基于外泌体的EGFR靶向NIR-Ⅱ荧光纳米探针构建及乳腺癌活体分子成像

Compared to near-infrared I fluorescence molecular imaging, near-infrared Ⅱ (NIR-Ⅱ) fluorescence molecular imaging has advantages including better tissue penetration, less autofluorescence interference, higher spatial resolution at deeper tissue penetration depths, thus possessing bright application prospect in early diagnosis and surgery navigation of breast cancer. However, further clinical translation of NIR-Ⅱ fluorescence molecular imaging faces with two big challenges: ① Quantum yields of most NIR-Ⅱ fluorescence probes are very low, which leads to weak fluorescence intensity; ② Most NIR-Ⅱ fluorescence nano-probes consist of inorganic materials, which results in weak biocompatibility. Herein, this project is to present a NIR-Ⅱ fluorescence molecular probe (Exo-CH1055-GE11) that with high biocompatibility, high fluorescence intensity, and high epidermal growth factor receptor (EGFR) specificity by using natural exosomes to load high dose of new renal-excreted NIR-Ⅱ dyes CH1055-PEG in cavity and large amounts of EGFR targeting molecules GE11 on membrane surface. Then, Exo-CH1055-GE11 will be used to evaluate breast cancer-bearing nude mouse models, aiming to establish a new breast cancer NIR-Ⅱ fluorescence molecular imaging strategy that with great clinical translation potentiality, high safety, and high accuracy. This project may provide a new approach for promoting the technology development of early diagnosis and surgery navigation of breast cancer.

相较于近红外I区，近红外Ⅱ区（NIR-Ⅱ）荧光分子成像组织穿透力更强、受组织自发荧光干扰更小、深层组织成像空间分辨率更高，在乳腺癌早期诊断及术中导航中极具应用潜力。但其进一步临床转化面临两大挑战：①大多数NIR-Ⅱ荧光探针量子产率低，荧光强度弱；②大多数NIR-Ⅱ荧光纳米探针由无机材料构成，生物相容性较差。本项目以外泌体为纳米平台，内腔荷载高剂量新型肾排泄小分子NIR-Ⅱ荧光染料CH1055-PEG，膜表面修饰大量表皮生长因子受体（EGFR）靶向分子GE11，构建具备高生物相容性、高荧光强度、高EGFR靶向性的NIR-Ⅱ荧光纳米探针（Exo-CH1055-GE11），并通过Exo-CH1055-GE11对乳腺癌荷瘤裸鼠模型进行评估，以期建立临床转化潜力大、安全、精确的新型乳腺癌NIR-Ⅱ荧光分子成像方法。本项目的展开可为促进乳腺癌早期诊断和术中导航技术的发展提供新的方案。

近红外II区（NIR-II）荧光成像组织穿透力强且受生物组织自发荧光干扰小，在肿瘤成像与术中导航中具有广阔的应用前景。但大多数水溶性NIR-II荧光有机分子和纳米材料因量子产率低荧光亮度低或潜在的长期生物毒性问题制约了其临床转化应用。本项目以外泌体尺寸生物质纳米载体小细胞外囊泡（small extracellular vesicles, sEV）为平台，内腔荷载一定量的水溶性NIR-II荧光分子CH1055-PEG，膜表面修饰EGFR靶向多肽GE11，构建具备良好生物相容性、高荧光亮度的EGFR靶向NIR-II荧光纳米探针sEV-CH1055-GE11，并评估其对EGFR高表达肿瘤的富集效果。结果显示，sEV与5倍质量的CH1055-PEG混合，通过电穿孔可得具有较强NIR-II荧光亮度的sEV-CH1055，且电穿孔过程对sEV形貌完整性及CH1055-PEG的吸收光谱均无显著影响。其后，通过亲脂作用将DSPE-PEG2k-GE11直接修饰于sEV-CH1055膜表面，并通过对比修饰前后sEV的ζ电势及粒径变化验证了GE11成功修饰。项目所构建的sEV-CH1055-GE11具有较强的EGFR高表达肿瘤细胞结合能力，细胞荧光实验结果显示与其孵育的肿瘤细胞NIR-II荧光强度约是对照组肿瘤细胞的两倍以上。sEV-CH1055-GE11静脉注射荷瘤鼠后，活体及离体组织NIR-II荧光成像结果显示探针可于肿瘤处实现有效富集。细胞活力及免疫组化结果均显示sEV-CH1055-GE11具有良好的生物相容性。综上，sEV-CH1055-GE11是一种具有一定临床应用前景的新型EGFR高表达肿瘤NIR-II荧光成像纳米探针。