短肽共组装功能化平台构建用于乳腺癌细胞三维培养及活性氧/氮物种实时监测

The change of the concentration of reactive oxygen species/reactive nitrogen species (ROS/RNS) in biological systems is often closely related to the occurrence and development of tumors. Therefore, monitoring of ROS/RNS released from cells is very important for pathological study and clinical diagnosis and treatment. However, traditional researches are mainly based on two-dimensional (2D) cell culture, but 2D culture model is difficult to accurately replicate cell function in three-dimensional (3D) tissues. Recently, 3D cell culture systems have gained increasing interest due to their evident advantages in providing more physiologically relevant information and more predictive data for in vivo tests. In this project, we will construct function hydrogel platforms based on co-assembly of short peptide and recognition element for 3D malignant breast cancer cell culture and real-time electrochemical monitoring of released ROS/RNS species. Through the screening of peptide sequences, the introduction of functional molecules and changing of the assembly conditions, the physical and electrochemical properties of hydrogels will be regulated. The platforms for both suitable cell 3D growth and effective and sensitive detection will be fabricated. Under the stimulation of drug molecules, ROS/RNS released from nontransformed and metastatic breast cells will be measured. The relevance between metastatic property and released ROS/RNS will be explored. Furthermore, the mechanism of action of drugs on breast cancer cells will be also researched. The project would provide an effective tool for real-time monitoring of released biointeresting molecules in 3D cell culture model, and possibly facilitate the development of early diagnosis and drug discovery of metastatic breast cancer.

生物体内活性氧/氮物种(ROS/RNS)浓度的变化常与肿瘤的发生、发展密切相关，监测细胞释放ROS/RNS对于病理学研究和临床诊疗都具有重要意义。然而传统研究是基于二维(2D)细胞培养开展，很难准确地复制出三维(3D)组织中细胞功能。鉴于3D细胞培养能更好地模拟体内环境，本项目拟开发基于短肽、识别元件等多组分共组装功能化水凝胶平台，用于恶性乳腺癌细胞3D培养和释放ROS/RNS物种的实时电化学监测研究。通过筛选肽序列、引入功能分子及改变组装条件，从而调控水凝胶的物理和电化学等性能，制备适合细胞3D生长及有效、灵敏的检测平台；开展不同类型（正常和转移性）乳腺癌细胞在药物等刺激下释放ROS/RNS实时动态监测研究，探究细胞转移性与释放ROS/RNS的关联及药物作用机制。项目实施将为3D培养细胞释放物的原位监测提供重要的技术及方法，并有望促进转移性乳腺癌早期诊断和药物研发等相关领域的发展。

监测细胞释放的ROS/RNS对于病理学研究和临床诊疗都具有重要意义。然而传统研究是基于二维细胞培养开展，很难准确地复制出三维(3D)组织中细胞的功能。鉴于3D细胞培养能更好地模拟体内环境，本项目通过短肽序列的调控、功能材料的复合等方式构建了系列传感平台，在此平台上实现3D细胞培养的同时，能原位、高灵敏、实时监测乳腺癌细胞释放的ROS/RNS物种。在应对复杂的3D细胞培养环境下电化学检测面临的污染问题，引入了Fmoc-FF: Fmoc-K(Fmoc)-D复合水凝胶，其良好的亲水性能够促进水化层的形成，从而显著地减轻了界面上非特异性的大分子吸附。基于此制备的修饰电极浸泡在DMEM细胞培养基中三天后，可保持对H2O2响应初始值93%以上，远高于目前报道的修饰电极维持在60%。基于肽复合水凝胶/Pt NPs/钛网修饰电极实现了两种乳腺癌细胞释放NO和H2O2的同时监测。实验结果发现，在PMA药物的刺激下，三阴性乳腺癌MDA-MB-231细胞释放的NO和H2O2总浓度高于MCF-7细胞，两种细胞氧化应激释放的物种都呈现出NO远高于H2O2，且转移性MDA-MB-231细胞释放的NO的量也远多于非转移性MCF-7细胞。另外，通过制备自愈合型水凝胶，可快速修复传感平台的响应，提高其稳定性。构建了高灵敏的含有ABEI肽水凝胶电致化学发光传感平台，实现了3D培养下三种亚型人乳腺癌细胞在药物刺激前后释放H2O2的实时监测及分型。在此基础上，联合纳米马达概念，还开发了细胞内自驱动型高效ROS清除剂，为ROS上调等相关疾病的治疗提供了新的策略。以上研究结果为3D培养细胞释放物的原位监测提供重要的技术及方法，并有望为高转移型三阴性乳腺癌的早期诊断和治疗提供理论基础。