三维网络结构水凝胶植入材料的抗蛋白表面修饰研究及生物相容性评价

Aim at the fibrosis triggered by protein adsorption on the surface of hydrogel microcapsules after transplanted in vivo, this proposal designed a modification method of “functionalized covalent grafting” for protein resistant surface and investigated innovatively its protein repellency in the complicated system of hydrogel microcapsules with a three-demensional (3D) network structure. First, the terminally functionalized methoxy poly (ethylene glycol) (MPEG) was covalently grafted on the backbone of natural polymer materials of hydrogel microcapsules to form “brush-like side chains” through layer-by-layer self-assembly method and in-situ covalent grafting method separately. This grafting method made a breakthrough of traditional physical modification such as interpenetration network modification and solved the problem of poor modification stability to improve the long-term efficacy. Second, different with the widely researched self-assembled monolayer system, the gel permeation chromatography (GPC) technique and the X-ray photoelectron spectroscopy (XPS) model were established to quantitatively analyze the graft density of MPEG on the 3D network structure. The quantitative relationship between the MPEG graft density and the protein adsorption extent on the hydrogel microcapsules was fully investigated by adjusting the architecture of MPEG graft copolymers and grafting conditions, which influenced the behavior of brush-like side chains interpenetrating into the 3D network or enriching on the surface of hydrogel. Third, to find out strategy of guiding more MPEG chains to gather on the surface rather than permeating into the porous structure for better protein repellency, the characterization platform combined the GPC technique, multiangle laser light scattering (MALLS) detector and the detecting method of 3D network structure was established respectively to disclose the conformation of MPEG graft copolymers and the 3D network structure of hydrogel microcapsules. Increase in MPEG surface graft density on hydrogel microcapsules was achieved by matching the conformation of MPEG graft copolymers with 3D network structure to solve the problem of MPEG diffusing into the hydrogel and improve the protein repellency. Finally, the in-vitro cytotoxicity and in-vivo biocompatibility of MPEG-modified hydrogel microcapsule were both fully evaluated to associate in-vitro protein repellency with in-vivo biocompatibility. This project laid a solid foundation for surface modification and quantitative analysis of the interaction between biomacromolecules and hydrogel with a three-dimensional network structure. This anti-fouling hydrogel biomaterial may have great potential for drug delivery and cell transplantation in vivo.

以作为药物控释及细胞移植载体的水凝胶微胶囊为研究对象，为解决其体内移植后因表面蛋白吸附引起纤维化包裹的严峻问题，进行抗蛋白表面修饰研究。首先，突破常用物理方法，采用“功能化共价接枝”修饰法，将末端功能化修饰分子共价接枝到微囊材料骨架上，形成“刷状侧链”，提高修饰稳定性和可控性；其次，区别于自组装单分子层体系，建立定量表征三维网络结构中修饰分子接枝密度的方法，研究结构参数和制备工艺对刷状侧链在水凝胶表面富集和内部扩散行为的影响，揭示接枝密度与抗蛋白吸附性能的定量规律；进而，搭建修饰分子空间构象与水凝胶网络结构测试平台，调控空间构象与三维网络结构相匹配，解决刷状侧链向微囊内部扩散难题，进一步增强蛋白排斥能力；最后，评价抗蛋白修饰材料体外细胞毒性和体内生物相容性，将抗蛋白吸附规律与生物相容性相关联，为开发具有良好生物相容性的微囊材料提供理论与实验依据，指导并促进水凝胶微胶囊在生物医学领域的应用。

本项目以作为药物控释及细胞移植载体的海藻酸钠/壳聚糖水凝胶微胶囊为研究对象，为解决其体内移植后因表面蛋白吸附引起纤维化包裹的严峻问题，进行抗蛋白表面修饰研究及生物相容性评价。首先，筛选Williamson亲核取代醛基化法将MPEG末端功能化共价修饰到壳聚糖骨架上，制备成不同取代度、MPEG分子量和壳聚糖分子量的CS-g-MPEG接枝共聚物，提高修饰稳定性和可控性，并对其进行红外、核磁及凝胶渗透色谱(GPC)表征；其次，分别建立GPC和X射线光电子能谱表征MPEG表观接枝密度和表面接枝密度的定量方法，并以免疫球蛋白(IgG)和纤维蛋白原(Fgn)为蛋白模型，研究微胶囊制备工艺和CS-g-MPEG结构参数对MPEG接枝密度与抗蛋白吸附性能的影响规律；进而，通过调控接枝共聚物空间构象与水凝胶三维网络结构相匹配以提高MPEG表面接枝密度，增强微胶囊蛋白排斥能力；最后，考察CS-g-MPEG的体外细胞毒性，并将MPEG修饰微胶囊进行小鼠腹腔移植，评价其体内生物相容性。研究表明，微胶囊制备工艺包括CS-g-MPEG溶液的pH、浓度和体积比对接枝密度及蛋白吸附量影响显著，且具有不同分子量和构象的蛋白在相同MPEG表观接枝密度下具有不同的吸附规律；MPEG表观接枝密度随取代度的升高而降低，抗蛋白吸附能力随取代度的升高呈先降低再提高的趋势，取代度为5%时可将IgG吸附量降低70%，Fgn吸附量降低48%；相同表观接枝密度下MPEG2K和MPEG5K的抗蛋白吸附能力要显著强于MPEG550；壳聚糖骨架分子量为79kDa比20kDa的CS-g-MPEG具有更强的抗蛋白能力；CS-g-MPEG不仅分布在微胶囊表面，还能扩散一定深度修饰在微胶囊三维网状水凝胶结构内部，且富集在微胶囊表面的MPEG链比扩散MPEG链具有更好的蛋白排斥效果，从而通过引入海藻酸钠缓冲层、增加微胶囊膜厚和原位共价接枝的方法，可将表面接枝密度显著提高，使IgG和Fgn吸附量降低97%；MPEG修饰显著降低了微胶囊表面的细胞黏附和纤维化包裹程度，提高了微胶囊的生物相容性。本项目研究结果为开发良好生物相容性微胶囊材料提供理论与实验依据，为水凝胶微胶囊作为药物控释及细胞移植载体应用于临床疾病的治疗奠定基础。