析表明，P-BPTI NPs 的体内血液循环半衰期长达 24.1 小时（单体仅为 2.9 小时）。基于病灶部位的高通透性和滞留效应，纳米颗粒在注射后 24 至 36 小时 于腹腔异位病灶处达到荧光富集峰值。
术中精准导航切除：在多发性腹腔子宫内膜异位症小鼠模型中，白光视野下病灶完全隐匿无法辨认，而在近红外二区成像指导下，微小且位置隐蔽的异位结节呈现高对比度明亮荧光。手术医生依据荧光边界实现了多发病灶的完整剥离切除。术后即刻荧光复查显示手术创面荧光完全消失，离体组织病理切片荧光与术前标记完全吻合，验证了切除的彻底性。

Fig.3 In vivo NIR-II fluorescence imaging of whole-body blood vessel. (a) NIR-II fluorescence whole-body vascular imaging of a mouse in different spectral regions (LP 900 nm, LP 1100 nm, and LP 1300 nm) after tail vein injection of P-BPTI NPs. The areas marked with yellow squares correspond to the original images used for FFT results. Scale bar: 10 mm. (808 nm laser excitation, 300 mW cm−2). (b) Cross-sectional fluorescence intensity profiles along the red lines of the blood vessel in panel a, e, and i, comparing mice injected with different materials (P-BPTI NPs, M-BPTI NPs, and ICG) under the same LP 1300 nm filter. (c) FFT results of the NIR-II fluorescence vascular images in panel a. Spatial frequency increases gradually from the center of the map outward, with the color bar indicating intensity. (d) The spatial frequency distribution of panel a, e, and i under the LP 1300 nm filter. (e) NIR-II fluorescence whole-body vascular imaging of a mouse in different spectral regions (LP 900 nm, LP 1100 nm, and LP 1300 nm) after tail vein injection of M-BPTI NPs. Scale bar: 10 mm. (808 nm laser excitation, 300 mW cm−2). (f) Comparing mice injected with different materials (P-BPTI NPs, M-BPTI NPs, and ICG) under the LP 1100 nm filter. (g) FFT results of the NIR-II fluorescence vascular images in panel e. (h) The spatial frequency distribution of panel a, e, and i under the LP 1100 nm filter. (i) NIR-II fluorescence whole-body vascular imaging of a mouse in different spectral regions (LP 900 nm, LP 1100 nm, and LP 1300 nm) after tail vein injection of ICG. Scale bar: 10 mm. (808 nm laser excitation, 300 mW cm−2). (j) Comparing mice injected with different materials (P-BPTI NPs, M-BPTI NPs, and ICG) under the LP 900 nm filter. (k) FFT results of the NIR-II fluorescence vascular images in panel i. (l) FFT results of the NIR-II fluorescence vascular images in panel a, e, and i under the LP 900 nm filter.#

Fig.4 NIR-II fluorescence angiograms of the femoral arteries and veins in mice obtained using P-BPTI NPs, M-BPTI NPs and ICG, respectively. NIR-II angiograms (left) and cross-sectional intensity curves (right) along the yellow dashed and red arrows on the main femoral artery and vein. (a–c) with P-BPTI NPs, M-BPTI NPs and ICG injection. Red and blue dashed curves represent Gaussian fits to the profiles. The mice were imaged under LP 900 nm filters. Excitation source: 808 nm laser, 150 mW cm−2. (d–f) Comparison of NIR-II fluorescence imaging quality between P-BPTI NPs, M-BPTI NPs and ICG and their corresponding cross-sectional intensity profile along the red dashed arrows. The mice were imaged under LP 1100 nm filter. Excitation source: 808 nm laser, 150 mW cm−2. (g–i) Comparison of NIR-II fluorescent imaging quality between P-BPTI NPs, M-BPTI NPs and ICG and their corresponding cross-sectional intensity profile along the red dashed arrows. The mice were imaged under LP 1300 nm filter. Excitation source: 808 nm laser, 150 mW cm−2. Scale bar: 5 mm.#

五、长期术后监测与病理安全性：长达49天的无复发追踪#

为了评估彻底切除的治疗效果并防范术后隐匿病灶复发，研究团队设计了周期性给药的术后长期追踪方案：

长达 49 天的周期性术后监测：术后小鼠以 7 天为间隔 周期性静脉注射 P-BPTI NPs，进行近红外二区荧光追踪扫描。在整整 49 天 的监测周期内，腹腔内均未出现异常荧光信号增强；终点剖腹检查证实腹腔内无任何新生结节、组织黏连或复发迹象。
组织病理与分子标志物逆转：术后第 21 天的组织学检测显示，未手术组小鼠病灶呈现典型的水泡样增生、异位腺体浸润与异常新生血管网；而在荧光手术组中，孕激素受体（PGR）、细胞增殖标志物 Ki67、血管新生酪氨酸激酶 EphB4 以及促炎细胞因子 TNF-α 的表达水平均大幅下降，证实了病理炎症与增殖活性的全面消退。
生物安全性评价：体外细胞毒性与溶血实验显示探针无生物毒性；小鼠注射 21 天后的血常规指标、肝肾功能生化指标（ALT、AST、总胆红素、肌酐等）以及主要器官（心、肝、脾、肺、肾）切片均未见病理损伤，展现了优秀的转化安全性。

Fig.5 Surgical resection of endometriosis in mice guided by the NIR-II imaging system. (a) Schematic illustration of the establishment of the endometriosis mouse model. (b) Bright-field images of endometriosis mouse and a locally magnified view of its abdominal cavity, with tail-vein injections of P-BPTI NPs, M-BPTI NPs, and ICG, respectively. (c) NIR-II region images of the endometriosis-bearing mice were obtained 36 h after the tail-vein injection of P-BPTI NPs (150 µL, 1 mg mL−1), M-BPTI NPs (150 µL, 1 mg mL−1), or ICG (150 µL, 0.25 mg mL−1), respectively, including a locally magnified view of the lesion site (808 nm laser excitation, 1300 nm long-pass filter, 200 mW cm−2 for P-BPTI NPs and M-BPTI NPs) and (808 nm laser excitation, 1000 nm long-pass filter, 100 mW cm−2 for ICG). (d) Anatomical bright field of mice with endometriosis and magnified diagrams of their lesion sites. (e) NIR-II imaging of endometriosis mice following abdominal dissection and its locally magnified image. (f) NIR-II imaging of the abdominal cavity after surgical resection in endometriosis mice. Original image Scale bar: 5 mm. Enlarged image Scale bar: 5 mm. (g) Bright field and NIR-II imaging of resected grafts. Scale bar: 2 mm.#

Fig.6 Longitudinal post-operative NIR-II fluorescence imaging performed every 7 days following repeated intravenous administration of P-BPTI NPs. (a) In vivo real-time NIR-II FL images of the endometriosis at different postinjection times of P-BPTI NPs (150 µL, 1 mg mL−1, 808 nm laser excitation, LP 1300 nm, 180 mW cm−2, Scale bar: 5 mm). (b) Corresponding quantitative analysis of fluorescence intensity at different time points (n = 3). (c) Bright-field and NIR-II fluorescence images of the endometriotic lesion site before and after NIR-II-guided surgical resection, including the anatomical suturing process (808 nm laser excitation, LP 1300 nm, 180 mW cm−2, Scale bar: 5 mm for mice, Scale bar: 2 mm for resected grafts). (d) To verify the thoroughness of the surgical excision and monitor for potential de novo lesions, P-BPTI NPs were intravenously administered every 7 days post-resection for long-term NIR-II fluorescence surveillance (150 µL, 1 mg mL−1, 808 nm laser excitation, LP 1300 nm, 180 mW cm−2, Scale bar: 5 mm). (e) Anatomical verification 49 days post-surgery. (Left) Macroscopic photograph of the whole endometriosis mouse after laparotomy at the 49-day endpoint. The dashed yellow rectangle indicates the surgical area of focus. (Right) Magnified view of the abdominal cavity. Original image Scale bar: 5 mm. Enlarged image Scale bar: 2 mm.#

Fig.7 In vivo evaluation of post-operative outcomes following P-BPTI NPs-assisted NIR-II fluorescence-guided surgery for endometriosis. (a) Hematoxylin-eosin (H&E) staining of peritoneal EMs lesion sites after 21 days of NIR-II fluorescence image-guided surgery by P-BPTI NPs. (b) Quantitative analysis of endometriotic lesion graft area in panel a. (c) Immunostaining of PGR in endometriosis lesions of mice that underwent NIR-II fluorescence image-guided surgery mediated by P-BPTI NPs and did not undergo surgery. (d) The IHC scores for PGR expression are presented in panel c. (e) Immunostaining of Ki67 in endometriosis lesions of mice that underwent NIR-II fluorescence image-guided surgery mediated by P-BPTI NPs and did not undergo surgery. (f) The IHC scores for Ki67 expression are shown in panel e. Original image Scale bar: 50 µm. Enlarged image Scale bar: 50 µm. (g) Immunofluorescence images of EphB4 in endometriosis lesions of mice that underwent NIR-II fluorescence image-guided surgery mediated by P-BPTI NPs and did not undergo surgery. Nuclei were stained with DAPI (blue), EphB4 (red). (h) Quantitative analysis of EphB4 expression in panel g. (i) Immunofluorescence images of TNF-α (red) and nuclei were stained with DAPI (blue) in endometriosis lesions of mice that underwent NIR-II fluorescence image-guided surgery mediated by P-BPTI NPs and did not undergo surgery. (j) Quantitative analysis of TNF-α expression in panel i. Original image Scale bar: 25 µm. Enlarged image Scale bar: 10 µm. Data shown are mean ± SD (n = 5) (p < 0.05, p < 0.01, p < 0.001 and ****p < 0.0001).#

六、研究总结与未来展望#

该研究成功构建了一种兼具高光稳定性、高量子产率与长血液循环寿命的近红外二区半导体聚合物纳米探针 P-BPTI。通过精巧的高分子链构象扭曲调控，打破了荧光染料聚集猝灭的固有局限，不仅在动物模型中完成了肉眼不可见的微小子宫内膜异位症病灶的高对比度实时手术导航切除，还开创性地建立了长达 49 天 的术后周期性荧光监测体系。

尽管该探针在动物实验中展现了卓越的临床前应用潜力，但由于目前探针主要依赖被动增强渗透滞留效应进行富集，未来研究若能进一步引入针对子宫内膜异位特异性受体的主动靶向配体，将有望进一步缩短成像窗口期、提高信噪比，加速向临床腹腔镜荧光手术导航系统的转化落地。

【Angew.Chem.】港中深唐本忠院士课题组|基于近红外二区荧光半导体聚合物探针实现子宫内膜异位症的高精度手术导航与49天长期监测