Kocholatá Michaela, Malý Jan, Kříženecká Sylvie, Janoušková Olga
Centre for Nanomaterials and Biotechnologies, Faculty of Science, Jan Evangelista Purkyně University in Ústí Nad Labem, Ústí Nad Labem, Czech Republic.
Department of Environmental Chemistry and Technology, Faculty of Environment, Jan Evangelista Purkyně University in Ústí Nad Labem, Ústí Nad Labem, Czech Republic.
Sci Rep. 2024 Dec 3;14(1):30111. doi: 10.1038/s41598-024-81940-8.
In recent years, there has been a growing interest in plant extracellular vesicles (pEVs) due to their immense potential for medical applications, particularly as carriers for drug delivery. To use the benefits of pEVs in the future, it is necessary to identify methods that facilitate their production in sufficient quantities while maintaining high quality. In this study, a comparative analysis of yields of tobacco pEV derived from apoplastic fluid, sterile calli, and suspension cultures, was performed to identify the most suitable plant material for vesicle isolation. Subsequent experiments focused on assessing the efficiency of small interfering RNA (siRNA) loading into callus-derived vesicles, employing various methods such as sonication, incubation, incubation supplemented with saponin, lipofection, and electroporation. Differences in loading efficiency among vesicles derived from apoplastic fluid, calli, and suspension cultures were observed. Moreover, our investigation extended to the presence of tobacco secondary metabolites, specifically anabasine and nicotine, within vesicles originating from three distinct tobacco sources. The outcomes of our study highlight variations not only in vesicle yields based on their source but also in their loadability and the presence of nicotine and anabasine. These findings contribute valuable insights into optimizing the production and application of pEVs for future medicinal purposes.
近年来，由于植物细胞外囊泡（pEVs）在医学应用方面具有巨大潜力，尤其是作为药物递送载体，人们对其兴趣与日俱增。为了在未来利用pEVs的优势，有必要确定能够在保持高质量的同时促进其大量生产的方法。在本研究中，对源自质外体流体、无菌愈伤组织和悬浮培养物的烟草pEV产量进行了比较分析，以确定最适合用于囊泡分离的植物材料。随后的实验重点评估了小干扰RNA（siRNA）加载到愈伤组织来源囊泡中的效率，采用了多种方法，如超声处理、孵育、添加皂苷的孵育、脂质体转染和电穿孔。观察到源自质外体流体、愈伤组织和悬浮培养物的囊泡在加载效率上存在差异。此外，我们的研究还扩展到了源自三种不同烟草来源的囊泡中烟草次生代谢产物（特别是新烟草碱和尼古丁）的存在情况。我们的研究结果不仅突出了基于来源的囊泡产量差异，还揭示了其装载能力以及尼古丁和新烟草碱的存在差异。这些发现为优化pEVs的生产和应用以用于未来医学目的提供了有价值的见解。
