具有多重刺激响应性的蛋白质聚合物纳米凝胶的制备及结构控制

Over the last few decades, stimuli-responsive protein nanocarriers have been researched intensively, especially the nanogel which is more stable compared to self-assembled nanocarriers due to its crosslinked structure. However, there are still some drawbacks to nanogels as protein nanocarriers. One important problem is that polyethylene glycol (PEG) and its derivatives used for protecting proteins and prolonging circulating time are not beneficial for cell uptake. In this proposal, a nanogel with temperature-induced reversible structure is proposed as a new kind of protein nanocarrier. The synthetic method is to crosslink temperature responsive PEG derivative polymers with proteins to afford temperature-responsive nanogels. When the temperature is below the phase transition temperature, the hydrophilic PEG derivative polymers are the protecting shells for proteins. And when the temperature is above the phase transition temperature, the polymers become hydrophobic and collapse to form the cores with hydrophilic proteins moving to the shells to stabilize the nanogels, which facilitate the cell uptake. Different cleavable covalent bonds have been chosen to connect polymers and proteins and the release of proteins can be triggered by the change of physiological environment after the cell uptake. The influence of reaction conditions on the size, phase transition temperature and structure will be researched thoroughly as well as the protein release and activity under different conditions. In vitro cytotoxicity assays and cell uptake assays will also be conducted to evaluate the nanogel. It is expected that this nanogel can balance circulating time, uptake efficiency and protein release and promote the development of protein nanocarriers.

近年来，运载蛋白质的环境响应型纳米载体得到深入研究，特别是具有稳定交联结构的纳米凝胶。但是目前纳米凝胶载体仍有待改善，针对纳米凝胶结构中常见的聚乙二醇及衍生物外层能延长循环时间但不利于细胞摄取的问题，本项目拟提出一种温度诱导结构可翻转的纳米凝胶蛋白质载体。将蛋白质作为交联剂与温敏性聚乙二醇衍生物反应制得温敏性纳米凝胶，当温度低于纳米凝胶的相变温度时，亲水的聚乙二醇衍生物在外层保护蛋白质；高于相变温度时，温敏性聚合物外层变为憎水内核，蛋白质处于外层便于细胞的摄取。选择多种可断裂化学键连接蛋白质与聚合物，细胞摄取后生理环境的变化可以触发蛋白质的释放。研究反应条件对纳米凝胶尺寸，相变温度及结构的影响，以及不同生理环境下蛋白质的释放和活性情况，并通过体外细胞培养对纳米凝胶的毒性和细胞摄取情况进行评价。该研究有望得到可以兼顾体内循环、细胞摄取和蛋白质释放的载体，对蛋白质载体的研究起推动和完善的作用。

在该青年基金的资助下，我们进行了以下研究：（1）利用蛋白质的胺基基团作为反应位点交联温敏性聚合物（P(DEGMA-co-HODMA-co-NPC)），制备得到带有触发式自降解共价键的多重响应性聚合物蛋白质杂化纳米凝胶，将脂肪酶负载于该纳米凝胶，可以使脂肪酶在高温或酸性条件下活性得到保护，在谷胱甘肽存在下触发纳米凝胶解离可以释放负载的脂肪酶，脂肪酶的胺基基团和活性都可以得到恢复。（2）调整体系（1）中制备纳米凝胶的聚合物的单体组成，得到聚合物（P(DEGMA-co-PEGMA-co-HODMA-co-NPC)）与蛋白质反应，使纳米凝胶的相变温度在37℃附近，高于相变温度可以使纳米凝胶尺寸减小，憎水部分比例增大，利于细胞摄取，两种模型蛋白质牛血清白蛋白和溶菌酶均可以成功的载入纳米凝胶，在谷胱甘肽存在下纳米凝胶解离，释放的蛋白质的胺基基团和活性可以得到恢复并且二级结构也得以基本保留。（3）制备带有二硫吡啶基团的温敏性聚合物（PDEGMA-b-P(PEGMA-co-PDSMA)），升温至最低临界溶解温度之上得到温敏性胶束组装体，利用带有多个巯基基团的牛血清白蛋白作为交联剂，通过巯基二硫吡啶交换反应交联胶束的壳层部分，得到具有蛋白质交联壳层结构的胶束，将其置于低于其临界溶解温度的环境下，胶束的核溶解，得到具有空心结构的蛋白质聚合物杂化纳米胶囊，该纳米胶囊在谷胱甘肽存在下可以发生解离。通过这些研究，我们构建了多种制备蛋白质纳米载体的方法，这些方法都具有操作简便，条件温和以及可适用于多种蛋白质的特点。得到的蛋白质载体具有多重刺激响应性，能较好保护蛋白质的官能团，二级结构和活性。该研究为进一步研发蛋白质药物载体打下了坚实基础。在实验工作基础上，我们发表4篇SCI二区文章。