Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl 2 , and AlCl 3 ) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant ( p < 0.05) lowering of the ZP. Moreover, the ZP was significantly ( p < 0.05) less negative in the presence of ions with higher valency (Al 3+ /Ca 2+ ) than in the presence of monovalent ones (Na + /K + ). Besides, the ZP of EVs became less negative at acidic pH, and vice versa . The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs.

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