Macropinocytosis-Inducible Extracellular Vesicles Modified with Antimicrobial Protein CAP18-Derived Cell-Penetrating Peptides for Efficient Intracellular Delivery

Extracellular Vesicles

The antimicrobial protein CAP18 (approximate molecular weight: 18 000), which was first isolated from rabbit granulocytes, comprises a C-terminal fragment that has negatively charged lipopolysaccharide binding activity. In this study, we found that CAP18 (106-121)-derived (sC18)2 peptides have macropinocytosis-inducible biological functions. In addition, we found that these peptides are highly applicable for use as extracellular vesicle (exosomes, EV)-based intracellular delivery, which is expected to be a next-generation drug delivery carrier. Here, we demonstrate that dimerized (sC18)2 peptides can be easily introduced on EV membranes when modified with a hydrophobic moiety, and that they show high potential for enhanced cellular uptake of EVs. By glycosaminoglycan-dependent induction of macropinocytosis, cellular EV uptake in targeted cells was strongly increased by the peptide modification made to EVs, and intriguingly, our herein presented technique is efficiently applicable for the cytosolic delivery of the biologically cell-killing functional toxin protein, saporin, which was artificially encapsulated in the EVs by electroporation, suggesting a useful technique for EV-based intracellular delivery of biofunctional molecules.

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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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