The mechanism of Hepatocyte-Targeting and safety profile of Phospholipid-Free small unilamellar vesicles

Extracellular Vesicles

Phospholipid-free small unilamellar vesicles (PFSUVs) composed of cholesterol and TWEEN80 (5:1 mol ratio), with an average diameter of 60 nm, displayed targeted delivery to the hepatocytes after intravenous (i.v.) injection. Here, we conducted a series of experiments to elucidate the hepatocyte targeting mechanism. The uptake of PFSUVs by HepG2 cells was increased by 3-fold in the presence of serum. The plasma protein corona adsorbed to PFSUVs was analyzed and subtypes of apolipoproteins were found enriched, specifically apolipoprotein AII (ApoA2). The cellular uptake was increased by 1.5-fold when the culture medium was supplemented with ApoA2, but not ApoC1 and ApoE. Furthermore, the cellular uptake of PFSUVs increased with increasing concentrations of ApoA2 in the medium and was almost completely blocked in the presence of BLT-1, an inhibitor for the scavenger receptor B-1 (SR-B1), which is a receptor for ApoA2. The data suggest that upon i.v. delivery, PFSUVs adsorbed plasma ApoA2 to the surface, which was recognized by SR-B1 expressed by the hepatocytes and then internalized. After internalization, mainly through the clathrin-mediated endocytosis, PFSUVs were found in the endosomes after 1-2 h post treatment and then lysosomes in 4 h. We also examined the cytotoxicity, hemolytic toxicity and complement activation of PFSUVs by incubating the formulation with HepG2 cells, red blood cells and human plasma, respectively, demonstrating no toxicity at concentrations higher than the therapeutic doses.

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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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