纳米荧光开关的构建及其乳腺癌早期诊断和分期系统建立研究

Early detection, diagnosis, and a reasonable treatment option for different stages are the key to improve the therapeutic efficacy of breast cancer. However, there are some flaws in the current methods for early diagnosis of breast cancer and defining its TNM stages. In this project, a "nanofluorescent switch" with a resting state in vitro and fluorescence recovery in breast cancer tissues and cells in vivo is designed and will be constructed. In this system, gold nanoparticles (GNPs) act as a structural core and fluorescence energy transfer acceptor; trastuzumab, specifically targeting the human epidermal growth factor receptor, plays the role of a targeting ligand; the expression levels of MMPs in breast cancer cells (or tissues) is chosen as the staging flag. GNPs and organic fluorescent molecules (FITC) is connected by a matrix metalloproteinase (MMP)-sensitive peptide. With the help of MMPs’ enzyme reaction, FITC fluorescence signal is triggered, which is caused by the dissociation of FITC from the surface of GNPs. The quantitative relationship between the intensity of fluorescence recovery and matrix metalloproteinase expression levels of the "nanofluorescent switch" is planned to be established. Through in-depth and systematic studies on the mechanism and quantitative analysis method of the switch system, it allows the establishment of a novel method for early diagnosis and staging of breast cancer, and provides theoretical and practical reference for the early diagnosis of other tumors.

乳腺癌的早期发现和诊断，针对不同的分期制定合理的治疗方案是提高其疗效的关键。然而，目前乳腺癌临床早期诊断方法和TNM分期的界定都存在一定缺陷。本项目设计和构建一种在体外处于荧光静息状态，在体内乳腺癌组织和细胞中荧光恢复的“纳米荧光开关”系统。该系统以金纳米粒子作为结构核心和荧光能量转移受体；特异性靶向人表皮生长因子受体的曲妥珠单抗作为靶向配体；乳腺癌细胞（或组织）中金属基质蛋白酶表达水平为肿瘤分期标志。金纳米粒子与有机荧光分子（FITC）通过对金属基质蛋白酶敏感的小肽相连。通过金属基质蛋白酶酶解反应，引发FITC从金纳米粒子表面解离所产生的荧光信号，建立“纳米荧光开关”荧光恢复强度与金属基质蛋白酶表达水平的定量关系。通过对该开关系统深入的机制和定量分析方法研究，以期建立一种新的乳腺癌早期诊断方法和分期系统，并为其他肿瘤的早期诊断提供理论和实践参考。

本项目构建了一种在体外处于荧光静息状态，在体内癌症组织和癌症细胞中荧光恢复的“纳米荧光开关”系统。该系统以金纳米粒（Gold nanoparticles，GNPs）作为结构核心和荧光能量转移受体；癌症组织或细胞中高表达的基质金属蛋白酶（MMPs）为肿瘤标志物；金纳米粒与有机荧光分子（FITC）通过对MMPs敏感的小肽相连。开关的表面修饰材料为巯基聚乙二醇单甲醚（SH-mPEG2000，PEG）或具有“反调理作用”的牛血清白蛋白（Bovine serum albumin，BSA）。借助在肿瘤内MMPs对小肽的识别促进荧光复燃，从而对肿瘤进行检测。.通过研究，我们（1）构建了两种不同表面修饰材料的纳米荧光开关：GNP-p-FITC/mPEG和GNP-p-FITC/BSA。（2）建立了纳米荧光开关的荧光恢复强度与MMPs表达水平之间的定量关系。（3）阐明了两种开关的荧光复燃的机制： GNP-p-FITC/mPEG（MMPs水解）；GNP-p-FITC/BSA（MMPs水解为主，GSH置换为辅）。（4）揭示了两种开关的细胞摄取机制均为网格蛋白介导的内吞，但PEG修饰显著降低了开关的肿瘤细胞摄取率，而BSA修饰的开关肿瘤细胞摄取率不受影响。（5）发现两种开关均可以抵达、蓄积、并深入肿瘤组织，在肿瘤内部荧光复燃，实现在体内对肿瘤组织和肿瘤细胞的检测。（6）BSA修饰的开关细胞毒性低，溶血性低，生物相容性更好，组织穿透力强，并具有更高的荧光复燃效率（复燃机理多样化，肿瘤细胞摄取率高），是更优的纳米荧光开关系统。本项目的研究推进了纳米结合物探针作为一种肿瘤早期诊断材料的基础研究，并且对采用反调理蛋白质作为纳米器件的表面修饰材料，提高纳米器件的体内生物效能进行了初步的探索。.