Mammadova Ramila, Maggio Serena, Fiume Immacolata, Bokka Ramesh, Moubarak Maneea, Gellén Gabriella, Schlosser Gitta, Adamo Giorgia, Bongiovanni Antonella, Trepiccione Francesco, Guescini Michele, Pocsfalvi Gabriella
Extracellular Vesicles and Mass Spectrometry Laboratory, Institute of Biosciences and BioResources, National Research Council of Italy, 80131 Naples, Italy.
Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 Urbino, Italy.
Pharmaceutics. 2023 Jan 19;15(2):333. doi: 10.3390/pharmaceutics15020333.
Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1β cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.
植物源纳米囊泡（PDNVs）作为治疗方法和药物递送载体，已成为哺乳动物细胞衍生的细胞外囊泡（EVs）颇具吸引力的替代品。在本研究中，我们分离了番茄果实衍生的纳米囊泡，并通过碘克沙醇密度梯度超速离心（DGUC）将它们分离成十二个组分。在密度为1.064±0.007 g/mL、1.103±0.006 g/mL和1.122±0.012 g/mL处观察到三条可见带。通过粒度分布、浓度、脂质和蛋白质含量以及基于质谱的蛋白质组学分析蛋白质组成，对粗制番茄PDNVs和DGUC组分进行了表征。在脂多糖（LPS）刺激的人单核细胞THP-1细胞培养物中评估了与这些条带相关的DGUC组分的细胞毒性和抗炎活性。番茄PDNVs的中密度和低密度可见DGUC组分显示LPS诱导的炎性IL-1β细胞因子mRNA产生显著降低。对这些组分中鉴定的蛋白质进行功能分析，发现存在与基因本体（GO）术语GO:0050794相关的14-3-3蛋白、内质网腔结合蛋白和GTP结合蛋白，以及包括炎症在内的几种细胞过程的调节。使用直接加载、超声处理和挤压方法将最丰富的中密度DGUC组分装载姜黄素，并比较抗炎活性。通过直接加载获得了最高的包封效率和载药量。超声处理加载的姜黄素增加了番茄PDNVs的基础抗炎活性。
