Harada Ayaka, Tsutsuki Hiroyasu, Zhang Tianli, Lee Ruda, Yahiro Kinnosuke, Sawa Tomohiro, Niidome Takuro
Faculty of Advanced Science and Technology, Kumamoto University.
Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University.
Chem Pharm Bull (Tokyo). 2020;68(4):363-368. doi: 10.1248/cpb.c19-00917.
Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.
聚（D，L-丙交酯-共-乙交酯）酸（PLGA）是一种合成共聚物，已被用于设计微/纳米颗粒，作为蛋白质和核酸等大分子的载体，这些大分子可通过内吞途径被内化。然而，难以控制细胞内递送至靶细胞器。在此，我们报告了一种用细菌细胞毒素修饰的纳米颗粒向内质网（ER）的细胞内递送系统以及该纳米颗粒的抗炎活性。枯草杆菌蛋白酶细胞毒素（SubAB）是某些肠出血性大肠杆菌（EHEC）菌株中的一种细菌毒素，它能切割宿主内质网伴侣蛋白BiP，并在亚致死浓度下抑制巨噬细胞中核因子-κB（NF-κB）的激活和一氧化氮（NO）的产生。PLGA纳米颗粒通过pH敏感连接用寡聚组氨酸标签（6×组氨酸标签）重组SubAB（SubAB-PLGA）进行修饰，并检测它们在巨噬细胞系J774.1细胞中向内质网的转运、对诱导型一氧化氮合酶（iNOS）的影响以及脂多糖（LPS）诱导的肿瘤坏死因子（TNF）-α细胞因子水平。与游离SubAB相比，SubAB-PLGA在J774.1细胞中切割BiP和诱导内质网应激标志物C/EBP同源蛋白（CHOP）方面显著有效。此外，SubAB-PLGA减弱了LPS刺激的iNOS和TNF-α的诱导。我们的研究结果为向巨噬细胞递送蛋白质提供了有用信息，并可能促进纳米颗粒在炎症性疾病治疗中的治疗应用。
