Critical considerations in determining the surface charge of small extracellular vesicles

Abstract

Small extracellular vesicles (EVs) have emerged as a focal point of EV research due to their significant role in a wide range of physiological and pathological processes within living systems. However, uncertainties about the nature of these vesicles have added considerable complexity to the already difficult task of developing EV‐based diagnostics and therapeutics. Whereas small EVs have been shown to be negatively charged, their surface charge has not yet been properly quantified. This gap in knowledge has made it challenging to fully understand the nature of these particles and the way they interact with one another, and with other biological structures like cells. Most published studies have evaluated EV charge by focusing on zeta potential calculated using classical theoretical approaches. However, these approaches tend to underestimate zeta potential at the nanoscale. Moreover, zeta potential alone cannot provide a complete picture of the electrical properties of small EVs since it ignores the effect of ions that bind tightly to the surface of these particles. The absence of validated methods to accurately estimate the actual surface charge (electrical valence) and determine the zeta potential of EVs is a significant knowledge gap, as it limits the development of effective label‐free methods for EV isolation and detection. In this study, for the first time, we show how the electrical charge of small EVs can be more accurately determined by accounting for the impact of tightly bound ions. This was accomplished by measuring the electrophoretic mobility of EVs, and then analytically correlating the measured values to their charge in the form of zeta potential and electrical valence. In contrast to the currently used theoretical expressions, the employed analytical method in this study enabled a more accurate estimation of EV surface charge, which will facilitate the development of EV‐based diagnostic and therapeutic applications.

The association between the size of adipocyte-derived extracellular vesicles and fasting serum triglyceride-glucose index as proxy measures of adipose tissue insulin resistance in a rat model of early-stage obesity

“…While we did not confirm a higher adEV count due to methodological differences, specifically having used the dynamic light scattering instead of nanoparticle tracking analysis, we did observe a lower negative zeta potential in the HFD group compared to the Ctrl group, but again this result was not statistically significant. Zeta potential, indicative of EVs surface charge, could affect their interactions with other vesicles or target cells, influencing content internalization ( 31 , 48 ). Notwithstanding this, zeta potential remains underreported in studies of EVs, and their alterations may depend on the cell of origin and membrane lipid composition ( 49 , 50 ).…”

Structurally Dynamic Monocyte–Liposome Hybrid Vesicles as an Anticancer Drug Delivery Vehicle: A Crucial Correlation of Microscopic Elasticity and Ultrafast Dynamics

“…The zeta potential of Monocyte-LP was 9.61 mV, lower than that of the unmodified LPs (20 mV), while the surface zeta potential of the Mons was −7.35 mV, lower than the hybrid Monocyte-LP (Figure e). The negative zeta potential observed for the monocyte membrane is attributed to the deprotonated COO– groups of proteins and acidic sugars (e.g., sialic acid) at physiological pH. , The colloidal stability of PC liposomes, monocytes, and hybrid Monocyte-LPs were further evaluated through monitoring the particle size and ζ potential over a period of 3 days. The results indicate that there is no significant change in charge and particle size observed for PC liposomes and hybrid Monocyte-LPs during this time frame (Figure S1).…”