上转换纳米颗粒（upconversion nanoparticles，UCNPs）因其独特的光学性质在生物检测和肿瘤诊疗等领域得到广泛应用。蛋白质/酶的亚细胞定位与肿瘤等疾病密切相关，但由于缺乏递送工具难以在亚细胞水平原位成像酶分子。另一方面，基于UCNPs的光动力治疗（photodynamic therapy，PDT）面临合成过程繁琐以及光敏剂装载含量低等问题，且缺乏有效方法原位成像PDT过程中关键分子及能量传递效率，限制了该体系在诊疗上的应用。本论文通过将UCNPs与DNA传感器设计及细胞器定位策略相结合，并引入光敏剂单元构筑了一系列近红外光调控的纳米器件，实现了细胞器内酶活性的精准成像以及PDT过程中线粒体内酶活性的动态监测，并提出了基于PDT的联合治疗及原位成像新策略。具体研究内容如下：（1）UCNPs介导的光激活型纳米传感器用于肿瘤细胞器内酶活性成像。通过引入光敏基团设计了光激活型DNA传感器，并与UCNPs及细胞器定位信号相结合，构建了一种细胞器靶向的近红外光激活型DNA纳米传感器，实现了对细胞器（细胞核和线粒体）内脱嘌呤/脱嘧啶核酸内切酶1（APE1）活性的精准成像分析。细胞和活体实验表明，该策略能够实现传感器的可控定位及近红外光介导的原位激活，有效地将成像功能限制在特定细胞器内，显著提高了分子成像的空间分辨率。（2）上转换发光调控的DNA纳米器件用于线粒体靶向型PDT过程中酶活性的动态成像。通过将DNA传感器与光敏剂及线粒体靶向配体组装于UCNPs表面构建了一种多功能DNA纳米器件。该器件能够精准定位于线粒体内，并将近红外光转换为绿光，以激发光敏剂产生活性氧，实现线粒体靶向型PDT；同时，利用装载的DNA传感器可实时监测治疗过程中线粒体内APE1酶活性的动态变化。研究表明，线粒体内APE1酶活性在PDT过程中逐渐增强，此变化与其氧化还原调节功能密切相关。（3）上转换发光调控的肿瘤PDT联合治疗及原位成像。本章通过对UCNPs进行表面修饰，可控合成了卟啉基UCNP-金属有机框架异质结构，实现了近红外光触发的PDT治疗。进一步将乏氧激活的化疗药物装载异质结构的纳米孔道中，并联合免疫检查点阻断疗法，实现了乏氧实体瘤的高效协同治疗。此外，通过上转换发光寿命成像对该体系的靶向递送及UCNPs与光敏剂间的能量传递效率进行了可视化原位成像，为基于PDT的联合治疗方案优化提供了指导。
Abstract: Upconversion nanoparticles (UCNPs) have been widely used in many fields such as biodetection and tumor therapy due to their unique optical properties. Subcellular localization of proteins/enzymes is tightly related to various diseases like tumor. Therefore, in situ imaging of enzymatic activity with subcellular resolution is helpful for evaluation of their functions. However, it remains a challenge to realize this goal. On the other hand, UCNPs-based photodynamic therapy (PDT) is limited by complex synthesis of materials and low loading rate of photosensitizers (PSs). Besides, in situ imaging of key molecules and energy transfer between UCNPs and PSs during PDT process is still challenging due to the lack of effective methods. In this dissertation, by combining UCNPs with DNA-based biosensors and organelle-targeting strategy, and further introduction of PSs and functional metal organic frameworks (MOFs) as building blocks, we designed and constructed a series of near infrared (NIR) light-regulated nanodevices, which allow to spatially-controlled imaging of enzymatic activity in specific organelles and further to monitor the mitochondrial enzymatic activity and energy transfer during PDT. In addition, we proposed a new PDT-based combinational therapy and imaging strategy against hypoxic tumors. The main research and results are as follows: (1) UCNPs-based DNA nanodevices for precise imaging of enzymatic activity in specific organelles. By introducing a photosensitive moiety, we designed a light-activated, DNA-based sensor. Then, based on the combination of it with UCNPs and organelle-targeting signals, we design and construct an organelle-targeted, NIR light-activatable DNA nanodevice for the imaging of the enzymatic activity of apurinic/apyrimidinic endonuclease 1 (APE1) in a chosen organelle (e.g., mitochondria or nucleus). Cellular and in vivo assays showed that this strategy achieves controllable localization of the system and permits NIR light-mediated in situ activation, thereby confining the sensing and imaging function to a specific organelle and significantly improving the spatial resolution of biodetection. Notably, this sensing strategy provides a general approach to dissect the important roles of key enzymes in tumorigenesis and progression at the subcellular level.(2) Upconversion luminescence (UCL)-regulated DNA nanodevices for dynamic imaging of enzymatic activity during the mitochondria-targeted PDT. By assembling a DNA-based sensor, PSs and mitochondria-targeting ligands on the surface of UCNPs, we design and construct a multifunctional DNA nanodevice. The system was able to target mitochondria and then convert NIR irradiation into green emission to excite PSs, generating reactive oxygen species for PDT. Furthermore, the loaded DNA sensor was capable of monitoring the dynamic APE1 activity during the PDT process. Benefiting from the versatility of the constructed nanodevice, a gradual accumulation of APE1 in mitochondria was observed during the PDT process, which was tightly associated with its redox function. This work provides technical tools for the study of anti-apoptotic mechanisms of tumor cells at the subcellular level.(3) UCL-mediated, PDT-based combinational therapy and in situ imaging of tumor. A porphyrinic UCNP@MOFs heterostructure was controllably synthesized through the conditional surface engineering of UCNPs and growth of MOFs, enabling NIR light-triggered PDT. Furthermore, by encapsulating a hypoxia-activated chemotherapeutic drug into the micro-porous of UCNP@MOFs and combining it with immune checkpoint blockade therapy, we develop an efficient synergistic treatment strategy against hypoxic solid tumors. In addition, by in situ lifetime imaging of UCL, the delivery processes of UCNP@MOFs and energy transfer efficiency between UCNPs and PSs are monitored, which allow to provid guidance for the optimization of PDT-based combinational therapy strategy.
