【作者】 张勇；

【导师】 高学云； 苏冬冬；

【作者基本信息】 北京工业大学 ， 化学工程与技术， 2023， 博士

【摘要】 疾病的早期诊断是提高治疗效果和治愈率的重要环节。激活型近红外（NIR）荧光探针拥有高选择性、高灵敏度和较深的成像深度,能够实现对活体内生理和病理过程的无创可视化检测,在疾病早期诊断和预后判断方面有极高的潜力。但是,由于生物体的复杂性,分子探针难以实现早期疾病标志物准确识别,并且原位病灶的位置深、体积小,很难实现疾病早期病灶的精准成像及准确定位。因此,迫切需要开发能够克服以上问题的NIR荧光探针,实现微小病灶的原位成像分析。结构定制性是小分子荧光探针的重要特性,以探针的功能性结构为主框架进行合理的修饰,可目地性地改变探针的光学性质、响应性能和代谢行为等性质,以优化探针对病灶的检测效果。本论文以分子荧光探针在活体成像中面临的问题为出发点,基于不同的设计策略来优化荧光探针,改善探针对相关疾病的检测能力,促进健康管理和精准医疗的发展。主要内容包括以下几方面:（1）肝靶向的激活型NIR荧光探针用于药物性肝损伤的可视化检测针对分子荧光探针在病灶部位积累不足和成像信号弱的问题,此部分采用探针的局部改造策略,引入靶向分子增强对病灶结构的识别,提高探针在原位病灶的滞留时间。结合对疾病标志物的激活型响应,最终实现高灵敏的原位成像分析。此部分设计了亮氨酸氨肽酶（Leucine aminopeptidase,LAP）特异性的激活型NIR荧光探针hCy-CA-LAP,用于检测药物性肝损伤（Drug-induced liver injury,DILI）的原位早期标志物LAP的水平。通过引入肝靶向配体胆酸,提高探针分子在肝脏区域靶向性和原位富集效果。动物实验发现hCy-CA-LAP可以在肝脏内有效富集,从而实现在活体水平上对DILI的准确无创的可视化检测。该研究发现具有原位靶向能力的激活型NIR荧光探针是提高DILI成像效果的关键,并实现对扑热息痛（Acetaminophen,APAP）诱导的急性肝损伤和利福平（Rifampicin,RFP）诱导的淤胆型肝损伤的原位成像和准确评估。（2）NIR荧光/光声双模态探针用于肝脏炎症的精确早诊针对单模态荧光成像难以在早期对疾病准确的定位和检测的问题,此部分采用多模态成像策略,结合不同成像模式的高灵敏度、高分辨率和深层组织成像能力,提供原位早期病灶更准确的定位以及更详细的解剖学或生物学信息。此部分综合荧光成像的高灵敏度与光声成像的深层组织高分辨率,以及两种成像的机制类似性,采用半花菁（hemi-Cyanine,hCy）染料作为双模态信号分子,设计了肝脏炎症早期ROS标志物O₂^·-特异性的激活型双模态探针hCy-Tf-CA。NIR荧光成像显示探针能够对肝脏炎症小鼠的肝脏部位的O₂^·-进行高灵敏度检测;同时,光声成像以更高的分辨率捕捉到了肝脏内部O₂^·-的分布信息。因此,hCy-Tf-CA显示了对早期肝脏炎症更加清晰的定位和准确的病情检测,同时一个分子实现两种成像模态,大大缩减了合成探针的工作量,为精准医疗提供一种非常有前景的策略。（3）亲水性的激活型NIR探针用于细胞和体内肿瘤标志物NQO1的高效检测针对小分子探针的理化性质差导致的活体成像灵敏度低、成像效果差的问题,采用光学性质优秀的成像探针平台,并基于理化性质优化策略设计成像探针,克服荧光探针面临的灵敏度低和准确性差的问题,促进探针对疾病标志物的高灵敏性检测和疾病的原位成像分析。本课题选择了斯托克斯位移大、光学亮度高的QCy7作为NIR荧光探针平台,期望设计背景干扰低、灵敏度高的成像探针。基于此,构建了肿瘤早期标志物醌氧化还原酶1（NAD（P）H:quinone oxidoreductase-1,NQO1）特异性的激活型NIR荧光探针QCy7-NQO1。针对探针在水体系内易聚集导致的成像效果差,本文在探针结构中引入两个亲水性磺酸基团,优化并设计了NQO1响应的亲水性荧光探针S-QCy7-NQO1,显著提高了其在生物体系中的响应速率和光学亮度,实现对活细胞内源性NQO1的灵敏检测和小鼠肿瘤的高对比度成像。因此,S-QCy7-NQO1不仅可以作为一种有效的工具用于以NOQ1水平升高为特征的早期癌症检测,并且提供了一种简单的思路来提高活体成像探针的成像性能。更多还原

【Abstract】 Early diagnosis of diseases is crucial for disease treatment.Due to the advantages of excellent selectivity,high sensitivity and deeper signal feedback depth,activatable near-infrared（NIR）fluorogenic probes can non-invasively monitor physiological and pathological processes in vivo,which have become an indispensable tool for the early diagnosis of disease.However,it is difficult for small molecular fluorogenic probes to accurately detect and map the early biomarkers with low concentration in the deep microlesion.Therefore,there is in urgent need of high-performance fluorogenic probes that can accurately in vivo detect tiny lesions of early diseases.Notably,thanks to the tailor-made features of small molecular fluorogenic probes,their photophysical properties,responsiveness and biodistribution can be customized systematically for specific purpose by the modification of molecular structure.In this thesis,the imaging performance of molecular probes was improved by different design strategies.We hope that the excellent probes are capable of achieving early diagnosis of the disease and further promote the development of precision medicine.（1）Enzyme-activated liver-targeted NIR fluorogenic probe for visualization of drug-induced liver injuryIn order to improve the insufficient fluorescence signal and low sensitivity caused by the insufficient accumulation of molecular probe in the lesion,active targeting strategies was adopted to promote the ability to target the region of interest.At the same time,by integrating highly targeted accumulation with activatable probe platform,diseased-related biomarker can be detected and mapped with high sensitivity,thereby finally achieving high-sensitive diagnosis of disease in situ.In this section,a LAP-activated hemi-cyanine fluorogenic probe hCy-CA-LAP was designed for the detection of LAP and fluorescence imaging of drug-induced liver injury（DILI）.The hCy-CA-LAP probe was modified with a high-affinity cholic acid to improve the liver-targeting capacity.Then the liver-targeting hCy-CA-LAP was used for in situ imaging of APAP-induced DILI.Compared with the non-targeting probe hCy-LAP,hCy-CA-LAP is able to effectively accumulate in the liver,thereby realizing precise imaging and evaluation of DILI in vivo.This study demonstrated that enzyme-activated fluorescent probes with in situ targeting are the key to improving the imaging performance,then hCy-CA-LAP was applied for accurate in vivo imaging of two different types of DILI（APAP-induced acute liver injury and RFP-induced cholestatic liver injury）.（2）NIR fluorescent/photoacoustic dual-modal probe for accurate imaging of hepatic inflammationFor the insufficient information and poor accuracy of single-modal fluorescence imaging,multi-modal imaging that combines the advantages of different modes was adopted for comprehensive and accurate detection of early lesions.By combining the high sensitivity of fluorescence imaging and the high resolution of photoacoustic imaging,an O₂^·--activated dual-modal probe hCy-Tf-CA was designed,which greatly improved the accuracy of the detection of early biomarker O₂^·-and liver inflammation.NIR fluorescence images showed that hCy-Tf-CA could sensitively detect hepatic O₂^·-in hepatic inflammation mouse modal.At the same time,photoacoustic images exhibited the distribution information of hepatic O₂^·-with high resolution.Therefore,hCy-Tf-CA can achieve more comprehensive and accurate assessment of early hepatic inflammation.Notably,this two-in-one strategy greatly reduce the synthetic workload,providing a promising strategy for precision medicine.（3）Activated NIR probe with hydrophilic modification for the detection and tracking of NQO1 in cells and in vivoIn view of the poor imaging performance of molecular fluorogenic probes in vivo,the physical properties were improved by the modification of probe structure,so as to facilitate the high sensitivity detection of biomarkers and in situ imaging of early lesions.First,QCy7 probe platform with large Stoke’s shift and excellent optical properties was selected to design sensitive imaging probes.Based on this platform,we constructed an early tumor biomarker NAD（P）H:quinone oxidoreductase-1（NQO1）specific activated NIR fluorogenic probe QCy7-NQO1.Then,by introducing two hydrophilic sulfonates,a hydrophilic NIR probe S-QCy7-NQO1 was designed and optimized,which significantly improved its response rate and optical brightness in biological systems,and realized sensitive detection of endogenous NQO1 activity in living cells and tumor-bearing mice.Therefore,S-QCy7-NQO1 not only serves as a practical tool for detecting abnormal NOQ1 levels in early tumor,but also provides a simple way to enhance the imaging performance of fluorogenic probes in vivo.更多还原