PDF(Pubmed)
Abstract：
RNA-based therapeutics, including siRNA, have obtained recognition in recent years due to their potential to treat various chronic and rare diseases. However, there are still limitations to lipid-based drug delivery systems in the clinical use of RNA therapeutics due to the need for optimization in the design and the preparation process. In this study, we propose adaptive focused ultrasound (AFU) as a drug loading technique to protect RNA from degradation by encapsulating small RNA in nanoliposomes, which we term nanoplexes. The AFU method is non-invasive and isothermal, as nanoplexes are produced without direct contact with any external materials while maintaining precise temperature control according to the desired settings. The controllability of sample treatments can be effectively modulated, allowing for a wide range of ultrasound intensities to be applied. Importantly, the absence of co-solvents in the process eliminates the need for additional substances, thereby minimizing the potential for cross-contaminations. Since AFU is a non-invasive method, the entire process can be conducted under sterile conditions. A minimal volume (300 μL) is required for this process, and the treatment is speedy (10 min in this study). Our in vitro experiments with silencer CD44 siRNA, which performs as a model therapeutic drug in different mammalian cell lines, showed encouraging results (knockdown > 80%). To quantify gene silencing efficacy, we employed quantitative polymerase chain reaction (qPCR). Additionally, cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM) techniques were employed to capture images of nanoplexes. These images revealed the presence of individual nanoparticles measuring approximately 100-200 nm in contrast with the random distribution of clustered complexes observed in ultrasound-untreated samples of liposome nanoparticles and siRNA. AFU holds great potential as a standardized liposome processing and loading method because its process is fast, sterile, and does not require additional solvents.
摘要：
基于RNA的疗法，包括siRNA,近年来，由于其治疗各种慢性和罕见疾病的潜力而获得了认可。然而,由于需要优化设计和制备过程,基于脂质的药物递送系统在RNA治疗剂的临床应用中仍然存在局限性.在这项研究中,我们提出了自适应聚焦超声（AFU）作为一种药物装载技术，通过将小RNA封装在纳米脂质体中来保护RNA免受降解，我们称之为纳米复合物。AFU方法是非侵入性和等温的，如纳米复合物在不与任何外部材料直接接触的情况下生产，同时根据所需的设置保持精确的温度控制。可以有效调节样品处理的可控性，允许广泛的超声强度的应用。重要的是,在该过程中不存在共溶剂消除了对额外物质的需要，从而最大限度地减少交叉污染的可能性。由于AFU是一种非侵入性方法，整个过程可以在无菌条件下进行。此过程需要最小体积（300μL），并且治疗迅速（在本研究中为10分钟）。我们用沉默子CD44siRNA进行的体外实验，在不同的哺乳动物细胞系中作为模型治疗药物，显示出令人鼓舞的结果(击倒>80%)。为了量化基因沉默的功效，我们采用定量聚合酶链反应（qPCR）。此外,采用低温电子显微镜（cryo-EM）和原子力显微镜（AFM）技术捕获纳米复合物的图像。这些图像揭示了测量约100-200nm的单个纳米颗粒的存在,与在脂质体纳米颗粒和siRNA的超声未处理样品中观察到的成簇复合物的随机分布形成对比。AFU具有作为一种标准化的脂质体加工和装载方法的巨大潜力，因为它的过程是快速的，无菌,并且不需要额外的溶剂。
