Red Blood Cells-derived Extracellular Vesicles as a novel RNA delivery system

Introduction Extracellular vesicles (EVs) are emerging and promising tools for drug delivery. They are physiologically produced by nearly all cell types and play significant role in many physiological and pathological processes. Among these, Red Blood Cells (RBCs) are particularly intriguing EV producers due to their easy accessibility, complete safety, non-immunogenicity, and lack of DNA content. However, Red Blood Cells-derived Extracellular Vesicle (RBCEV) production is still challenging because of issues related to yield, clinical translatability, and loading efficiency. Materials and method We started from pre-loaded RBCs to obtain RNA-loaded RBCEVs with a newly developed and patented method called “soft extrusion”. This non-disruptive physical vesiculation technique mimics the natural processes occurring during RBC passage through the spleen. The encapsulation was conducted through a hypotonic dialysis, using a synthetic mRNA as cargo. This is a modified mRNA with methyl-pseudouridine for enhanced stability and with a codon optimization for murine models. Our RBCEVs were then purified through a ultracentrifugation and characterized by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Flow Cytometry (FC). Finally, RBCEVs underwent total RNA extraction followed by qRT-PCR to assess the loading efficiency. Results Thanks to their unique properties, RBCs were efficiently loaded with the above-mentioned synthetic mRNA encoding for the GAMT enzyme, leading to the RBCEV production that effectively retained molecules within them. First, a dose-dependent loading was set up with 4 different concentrations of long RNA that revealed that the two highest concentrations were very similar to each other, likely due to a saturation effect. Then, we successfully obtained GAMT mRNA-loaded, both RBCs and RBCEVs, as reported by qRT-PCR quantification. Thus, we demonstrated the feasibility of producing RBCEVs loaded with long RNAs and with great features in terms of size distribution, and biochemical features. Moreover, the whole process has been monitored at each step through the use of a cell counter to demonstrate that our soft extrusion is non-disruptive. Indeed, our findings showed that we are able to induce RBCs to vesiculate without their breakage. Finally, preliminary pharmacokinetic and efficacy studies are currently ongoing. Conclusion Our RBCEV mimetics exhibit efficient loading and delivery of biological molecules, making them a promising tool for the development of RNA-based therapies. Their advantages include easy translation into clinical applications and the ability to be fully automated, providing a robust and scalable solution for therapeutic delivery. Furthermore, the absence of chemicals makes them non-toxic; thus, confirming their potential use for therapeutic applications, such as for metabolic disorders.