Systematic process evaluation of the conjugation of proteins to gold nanoparticles

Extracellular Vesicles

The present work addresses some fundamental aspects in the preparation of protein-conjugated gold nanoparticles, in order to ensure an appropriate final product. Ten broadly available and/or easy to implement analytical tools were benchmarked and compared in their capacity to provide reliable and conclusive information for each step of the procedure. These techniques included transmission electron microscopy, UV/VIS spectroscopy, dynamic light scattering, zeta-potential, Fourier-transformed infrared spectroscopy, colloidal stability titration, end-point colloidal stability analysis, cyclic voltammetry, agarose gel electrophoresis and size-exclusion chromatography (SEC). Four different proteins widely used as adaptors or blocking agents were tested, together with 13 nm gold nanoparticles containing different surface chemistries. Among all tested techniques, some of the least popular among nanomaterial scientists probed to be the most informative, including colloidal stability, gel electrophoresis and SEC; the latter being also an efficient purification procedure. These three techniques provide low-cost, low time consuming, sensitive and robust ways to assess the success of the nanoparticle bioconjugation steps, especially when used in adequate combinations.

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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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