胃癌转移淋巴结微环境响应性诊疗一体化智能探针构建及术中荧光导航下精准光热治疗

It is a long-sought goal for human to achieve the accurate dissection of metastatic lymph nodes and to avoid the normal tissue injuries in surgery of gastric cancer. However, the key problem is how to differentiate benign from malignant lymph nodes accurately during surgery, and how to remove those malignant ones fault-freely and non-surgically. In this project, CuS with favorable thermal effect will be adopted as the carrier, modified with both gastric cancer cell targetd group (RGD) and matrix metalloproteinase (MMP) substrate peptide, whose bilateral ends will be crosslinked with fluorophore (Alex647) and fluorescent quenching group (BHQ-3), to construct a gastric cancer cell recognizable (through RGD) and tumor microenvironment responsive (through MMP) dual targeting intelligent fluorescent probe called CuS-647. Orthopotic nude mice modle of gastric carcinoma with lymph node metastasis will be established as the research object. The diagnostic performance of CuS-647 based tumor microenvironment responsive intelligent fluorescent probe in differitiating metastatic lymph nodes from benign ones intraoperatively will be evaluated. And the treatment effect of this intelligent theranostic probe in photothermal ablation of metastatic lymph nodes precisely under the near infrared laser irradiation will be verified. This study will provide a technical support for accurate intraoperative diagnosis and simultaneous noninvasive removal of metastatic lymph nodes of gastric cancers intraoperatively.

努力开展淋巴结精准无创清扫，最大限度地保护周围正常组织，实施胃癌最优个体化手术治疗一直是人们长期追求的目标，而能否在术中精准地判断转移淋巴结，并给予非手术的“无创切除”是实现这一目标的关键。本研究以具有良好光热效应的CuS为载体，通过胃癌细胞靶向基团RGD和基质金属蛋白酶MMP的作用底物对其进行修饰，并在MMP底物多肽两端分别交联荧光基团Alex647和荧光淬灭基团BHQ-3，构建出由RGD识别胃癌细胞和肿瘤微环境响应性MMP双靶向智能荧光探针。以裸鼠胃原位癌淋巴结转移模型为研究对象，评估CuS肿瘤微环境响应性智能荧光探针术中诊断胃癌转移淋巴结的效能；研究近红外激光照射下该智能探针选择性“光热切除”转移淋巴结的能力，为实现胃癌转移淋巴结术中诊断和无创清扫的一体化提供技术支撑。

我国胃癌高发，且具有病死率高、早期胃癌比例低、进展期病例（约占90%）为主要诊治对象的特点。淋巴转移是胃癌的重要生物学特性，是胃癌术后复发的决定性因素。对于进展期胃癌，即便在D2手术基础上进行预防性腹主动脉周围淋巴结清扫也不能降低胃癌局部淋巴结的复发率，却会带来较高的手术并发症及其相关死亡率，同时胃癌扩大根治术也损害了正常淋巴组织对肿瘤转移的屏障作用和免疫监视。因此，精准和无创地清扫转移淋巴结，实施胃癌最优个体化手术，避免清扫范围的过度或不足无疑是减轻手术创伤，防范手术风险，提高疗效和改善生活质量最为理想的治疗目标，而能否在术中精准地判断转移淋巴结，并给予非手术的“无创切除”是实现这一目标的关键。为此，本研究设计出在光热纳米材料硫化铜表面修饰基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）底物肽段，通过激活近红外荧光信号及cRGD基团靶向肿瘤细胞表面受体的方法，构建出具备荧光成像和光热治疗双重功效的纳米诊疗一体化探针。.我们通过将酶激活的双模态成像探针靶向递送到肿瘤细胞中，以促进对胃癌原发灶及其转移淋巴结的精准成像和选择性光热治疗。本研究以肿瘤细胞靶向基团cRGD和在胃癌细胞中高表达的基质金属蛋白酶MMP-2的作用底物共同修饰的Gd/CuS为载体，在MMP-2底物多肽两端分别交联荧光基团Cy5.5和荧光猝灭基团QSY21，构建出由cRGD识别胃癌细胞和肿瘤微环境响应性MMP-2双靶向智能纳米探针T-MAN。实验结果显示T-MAN在MR下显示出较高的r1弛豫率（约60.0 mM-1s-1/1T）和较强的近红外荧光激活（激活倍数约185倍），可用于裸鼠胃癌原发灶及其转移淋巴结的高空间分辨率MR和低背景荧光活体成像。T-MAN在波长808 nm激光照射下具有较高的光热转换效率（photothermal conversion efficacy，PCE，  70.1%），所产生的热量能有效杀伤肿瘤细胞。在MR/荧光双模态成像指导下，T-MAN可以对胃癌原发灶及其转移淋巴结实施精准光热治疗。