Characterization Challenges of Self-Assembled Polymer-SPIONs Nanoparticles: Benefits of Orthogonal Methods

Extracellular Vesicles

Size and zeta potential are critical physicochemical properties of nanoparticles (NPs), influencing their biological activity and safety profile. These are essential for further industrial upscale and clinical success. However, the characterization of polydisperse, non-spherical NPs is a challenge for traditional characterization techniques (ex., dynamic light scattering (DLS)). In this paper, superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyvinyl alcohol (PVAL) exhibiting different terminal groups at their surface, either hydroxyl (OH), carboxyl (COOH) or amino (NH2) end groups. Size, zeta potential and concentration were characterized by orthogonal methods, namely, batch DLS, nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), asymmetric flow field flow fractionation (AF4) coupled to multi-angle light scattering (MALS), UV–Visible and online DLS. Finally, coated SPIONs were incubated with albumin, and size changes were monitored by AF4-MALS-UV-DLS. NTA showed the biggest mean sizes, even though DLS PVAL-COOH SPION graphs presented aggregates in the micrometer range. TRPS detected more NPs in suspension than NTA. Finally, AF4-MALS-UV-DLS could successfully resolve the different sizes of the coated SPION suspensions. The results highlight the importance of combining techniques with different principles for NPs characterization. The advantages and limitations of each method are discussed here.

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Recent Publications

Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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