Characterization of Kluyveromyces marxianus Extracellular Vesicles as a New Potential Delivery System for Biologically Active Compounds

Extracellular vesicles (EVs) released by probiotic microorganisms may be involved in intercellular communication via transferring their cargo to other cells. Hence, there is growing interest in their application as a drug delivery system. This article presents studies on EVs secreted by three strains of Kluyveromyces marxianus (B0399, WUT240, and WUT216) under standard growth conditions and in fluids simulating a gastrointestinal environment. Nanoparticle tracking analysis and transmission electron microscopy revealed spherical EVs with modal diameters ranging from 79.8 to 148.2 nm, depending on the growth conditions. Research conducted on human intestinal cell lines did not reveal cytotoxic effects of EVs. Moreover, EVs exhibited mild antioxidant properties in normal cells. K. marxianus EVs did not stimulate the release of the pro-inflammatory cytokine IL-8 in the HT-29 model but induced a tailored immunomodulatory response in Galleria mellonella hemocytes, characterized by elevated levels of IL-8-like proteins in the absence of a TNF-α-like response. Proteomic analysis identified 161 core proteins common to all strains, yielding 33 potential biomarkers lacking significant human homology, which would be useful for tracking yeast EV carriers in subsequent studies. In addition, yeast EVs were found to have a high affinity for human cells, transporting biologically active molecules (doxorubicin) and the Nile Red dye into recipient cells. Altogether, this study provides clear evidence that the EVs of K. marxianus could be considered a drug delivery system.