Enzyme-catalysis self-assembled oligopeptide hydrogel holds great interest in drug delivery, which has merits of biocompatibility, biodegradability and mild gelation conditions. However, its application for protein delivery is greatly limited by inevitable degradation of enzyme on the encapsulated proteins leading to loss of protein activity. Moreover, for the intracellularly acted proteins, cell membrane as a primary barrier hinders the transmembrane delivery of proteins. The internalized proteins also suffer from acidic and enzymatic degradation in endosomes and lysosomes. We herein develop a protease-manipulated hybrid nanogel/nanofiber hydrogel for localized delivery of intracellularly acted proteins. The embedded polymeric nanogels (CytoC/aNGs) preserve activity of cytochrome c (CytoC) that is an intracellular activator for cell apoptosis as a model protein against proteolysis, and do not affect the gelation properties of the protease-catalysis assembled hydrogels. The injectable hydrogel (CytoC/aNGs/Gel) serves as a reservoir to enhance intratumoral retention and realize sustainable release of CytoC/aNGs. The released CytoC/aNGs increase cellular uptake of CytoC and enhance its intracellular delivery to its target site, cytoplasm, resulting in favorable apoptosis-inducing and cytotoxic effects. We show that a single local administration of CytoC/aNGs/Gel efficiently inhibit the tumor growth in the breast tumor mouse model.

酶催化自组装寡肽水凝胶在药物递送方面引起了极大的兴趣，其具有生物相容性、生物降解性和温和的凝胶条件等优点。然而，其在蛋白质递送中的应用受到封装蛋白质上不可避免的酶降解导致蛋白质活性丧失的极大限制。此外，对于细胞内作用的蛋白质，细胞膜作为主要屏障阻碍了蛋白质的跨膜传递。内化蛋白质在内体和溶酶体中也遭受酸性和酶促降解。我们在此开发了一种蛋白酶操纵的混合纳米凝胶/纳米纤维水凝胶，用于细胞内作用蛋白的局部递送。嵌入的聚合物纳米凝胶 (CytoC/aNGs) 保留了细胞色素c 的活性(CytoC) 是细胞凋亡的细胞内激活剂，作为抗蛋白酶解的模型蛋白，不影响蛋白酶催化组装的水凝胶的凝胶特性。可注射水凝胶（CytoC/aNGs/Gel）作为一种储库，可增强肿瘤内保留并实现CytoC/aNGs的可持续释放。释放的 CytoC/aNG 增加了细胞对 CytoC 的摄取，并增强了其向靶位点、细胞质的细胞内递送，从而产生有利的细胞凋亡诱导和细胞毒性作用。我们表明，单次局部给药 CytoC/aNGs/Gel 可有效抑制乳腺肿瘤小鼠模型中的肿瘤生长。