The Endocytic Pathway Of Giant Pinocytosis Mediates The Dandelion Decoction Body Into The Cell

Exosomes are nano-sized particles that are actively secreted by a variety of mammalian cells to efficiently deliver their cargos to recipient cells.It has been reported that plant-derived exosome-like nanoparticles(ELNs)have been isolated from grapefruit,grapes,ginger and carrots and that ELNs are similar to those mammalian-derived exosomes both in structure and size.Our group also isolated and characterized a class of ELNs from herbal decoctions of Pu Gong Ying(PGY,Taraxacum mongolicum),named decoctosome.Decoctosome,which consists of lipids,small molecules,proteins and RNAs,may be the main active component in herbal decoction.Plant-derived small RNAs could ameliorate acute lung injury mouse models by oral administration,which will provide new supporting evidence for cross-kingdom regulation.However,the detailed mechanism of cellular uptake of decoctosome is still unknown.Here,we sought to characterize cellular mechanisms involved in PGY decoctosome uptake.Internalization of fluorescently-labeled PGY decoctosome was evaluated in A549 cells.By using a range of chemical inhibitors to block specific pathways as well as RNA interference to knockdown key proteins involved in individual endocytic pathways,cellular uptake of PGY decoctosome was assessed using flow cytometry and confocal microscopy.Our results showed that lysosomotropic agents could significantly inhibit the entry of PGY decoctosome into cells.Moreover,inhibitors of macropinocytosis,including cytochalasin D and amiloride significantly reduced the internalization of PGY decoctosome in a dose dependent manner.Consistently,knockdowning of Racl and PAK1 by RNAi impeded the cell entry of PGY decoctosome.These results indicate that PGY decoctosome enters cells via endocytosis and predominantly via macropinocytosis.As the ability of nanoparticles to deliver their cargo and exert a biological function is largely dependent on the mechanism through which they are taken up,our findings will contribute to further development of intracellular delivery systems and provide a new perspective for understanding the intercellular communication mediated by decoctosome.