自给H2O2型超声响应牛血清白蛋白（BSA）基NO释放系统用于肝癌靶向治疗及机制研究

Currently, the common treatment methods against liver tumor (surgery and chemotherapy) inevitably suffer from extremely severe trauma. Thus, as one gas therapy, nitric oxide therapy sharing high efficiency and high safety can be regarded as a potential candidate, but the design of NO release system remains a challenge. In previous work, we engineered an ultrasound-enhanced H2O2-responsive NO release system using hollow mesoporous silica nanoparticles (HMSNs) as carriers, but the poor degradability of inorganic carrier and insufficiency of H2O2 in tumor remain two obstacles that limits practical application of this NO release system. To address the bottleneck, basing on the previous work, bovine serum albumin (BSA) of excellent biosafety and degradability will be employed as the carrier instead of inorganic HMSNs to encapsulate NO donor (L-arginine, LA) and H2O2 donor (Vitamin C, VC), ultimately constructing a BSA-based nanosystem that is also simultaneously modified with docosahexaenoic acid (DHA) ligands. The combined targeting ability of DHA active targeting, ultrasound and NO-enhanced permeability for promoting accumulation of this targeted nanoplatform in liver tumor will be evaluated via confocal microscopy, flow cytometry, animal fluorescence imaging etc. Many methods including quantitative detection of H2O2 and NO, electron spin resonance (ESR), confocal microscopy etc., will be adopted to ascertain the principle and the influence of VC degradation into H2O2 in tumor on the ultrasound-responsive NO release. Furthermore, the principle and outcome of NO/H2O2 synergistic therapy against liver tumor will be evaluated and investigated via pathological examination and molecular characterization, etc., which paves a new route to the treatment of liver tumor.

肝癌常用的治疗手段（手术和化疗）创伤性极大，高效、安全的NO气体治疗成为一个潜在的替代方法，但NO释放系统设计仍是个难题。前期我们以中空介孔氧化硅为载体构建了超声增强H2O2响应的NO释放系统，但无机载体降解性差和肿瘤H2O2含量不足限制了其应用。为克服这一瓶颈，本项目以前期工作为基础，以生物安全性、降解性好的BSA为载体包裹L-精氨酸（NO供体，LA）和H2O2供体（维他命C，VC），构建新型BSA基NO释放系统，同时表面螯合肝癌靶向配体（二十二碳六烯酸，DHA）。通过共聚焦、流式细胞仪、小动物荧光成像等手段考察DHA主动靶向与超声/NO介导渗透性结合的靶向策略增强富集机制。通过H2O2和NO测定、电子自旋共振、共聚焦等手段研究肿瘤处VC降解产生H2O2的机制以及对NO释放的影响。进一步利用病理学、分子生物学等手段研究肝癌NO/H2O2协同治疗效果及机制，为临床肝癌治疗提供新思路。

精准、可控NO释放是阻断瘤免疫逃逸、减轻免疫抑制微环境，抑制肿瘤生长、转移、复发的前提，但目前仍是一个难题。此外，肿瘤的发生、发展、转移与肿瘤微环境、肿瘤异质性密切相关；因此，如何通过调控肿瘤微环境以及精准监控疗效成为肿瘤精准诊疗的关键科学问题。而纳米技术的发展为这些问题的解决提供了可能性。我们围绕肿瘤治疗效能提升及疗效评估开展了多个方面工作：一是发现了一种肺癌免疫逃逸靶点，并设计了一种NO调控的肿瘤自体疫苗。该疫苗可通过L-精氨酸增强PDE5介导的NO路径激活，除直接抗肿瘤外，还可以阻断PDE5免疫逃逸靶点；同时包覆的肿瘤细胞膜，除了靶向富集外，可作为抗原，进一步激活系统性免疫响应，从而进一步增强抗肿瘤的免疫响应。为肿瘤免疫治疗提供新的靶点和思路。二是设计并制备一种可释放NO和活性氧（ROS）的治疗剂，释放的ROS半衰期极短，但与NO结合后，可转化成RNS，其半衰期大大增加，从而提高自由扩散行程，增强抗肿瘤效果，并提供了一种行之有效的解决策略。三是设计并构建多种基于代谢微环境、免疫微环境、乏氧微环境、氮/氧平衡微环境、氧化应激微环境、酸/碱平衡微环境等肿瘤微环境调控的新型生物材料，并建立相应的治疗体系、方法，增强低强度超声治疗的效能，抑制肿瘤转移、复发并研究其潜在机制，同时指导其他治疗方式的新型治疗剂设计，为实现肿瘤的精准、安全、高效以及综合治疗提供新思路。四是基于肿瘤治疗过程中分子探针结构、性能的变化以及肿瘤微环境的变化，从而构建出多种可反映出这种变化的结构化、智能化分子探针，以期实现前述肿瘤过程的引导、监控以及评价治疗过程，为提高前述肿瘤治疗精准性、安全性、有效性提供强有力保障。