Optimised Electroporation for Loading of Extracellular Vesicles with Doxorubicin

Extracellular Vesicles
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The clinical use of chemotherapeutics is limited by several factors, including low cellular uptake, short circulation time, and severe adverse effects. Extracellular vesicles (EVs) have been suggested as a drug delivery platform with the potential to overcome these limitations. EVs are cell-derived, lipid bilayer nanoparticles, important for intercellular communication. They can transport bioactive cargo throughout the body, surmount biological barriers, and target a variety of tissues. Several small molecule drugs have been successfully incorporated into the lumen of EVs, permitting efficient transport to tumour tissue, increasing therapeutic potency, and reducing adverse effects. However, the cargo loading is often inadequate and refined methods are a prerequisite for successful utilisation of the platform. By systematically evaluating the effect of altered loading parameters for electroporation, such as total number of EVs, drug to EV ratio, buffers, pulse capacitance, and field strength, we were able to distinguish tendencies and correlations. This allowed us to design an optimised electroporation protocol for loading EVs with the chemotherapeutic drug doxorubicin. The loading technique demonstrated improved cargo loading and EV recovery, as well as drug potency, with a 190-fold increased response compared to naked doxorubicin.

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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.

2023
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