Impact of the production method on the properties of erythrocyte-derived extracellular particles: a quantitative and qualitative evaluation

: Red blood cell-derived extracellular particles (RBCEPs) possess many benefits in healthcare applications representing a simple and powerful platform for drug delivery. Nowadays, whether the different methods proposed to produce them could influence their properties remains poorly investigated. We compared three main types of RBCEPs: (i) naturally released during the blood bag storage (EryErythrosomes, EryEs), (ii) produced artificially through RBC sonication (NanoErythrosomes, NanoEs), and (iii) released after RBC chemical stimulation (Vesiculation-Induced EryEs, VI-EryEs). Concentration, protein amount, size distribution, and morphology (TEM) were preliminary evaluated. Then the expression of EV- and erythrocyte-associated markers was assessed by flow cytometry and western blot while proteomic analyses estimated the differences in total protein content. The efficiency of membrane functionalization was assessed by copper-free click chemistry. 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. The traditional EV-associated rounded shape was found to coexist with a tubular shape. Tetraspanin expression was absent while all three formulations expressed Alix, Flotillin-1, and Tsg101. On the other hand, Glycophorin A was the only RBC marker expressed ubiquitously. The analyzed subpopulations showed three distinct proteomic signatures which defined distinct enrichment profiles of molecular functions. Finally, NanoEs turned out to be the most efficient in surface functionalization adopting a copper-free click chemistry strategy. This work provides a pioneering contribution to the study and application of RBCEPs, indicating that the isolation method can significantly impact both the quantitative and qualitative properties of these natural nanoparticles.