Physicochemical properties of intact fungal cell wall determine vesicles release and nanoparticles internalization

Our previous microscopic observations on the wet mount of cultured Candida yeast showed release of large extracellular vesicles (EVs) that contained intracellular bacteria (∼500-5000 nm). We used Candida tropicalis, to examine the internalization of nanoparticles (NPs) with different properties to find out whether the size and flexibility of both EVs and cell wall pores play role in transport of large particles across the cell wall. Candida tropicalis was cultured in N-acetylglucoseamine-yeast extract broth (NYB) and examined for release of EVs every 12 h by the light microscope. The yeast was also cultured in NYB supplemented with of 0.1%, 0.01% of Fluorescein isothiocyanate (FITC)-labelled NPs; gold (0.508 mM/L and 0.051 mM/L) (45, 70 and 100 nm), albumin (0.0015 mM/L and 0.015 mM/L) (100 nm) and Fluospheres (0.2 and 0.02%) (1000 and 2000 nm). Internalization of NPs was recorded with fluorescence microscope after 30 s to 120 min. Release of EVs mostly occurred at 36 h and concentration of 0.1% was the best for internalization of NPs that occurred at 30 s after treatment. Positively charged 45 nm NPs internalized into >90% of yeasts but 100 nm gold NPs destroyed them. However, 70 nm gold and 100 nm negatively-charged albumin were internalized into <10% of yeasts without destroying them. Inert Fluospheres either remained intact on the surface of yeasts or became degraded and internalized into ∼100% of yeasts. Release of large EVs from the yeast but internalization of 45 nm NPs indicated that flexibility of EVs and cell wall pores as well as physicochemical properties of NPs determine transport across the cell wall.