荧光信号释放受分子逻辑门机制控制的新型靶向智能探针及荧光标记成像

1. Molecular Design of the Smart Bioprobes with High Selectivity and Sensitivity. .To obtain novel tumor targeting bioprobes for labelling and fluorescence optical imaging with high selectivity and sensitivity, this project will focus our efforts on the molecular design of smart fluorescence bioprobes by taking advantage of the cellular pH differences between health tissue and tumor. Our strategy is taking the following three important factors into account for the molecular design of the bioprobes to endue them with smart natures. Namely, the specific labelling of tumors is achieved by modification of the bioprobes by arginine-glycine-aspartic acid (RGD) (or octreotide) peptide according to the antibody- antigens binding mould; the signal silence fluorescence reporters are activable by the reactive high oxygen species (rHOS) in cells; and the signal release of the activated bioprobes is controlled by cellular pH value. ..2. Specific Labeling, the Activation of the Reporter and the Optical Signal Release of the Activated Reporter. .Firstly, the bioprobes are accumulated selectively on the membrane of tumor cells by using an integrin (or somatostatin) targeted strategy by the moiety of RGD (or octreotide) peptide within the bioprobes. After the bioprobes being endocytosed by cells, the signal silence fluorescence reporter is activated by rHOS in cells. The release of the optical signal of the activated reporter is controlled by cellular pH value. The molecular events about the bioprobes in cells including the activation of the reporter by rHOS and the release of the reporter's optical signal being controlled by cellular pH are similar to a mould of dual input AND logic gates, which shall lead to high specific smart bioprobes for tumor labelling and fluorescence optical imaging by using such a dual input logic gates paradigm in cells....3. The Goals of the Project..The aims of this project are as follows: 1) obtaining 3-5 smart bioprobes which can differentiate tumors from health tissue with high selectivity and sensitivity; 2) obtaining the most important scientific data, such as the optimized detection time after labelling of tumors and the optimized labelling dosage of the bioprobes, etc. by a detailed investigation of the labelling and fluorescence optical imaging properties of the novel smart bioprobes toward the models of implanted tumors; and 3) establishing the detection models of the specific labelling and fluorescence optical imaging of tumors with high selectivity and sensitivity.

围绕开发量子效率高、标记特异性强，光学标记后可从健康组织中高灵敏、高选择性的区分出肿瘤组织的荧光探针。本项目拟利用健康组织和肿瘤组织的生理pH值差异，从探针①满足靶向选择性标记功能；②荧光信号报告基团由细胞活性物质激活和③光学信号释放受细胞pH值控制这三个基本要素切入，在探针的设计方面充分利用抗体-受体结合原理对探针进行靶向修饰来实现对肿瘤细胞光学标记的一级选择性；同时，利用细胞活性物激活探针的光学信号报告基团和细胞pH调控光学信号释放这两种细胞内分子激活事件来控制光学信号到达二级选择性的目的，设计出荧光探针在细胞内激活和光学信号释放受类似AND逻辑门输入机制控制的新型靶向智能荧光探针。通过这类探针对肿瘤模型的光学标记成像研究，获得肿瘤-本底光学信号对比度高和成像清晰度高的荧光标记检测模型，发展从健康组织中区分出肿瘤组织的新型智能荧光探针标记技术,并建立对肿瘤进行荧光标记成像检测新模型。

围绕开发量子效率高、标记特异性强，光学标记后可从健康组织中高灵敏、高选择性的区分出肿瘤组织的荧光探针。在国家基金的支持资助下，我们主要从满足荧光成像要求的新型近红外荧光染料设计合成，与癌症相关的物种检测探针设计、探针的靶向定位修饰、探针的光谱检测和荧光标记成像检测等多方位、多角度开展了系统、全面的探索工作。在本项目的研究中，取得的主要相关进展包括如下内容：1）设计并合成了多类发光处于近红外区域（650 nm-860 nm）的新型荧光染料，与传统菁类近红外荧光染料和最近报道的Si-罗丹明系列近红外荧光染料相比，这些染料在Stokes位移和光稳定性上都有很大突破，这些新染料的Stokes位移超过100 nm，均远远超过上述两类染料。同时，在细胞中采用635 nm的激光器连续照射30分钟不产生光信号衰减，抗漂泊能力大大超过传统菁类近红外荧光染料，这类新型染料将逐渐形成我们在本领域独特的特色；2）获得了50多个对重金属阳离子、阴离子、高氧化活性物种、生物小分子和生物大分子具有高灵敏度、高选择性检测功能的荧光探针材料，这些荧光探针材料在环境监控、实际样品分析中具有重要的价值；并从这些探针中获得了20多个可在癌细胞中实现荧光成像检测的荧光探针材料。3）突破了自补偿阴离子荧光探针设计策略，发展了新型诱导聚集设计策略；4）执行期新申报发明专利10项，获授权发明专利3项；5）执行期已发表标注基金号的SCI论文19篇，其中SCI影响因子大于3.0的论文13篇; SCI影响因子小于3.0的论文6篇，发表论文的SCI论文总影响因子超过65，部分发表的研究工作被Nature Index 2014, China，Tianjin，作为天津理工大学的代表工作进行了评价 (Nature, 2014, 516, S74)；6）项目执行期，本方向搭建了完善的学科创新平台，为本方向的后续发展奠定了坚实的基础。