PDF(Pubmed)
Abstract：
The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) protein plays an essential role in the cisplatin (CDDP)-induced generation of reactive oxygen species (ROS). In this study, we evaluated the suitability of ultrasound-mediated lysozyme microbubble (USMB) cavitation to enhance NOX4 siRNA transfection in vitro and ex vivo. Lysozyme-shelled microbubbles (LyzMBs) were constructed and designed for siNOX4 loading as siNOX4/LyzMBs. We investigated different siNOX4-based cell transfection approaches, including naked siNOX4, LyzMB-mixed siNOX4, and siNOX4-loaded LyzMBs, and compared their silencing effects in CDDP-treated HEI-OC1 cells and mouse organ of Corti explants. Transfection efficiencies were evaluated by quantifying the cellular uptake of cyanine 3 (Cy3) fluorescein-labeled siRNA. In vitro experiments showed that the high transfection efficacy (48.18%) of siNOX4 to HEI-OC1 cells mediated by US and siNOX4-loaded LyzMBs significantly inhibited CDDP-induced ROS generation to almost the basal level. The ex vivo CDDP-treated organ of Corti explants of mice showed an even more robust silencing effect of the NOX4 gene in the siNOX4/LyzMB groups treated with US sonication than without US sonication, with a marked abolition of CDDP-induced ROS generation and cytotoxicity. Loading of siNOX4 on LyzMBs can stabilize siNOX4 and prevent its degradation, thereby enhancing the transfection and silencing effects when combined with US sonication. This USMB-derived therapy modality for alleviating CDDP-induced ototoxicity may be suitable for future clinical applications.
摘要：
烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶4（NOX4）蛋白在顺铂（CDDP）诱导的活性氧（ROS）产生中起着至关重要的作用。在这项研究中,我们评估了超声介导的溶菌酶微泡（USMB）空化在体外和离体增强NOX4siRNA转染的适用性。构建了溶菌酶壳微泡（LyzMB），并将其设计为siNOX4负载为siNOX4/LyzMB。我们研究了不同的基于siNOX4的细胞转染方法，包括裸siNOX4、LyzMB混合siNOX4和负载siNOX4的LyzMB，并比较了它们在CDDP处理的HEI-OC1细胞和Corti外植体的小鼠器官中的沉默作用。通过定量花青3(Cy3)荧光素标记的siRNA的细胞摄取来评估转染效率。体外实验表明，US和负载siNOX4的LyzMB介导的siNOX4对HEI-OC1细胞的高转染效率（48.18％）显着抑制CDDP诱导的ROS生成至几乎基础水平。在用US超声处理的siNOX4/LyzMB组中，小鼠Corti外植体的离体CDDP处理的器官显示出更强大的NOX4基因沉默作用，明显消除了CDDP诱导的ROS产生和细胞毒性。在LyzMBs上负载siNOX4可以稳定siNOX4并防止其降解，因此，当与US超声处理结合时，增强了转染和沉默效果。这种用于减轻CDDP诱导的耳毒性的USMB衍生的治疗方式可能适合于未来的临床应用。
