聚(L-谷氨酸)水凝胶的Diels-Alder制备及其生物分子响应性研究

Preparation of Poly(L-glutamic acid)-based Hydrogels via Diels-Alder Reaction and Study on Their Biomolecule-responsive Properties

报道了一种含有二硫键的聚L-氨基酸共价交联网络,制备了能对含巯基生物分子与蛋白酶产生响应的新型聚L-氨基酸水凝胶.通过二硫键将降冰片烯基团键合在聚(L-谷氨酸)侧链,所得到的聚合物与末端修饰四嗪基团的四臂聚乙二醇在水溶液中混合,通过降冰片烯与四嗪基团之间发生Diels-Alder反应形成分子间共价交联,获得了聚(L-谷氨酸)/聚乙二醇水凝胶.研究了水凝胶在含巯基生物活性分子谷胱甘肽(GSH)作用下的性质变化.结果表明,2种官能化聚合物混合后可快速形成稳定的水凝胶,其力学性质随聚合物浓度、2种聚合物比例和降冰片烯基团的取代度的改变而变化.体外降解实验结果表明,在GSH或弹性蛋白酶存在的条件下,水凝胶的降解速率显著增加.同时,经GSH处理的水凝胶机械强度也显著降低.大鼠体内实验表明,在交联点引入GSH响应性的二硫键会明显加速聚氨基酸水凝胶的体内降解.进一步体外细胞实验与组织学分析结果表明,所获得聚氨基酸/聚乙二醇水凝胶具有良好的体外细胞相容性和动物体内组织相容性.

A novel type of biomolecule-responsive polypeptide hydrogels were fabricated by inverse electron demand Diels-Alder reaction between disulfide-linked, norbornene-conjugated poly（L-glutamic acid）（PLG-SS-Norb） and tetrazine-teminated 4-armed poly（ethylene glycol）（PEG-T）. Mono-norbornene group modified cystamine（Norb-SS-NH2） was synthesized and then introduced into the side chains of PLG through EDC/NHS chemistry. The obtained polymer was mixed with PEG-T to form the hydrogels in phosphate buffer saline（PBS） at various polymer concentrations and PLG-SS-Norb/PEG-T mass ratio. The gelation was accomplished in several minutes via inverse electron demand Diels-Alder reaction between the norbornene and tetrazine groupswith nitrogen as the byproduct. The mechanical properties were investigated by dynamic mechanical analysis, which indicated that the storage moduli of the hydrogels were influenced by polymer concentration and the molar ratio of two functional groups. Due to the peptide linkage in poly（L-glutamic acid）（PLG） backbone and disulfide bonds in the crosslinking points, the hydrogels were sensitive to proteolitic enzymes and thiol-containing reductive biomolecules. The degradation of the hydrogel was markedly accelerated in the presence of glutathione（GSH） or elastase in vitro. Particularly, dynamic mechanical analysis revealed a remarkabe decrease in the storage moduli from 3.3 k Pa to 0.51 k Pa within 12 h in the presence of 0.8 mmol/L GSH, and the influence of GSH treatment on the hydrogel was also clearly demonstrated by changing pore structure in scanning electron microscopy（SEM） images. After subcutaneous injection into rats, PLG/PEG hydrogel linked with disulfide bonds completely degraded in 6 days and displayed obviously enhanced degradation rate compared to the counterpart without disulfide bonds. Moreover, MTT assay showed that nearly 90% of L929 cells remained viable after incubation with PLG-SS-Norb or PEG-T at concentrations up to 1 g/L. The live/dead cell staining assay and the histology analysis showed that the polypeptide hydrogel exhibited good cyt℃ompatibility in vitro and hist℃ompatibility in vivo.