Cryo-TEM and NTACharacterisation ofHyperglycemia-InducedVariability in Morphology andSize Distribution of EVs

<ns3:p>&lt;br&gt;&lt;b&gt;Introduction:&lt;/b&gt; Extracellular vesicles (EVs) are membrane-bound structures that playa crucial role in intercellular communication and molecular transport. Due to theirbiological functions, EVs hold great potential as a novel diagnostic tool or as the targeteddrug carriers in anticancer therapies. Elevated glucose concentrations affect cellularmetabolism, thereby modifying the composition of the EVs and their lipid bilayer. This, inturn, can influence EVs' properties and morphology, which is important in their function.&lt;/br&gt;&lt;br&gt;&lt;b&gt;Hypothesis:&lt;/b&gt; Hyperglycemic conditions, by altering the composition and metabolism ofEVs, influence their size distribution and physicochemical properties.&lt;/br&gt;&lt;br&gt;&lt;b&gt;Objective:&lt;/b&gt; Visualisation and size distribution comparison of EVs isolated fromnormoglycemic (NG) and hyperglycemic (HG) conditions.&lt;/br&gt;&lt;br&gt;&lt;b&gt;Methods:&lt;/b&gt; Two complementary methods based on different physical principles were appliedand compared in terms of their applicability: nanoparticle tracking analysis (NTA) and cryo--transmission electron microscopy (Cryo-TEM). In this study, 1.1B4 cells were cultured innormo- (NG, 5 mM glucose) and hyperglycemic (HG, 25 mM glucose) conditions.&lt;/br&gt;&lt;br&gt;&lt;b&gt;Results:&lt;/b&gt; The mean hydrodynamic EV size under hyperglycemic conditions (236 28 nm)was slightly higher than the control (209 43 nm; Paired Sample Wilcoxon Signed Test,p = 0.05). Physical EV sizes (Cryo-TEM), as well as mode hydrodynamic sizes (NTA) wereconsistent and not affected by glucose concentration. In contrast to NTA, Cryo-TEM isa more precise technique for real-size distribution analysis, whereas NTA provides a rapidand cost-effective assessment of hydrodynamic radii. The theoretical level of detection ofNTA for EVs was close to what was observed in results.&lt;/br&gt;&lt;br&gt;&lt;b&gt;Conclusions:&lt;/b&gt; Hyperglycemic conditions slightly influenced only the mean hydrodynamic radius of EVs, indicating effects on EV corona size and the biogenesis of large EVs (lEVs).&lt;/br&gt;</ns3:p>