Sosnowska Malwina, Wierzbicki Mateusz, Nasiłowska Barbara, Bakalova Totka Nikolaeva, Piotrowska Klara, Strojny-Cieślak Barbara, Sawosz Ewa, Kutwin Marta
Department of Nanobiotechnology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland.
Biomedical Engineering Center, Institute of Optoelectronics, Military University of Technology, Warsaw, Poland.
Int J Nanomedicine. 2024 Nov 21;19:12221-12255. doi: 10.2147/IJN.S482652. eCollection 2024.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the nasal cavity, penetrates the nasal epithelial cells through the interaction of its spike protein with the host cell receptor angiotensin-converting enzyme 2 (ACE2) and then triggers a cytokine storm. We aimed to assess the biocompatibility of fullerenol nanoparticles C(OH) and ectoine, and to document their effect on the protection of primary human nasal epithelial cells (HNEpCs) against the effects of interaction with the fragment of virus - spike protein. This preliminary research is the first step towards the construction of a intranasal medical device with a protective, mechanical function against SARS-CoV-2 similar to that of personal protective equipment (eg masks).
We used HNEpCs and the full-length spike protein from SARS-CoV-2 to mimic the first stage of virus infection. We assessed cell viability with the XTT assay and a spectrophotometer. May-Grünwald Giemsa and periodic acid-Schiff staining served to evaluate HNEpC morphology. We assessed reactive oxygen species (ROS) production by using 2',7'-dichlorofluorescin diacetate and commercial kit. Finally, we employed reverse transcription polymerase chain reaction, Western blotting and confocal microscopy to determine the expression of angiotensin-converting enzyme 2 (ACE2) and inflammatory cytokines.
There was normal morphology and unchanged viability of HNEpCs after incubation with 10 mg/L C(OH), 0.2% ectoine or their composite for 24 h. The spike protein exerted cytotoxicity via ROS production. Preincubation with the composite protected HNEpCs against the interaction between the spike protein and the host membrane and prevented the production of key cytokines characteristic of severe coronavirus disease 2019, including interleukin 6 and 8, monocyte chemotactic protein 1 and 2, tissue inhibitor of metalloproteinases 2 and macrophage colony-stimulating factor.
In the future, the combination of fullerenol and ectoine may be used to prevent viral infections as an intranasal medical device for people with reduced immunity and damaged mucous membrane.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）进入鼻腔，通过其刺突蛋白与宿主细胞受体血管紧张素转换酶2（ACE2）相互作用穿透鼻上皮细胞，进而引发细胞因子风暴。我们旨在评估富勒醇纳米颗粒C(OH)和依克多因的生物相容性，并记录它们对保护原代人鼻上皮细胞（HNEpCs）免受与病毒刺突蛋白片段相互作用影响的效果。这项初步研究是构建一种具有类似个人防护装备（如口罩）的针对SARS-CoV-2的防护性机械功能的鼻内医疗器械的第一步。
我们使用HNEpCs和SARS-CoV-2的全长刺突蛋白来模拟病毒感染的第一阶段。我们用XTT法和分光光度计评估细胞活力。May-Grünwald Giemsa染色和过碘酸-希夫染色用于评估HNEpC的形态。我们使用2',7'-二氯荧光素二乙酸酯和商业试剂盒评估活性氧（ROS）的产生。最后，我们采用逆转录聚合酶链反应、蛋白质免疫印迹法和共聚焦显微镜来确定血管紧张素转换酶2（ACE2）和炎性细胞因子的表达。
用10 mg/L C(OH)、0.2%依克多因或它们的复合物孵育24小时后，HNEpCs的形态正常且活力未改变。刺突蛋白通过产生ROS发挥细胞毒性作用。与复合物预孵育可保护HNEpCs免受刺突蛋白与宿主膜之间的相互作用，并防止产生2019年严重冠状病毒病的关键细胞因子，包括白细胞介素6和8、单核细胞趋化蛋白1和2、金属蛋白酶组织抑制剂2和巨噬细胞集落刺激因子。
未来，富勒醇和依克多因的组合可作为一种鼻内医疗器械用于免疫力低下和黏膜受损的人群，以预防病毒感染。
