Assembly of Genetically Engineered Ionizable Protein Nanocage-based Nanozymes for Intracellular Superoxide Scavenging

Nanozymes play a pivotal role in mitigating excessive oxidative stress, however, determining their specific enzyme-mimicking activities for intracellular free radical scavenging is challenging due to endo-lysosomal entrapment. In this study, we employed a genetic engineering strategy to generate ionizable ferritin nanocages (iFTn), enabling their escape from endo-lysosomes and entry into the cytoplasm. Specifically, ionizable repeated Histidine-Histidine-Glutamic acid (9H2E) sequences were genetically incorporated into the outer surface of human heavy chain FTn, followed by the assembly of various chain-like nanostructures via a two-armed polyethylene glycol (PEG). Utilizing endosome-escaping ability, we designed iFTn-based tetrameric cascade nanozymes with high superoxide dismutase- and catalase-mimicking activities. The in vivo protective effects of these ionizable cascade nanozymes against cardiac oxidative injury were demonstrated in mouse models of cardiac ischemia-reperfusion (IR). RNA-sequencing analysis highlighted the crucial role of these nanozymes in modulating superoxide anions-, hydrogen peroxide- and mitochondrial functions-relevant genes in IR injured cardiac tissue. These genetically engineered ionizable protein nanocarriers provide opportunities for developing ionizable drug delivery systems. Overall design: Mouse ischemia-reperfusion (IR) models were established following treatment with PBS, iFTn-M4, iFTn-R4 and iFTn-MR4 for two doses (equivalent protein concentration of 2.5 mg/kg) given every other day. After three days, RNA was extracted from the isolated cardiac IR tissue.

纳米酶（Nanozymes）在缓解过度氧化应激方面发挥着关键作用，然而，由于内涵体-溶酶体（endo-lysosomal）捕获效应，精准测定其用于细胞内自由基清除的特异性模拟酶活性仍极具挑战。本研究采用基因工程策略，构建了可电离铁蛋白纳米笼（ionizable ferritin nanocages, iFTn），使其能够逃逸内涵体-溶酶体并进入细胞质。具体而言，研究人员将可电离的组氨酸-组氨酸-谷氨酸重复序列（9H2E, ionizable repeated Histidine-Histidine-Glutamic acid）基因整合到人重链铁蛋白的外表面，随后通过双臂聚乙二醇（polyethylene glycol, PEG）组装得到多种链状纳米结构。借助内涵体逃逸能力，我们设计了基于iFTn的四聚体级联纳米酶，该酶兼具优异的超氧化物歧化酶与过氧化氢酶模拟活性。在心肌缺血再灌注（ischemia-reperfusion, IR）小鼠模型中，验证了这类可电离级联纳米酶对心脏氧化损伤的体内保护作用。RNA测序（RNA-sequencing）分析显示，此类纳米酶可调控缺血再灌注损伤心肌组织中与超氧阴离子、过氧化氢及线粒体功能相关的基因表达，揭示了其关键作用机制。这类基因工程改造的可电离蛋白质纳米载体，为开发可电离药物递送系统提供了新的契机。总体实验设计：构建小鼠心肌缺血再灌注模型，分别经PBS、iFTn-M4、iFTn-R4及iFTn-MR4处理，给药方案为每隔一天给药一次，共给药2次（等效蛋白浓度为2.5 mg/kg）。给药3天后，从分离得到的缺血再灌注损伤心肌组织中提取RNA。