多功能多级孔环氧水凝胶的制备及对神经细胞的保护和生长行为的调控

Successful CNS repair remains challenging, as the pathological microenvironment formed post injury is complex and hostile, with a library of factors hindering the process of repair and regeneration. These include, for example, depletion of neurotrophic factors, scar formation, over production of neural growth inhibitory molecules, and excess reactive oxygen species (ROS). While some of these aspects have been taken into consideration in neural repair strategies, the issue related with ROS yet to be actively addressed. This proposal seeks to rationally design and develop a novel class of multifunctional epoxy hydrogels, which can directly counteract the deleterious effects of excess ROS and offer neural protection. Specifically, the protection will be achieved as a result of the following actions: a) the vicinal diol groups in the designed epoxy hydrogel network is ROS cleavable, thus consuming ROS radicals; b) the hydrophobic nanopores formed by temperature-induced phase separation and incorporated into the hydrogel structure during the preparation process allows encapsulation of natural antioxidants (e.g. resveratrol or curcumin) to remove excess ROS. Conversely, ROS cleavage of the diol groups leads to modification of the microstructure of hydrogels, as our preliminary study revealed a transition from macroporous beaded structure to fibrous network. Such ROS-induced restructuring of the hydrogel could benefit neural growth due to structural resemblance to natural ECM, promoting neural integration with the hydrogel. Besides the aforementioned functionalities, the tailor designed epoxy hydrogel comes with neural adhesiveness, as the adopted epoxy chemistry naturally makes amine groups readily available in the hydrogel. Together, these features of the epoxy hydrogels will offer a multi-pronged approach toward establishing a better microenvironment for CNS repair. In the proposed four-year project, an in-depth, systematic investigation of the effects of various reaction parameters, such as reactant chemical structure, concentration, time and temperature, on properties of the hydrogels will first be conducted. The ROS scavenging ability of the hydrogels will be examined along with the effects of ROS on the modulus and morphology of the hydrogel. Antioxidant loading into and release from the hydrogel will be studied, and a correlation between the hierarchical porous structure and ROS triggered release will be established. Neural protection and modulation of neuronal cell growth with the epoxy hydrogels will be investigated both in vitro via primary cell culture and in vivo using animal model of cortex lesion. Impact of the epoxy hydrogel developed in this project could be extended beyond neural repair, due to the significance of ROS overproduction in other pathological conditions such as myocardial infarction and chronic inflammation.

中枢神经修复极具挑战，主要原因是损伤部位病变造成的复杂且险恶的微环境对神经细胞极为不利。尽管水凝胶在营造神经修复微环境方面具有潜力，针对环境中过量存在活性氧自由基（ROS）这一重要问题仍缺乏直接且有效应对方案。本项目提出理性设计与制备含ROS响应的邻位二醇化学结构和负载抗氧化剂的疏水性纳米孔洞的多级孔新型环氧水凝胶，通过上述双重手段消除ROS对神经细胞的伤害。同时，ROS响应性赋予水凝胶微观结构和形貌动态重塑的功能，从而达到对神经细胞生长的调控。此外，含有支持神经元细胞黏附和生长的氨基官能团是环氧化学反应为本体系带来的另一优势。本项目将系统研究环氧水凝胶的可控制备，建立环氧水凝胶多级孔结构对于凝胶的弹性模量、抗氧化药物释放以及ROS响应性影响的内在规律，并通过细胞实验和大鼠皮质损伤模型的体内实验来评价该材料对神经细胞的保护和生长的调控。此材料对其它富含ROS微环境的局部修复也具有应用价值。

本项目围绕环氧-胺反应，秉承绿色的制备理念，研究了多功能多级孔结构的新型生物医用环氧水凝胶的可控制备。以水为溶剂，考察了小分子型和大分子型的双环氧单体与多种聚醚胺单体的反应，评价了不同组合的成胶能力，对构建的水凝胶体系的微观结构、形貌、力学和生物学性能等进行了表征。本研究揭示了环氧-胺反应得到的预聚物中间体水溶液的温敏行为对水凝胶微结构的关键作用，建立了单体亲疏水性对预聚物温敏性的影响规律。通过合理利用温敏行为介导的相分离现象，便捷实现该环氧水凝胶体系的微观形貌多元化，包括由微凝胶组合而成的多孔凝胶，其中的微凝胶大小可以是相对均匀的，也可呈现一定的梯度化；或是微凝胶以不同密度随机分散在均质凝胶中的杂化结构。相应的，水凝胶的模量展示了可供多种软组织（如神经、皮肤、软骨等）应用的力学适配性。研究表明，微凝胶的形成机理与相分离密不可分，其内部的疏水性纳米孔洞也赋予了相应的水凝胶材料内在的容纳疏水性抗氧化物质的功能。在所研究的环氧单体中，双环氧化丁二烯的应用为水凝胶带来了具有活性氧响应性的邻位二醇的化学结构，使得水凝胶的微观结构在活性氧环境中可由微球状向纤维状演变，模量降低，亲水性提高。水凝胶展示了良好的生物相容性，对活性氧的响应性则改善了神经细胞在氧化应激下的存活率。尽管神经细胞在其表面未达到预期的生长效果，但水凝胶中的胺基为后续的修饰来改善其作为神经细胞亲和基底提供可能。此外，环氧水凝胶及其微凝胶具有原位金杂化和硅杂化，复合天然多酚和止血的能力，凸显其功能的多样性。