Red blood cells-derived extracellular vesicles (RBCEVs): A novel drug delivery system for efficient loading and delivery of RNA molecules.

English

Red blood cell-derived extracellular vesicles (RBCEVs) have acquired increasing attention for therapeutic applications because of their high biocompatibility and safety. However, large-scale production and detailed characterization of such lab-scale drug-loaded RBCEVs are highly needed. The current study, therefore, aimed at high-yield production of cargo-loaded RBCEVs that could be suitable for their various clinical applications. RBCs were purified from whole blood and subjected to leukodepletion. RBCs were loaded by hypotonic dialysis, and isotonic resealing and reannealing. Several cargoes were tested such as fluorescent polymers or miRNA 210. Loaded RBCs then underwent our newly developed physical vesiculation method, called “soft extrusion” to produce RBCEVs. Obtained RBCEVs were deeply characterized according to the new guidelines MISEV2023. For uptake studies, we labeled our RBCEV samples with PKH26 to follow EV trafficking in human umbilical vein endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMC). Moreover, production of miR-210-loaded RBCEVs and their biological effects were analyzed by quantitative PCR (qPCR). Our results demonstrated that cell recovery was high (60.07 ± 5.39% and 60.63 ± 6.03% for RBCs UL and L, respectively), which indicates that the loading procedure is safe. DLS analyses of raw products from extrusion were 202.9 ± 27.9 nm (UL) and 207.1 ± 14.2 nm (L). NTA analysis demonstrates that both the UL and L RBCEV populations were quite homogenous, with a size of 130 nm (mode) and a yield of 4-5 × 1012 particles/ml. TEM analysis demonstrates that our RBCEVs are round and lipid-bound with a size of 50–200 nm, which proves that the process is suitable and very efficient for vesiculation. Regarding flow cytometry analysis, the high LCD mean fluorescence intensity (MFI) proves that our EVs are intact and resealed. High positivity to Glycophorin and CD47, and low positivity to Annexin V were detected in both UL and L samples, showing that these RBCEVs are correctly oriented and mirror mother cells. Furthermore, FITC dextran was clearly detected in our loaded L sample. In vitro analyses of PKH26-labeled RBCEVs by HUVEC and PBMC demonstrates no toxicity and maximum uptake at 24hr. Moreover, our results highlighted efficient loading and efficient delivery of miR-210 by RBCEVs to HUVEC, alongside the inhibition of a known target (PTP1B protein). Hence, it can be used in the treatment of several diseases involving miR-210 dysregulation. In conclusion, our newly developed platform can produce a highly homogeneous population of drug-loaded RBCEVs and can overcome challenges in the field of drug delivery by EVs, such as low yield of production and no translatability to clinics.