基于靶向氧气纳米传感器的高分辨荧光成像对组织缺氧下肿瘤光动力疗法优化的研究

Molecular oxygen is indispensible to the efficacy of photodynamic therapy (PDT), while tumor hypoxia restricts the supply of oxygen. Moreover, PDT can further decrease the concentration of oxygen and increase the proliferation and resistant of tumor cells. Therefore, the PDT efficacy can be greatly improved if the concentration of dissolved oxygen, during the process of PDT treatment, within tumor microenvironment is monitored with high spatial and temporal resolution. Although many studies have been reported to image hypoxia, most of them are just whole-body imaging with the aim to detect tumor. Up to now, oxygen concentration in tumor microenvironments under PDT treatment has rarely been studied. In this proposal, (multicellular tumor spheroids, MCTs) are adopted to simulate solid tumors, and mitochondrial targeted oxygen nanosensors are used to image the intracellular area with severely varied oxygen based on the high-resolved confocal microscope. All those make it is possible to image the dissolved oxygen within tumor microenvironments with high resolution. At the same time, the PDT parameters will be timely adjusted according to the concentration of oxygen, which are optimized through monolayer tumor cells, MCTs and in vivo tumor in sequence. With the performance of this proposal, high-resolved imaging of dissolved oxygen within tumor microenvironments up to cellular level might be obtained, and a model about the diffusion and consumption of oxygen during the PDT process will be established, and optimized PDT parameters obtained finally.

肿瘤的光动力治疗（PDT）需要分子氧参与，但肿瘤组织中溶氧量过低（组织缺氧）限制氧的供给而削弱了PDT疗效。并且，随着氧的消耗，PDT会加剧组织的缺氧水平，增强癌细胞的增殖能力和耐药性。因此，实时了解PDT作用下肿瘤微环境中溶氧分布对于提高治疗效果具有重要意义。目前对组织缺氧的荧光成像已经开展了很多工作，但大都局限于整体成像（分辨率低）以检测肿瘤，对PDT作用下肿瘤微环境内溶氧的高分辨成像则鲜有报道。本项目拟利用肿瘤多细胞球模拟实体肿瘤，利用靶向线粒体的纳米传感器探测细胞内溶氧变化最剧烈的区域，使用激光共聚焦显微镜对PDT作用下肿瘤微环境的溶氧进行高分辨荧光成像。并在PDT过程中基于探测到的溶氧量及时调整治疗参数，并通过单层细胞系、肿瘤多细胞球和活体肿瘤依次优化筛选。该项目的开展有望得到肿瘤多细胞球内溶氧分布的高分辨荧光成像，建立起溶氧扩散和消耗的理论模型，并获取优化的肿瘤PDT治疗参数。

肿瘤的光动力治疗（PDT）过程中需要氧的参与，但肿瘤组织本身乏氧，且PDT过程中氧的消耗会加剧组织的缺氧水平；此外，组织乏氧还会增强癌细胞的增殖能力和耐药性。上述因素会削弱PDT的实际疗效。因此，基于纳米技术研究组织缺氧下的PDT疗法对于提高治疗效果具有重要意义。本项目的主要研究内容是基于功能性荧光纳米粒子，分别用于光敏药剂的输运、细胞荧光标记、溶解氧检测和细胞损伤评估等，以最终获得优化的PDT治疗参数。所取得的重要结果有：（1）利用疏水性光敏剂ZnPc制备了生物兼容性良好的纳米光敏剂，其表面为荷正电的多聚赖氨酸分子，从而可以被细胞高效吞噬。通过体外和小动物体内实验证明，当纳米粒子基质材料为PFO且负载率为4%光敏剂分子的相对聚集度最低，具有最优的PDT功效。（2）利用具有近红外发射特性的(DPQ)2Ir(acac)分子制备纳米粒子， 研究结果表明10%的Ir纳米粒子具有最强的荧光强度和高的单线态氧产生率。该纳米粒子既具有良好的透气性，又可抑制氧气猝灭，可同时进行细胞标记和PDT治疗。（3）制备了Pt配合物掺杂的荧光氧气纳米传感器，利用其可实时监测细胞的呼吸速率，据此不仅可评估PDT所导致的细胞活性的损伤，更能对细胞的瞬时损伤进行评估。（4）基于氧气纳米传感器制备了葡萄糖酶传感器，可以通过比率荧光强度和时间分辨荧光两种模式实现对葡萄糖的灵敏检测，相应的动态检测范围为（2-10 mM）和（1-6 mM），检测下限为（1-2 mM）和（0.1-0.2 mM）。上述研究成果分属于组织缺氧检测、高效纳米光敏剂的制备、PDT疗效评估和近红外PDT治疗4个子领域，对于克服肿瘤组织缺氧对PDT的制约、提高PDT功效具有重要的指导意义和实践支持。