ABSTRACT: The absence of DNA, their abundance, and ease of acquisition make erythrocytes an excellent source for EV production. Accordingly, red blood cell-derived extracellular particles (RBCEPs) possess many benefits in healthcare applications representing a simple and powerful platform for drug delivery. Nowadays, how the different methods proposed to produce them could influence their properties remains poorly investigated. We compared three main types of RBCEPs: (i) those naturally released during the blood bag storage (EryErythrosomes, EryEs, E), (ii) those produced artificially through RBC sonication (NanoErythrosomes, NanoEs, N), and (iii) those released after RBC chemical stimulation, in this case with calcium ionophore (Vesiculation induced-EryEs, VI-EryEs, R). We evaluated the characteristics of the different subtypes and the efficiency of membrane functionalization by copper-free click chemistry. Concentration and protein amount were analyzed by NTA and BCA, respectively, while transmission electron microscopy (TEM) was employed to evaluate size distribution and morphology. The expression of both EV- and erythrocyte-associated markers was evaluated by flow cytometry and western blot while proteomic analyses estimated the differences in total protein content. As expected, all three subpopulations were able to be produced on a large scale, with NanoEs being the ones with the highest concentration, polydispersity, and largest dimension. TEM analysis revealed not only the traditional EV-associated rounded shape but also the presence of a tubular shape. Tetraspanins were universally absent while all three subpopulations expressed Alix, Flotillin-1, and Tsg101. On the other hand, Glycophorin A was expressed ubiquitously while BAND 3 was uniquely found in NanoEs and EryEs. In addition, the analyzed subpopulations showed three distinct proteomic signatures and a significant amount of differentially expressed proteins that indicated distinct enrichment profiles of molecular functions. In vitro experiments on both a human fibroblast cell line and THP-1 macrophages revealed that the different subpopulations did not significantly influence internalization efficiency. Finally, NanoEs turned out to be the most efficient in surface functionalization with almost 90% of Click-EPs. This work provides a pioneering contribution to the study and application of RBCEPs, indicating that the isolation method can significantly impact both the qualitative and quantitative properties of these natural nanoparticles. Among the three subpopulations examined, NanoEs turned out to be the one suiting better the role of targeted drug delivery vehicles.
Martina Bartolucci 
