Red Blood Cell Membrane Vesicles for siRNA Delivery: A Biocompatible Carrier With Passive Tumor Targeting and Prolonged Plasma Residency

Giulia Della Pelle,1,2 Bostjan Markelc,3 Tim Bozic,3 Jernej Šribar,4 Igor Krizaj,4 Kristina Zagar Soderznik,1 Samo Hudoklin,5,* Mateja Erdani Kreft,5,* Iztok Urbančič,6 Matic Kisovec,7 Marjetka Podobnik,7 Nina Kostevšek1 1Department for Nanostructured Materials, Jožef Stefan Institute, Ljubljana, 1000, Slovenia; 2Jožef Stefan International Postgraduate School, Ljubljana, 1000, Slovenia; 3Department of Experimental Oncology, Institute of Oncology Ljubljana, Ljubljana, 1000, Slovenia; 4Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, 1000, Slovenia; 5Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, 1000, Slovenia; 6Laboratory of Biophysics, Condensed Matter Physics Department, Jožef Stefan Institute, Ljubljana, 1000, Slovenia; 7Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, 1000, Slovenia*These authors contributed equally to this workCorrespondence: Giulia Della Pelle, Jozef Stefan Institute, Jamova Cesta 39, Ljubljana, 1000, Slovenija, Tel +393356577520, Email [email protected]: Despite many advances in gene therapy, the delivery of small interfering RNAs is still challenging. Erythrocytes are the most abundant cells in the human body, and their membrane possesses unique features. From them, erythrocytes membrane vesicles can be generated, employable as nano drug delivery system with prolonged blood residence and high biocompatibility.Methods: Human erythrocyte ghosts were extruded in the presence of siRNA, and the objects were termed EMVs (erythrocyte membrane vesicles). An ultracentrifugation-based method was applied to select only the densest EMVs, ie, those containing siRNA. We evaluated their activity in vitro in B16F10 cells expressing fluorescent tdTomato and in vivo in B16F10 tumor-bearing mice after a single injection.Results: The EMVs had a negative zeta potential, a particle size of 170 nm and excellent colloidal stability after one month of storage. With 0.3 nM siRNA, more than 75% gene knockdown was achieved in vitro, and 80% was achieved in vivo, at 2 days PI at 2.5 mg/kg. EMVs mostly accumulate around blood vessels in the lungs, brain and tumor. tdTomato fluorescence steadily decreased in tumor areas with higher EMVs concentration, which indicates efficient gene knockdown. Approximately 2% of the initial dose of EMVs was still present in the plasma after 2 days.Conclusion: The entire production process of the purified siRNA-EMVs took approximately 4 hours. The erythrocyte marker CD47 offered protection against macrophage recognition in the spleen and in the blood. The excellent biocompatibility and pharmacokinetic properties of these materials make them promising platforms for future improvements, ie, active targeting and codelivery with conventional chemotherapeutics.Keywords: biomimetic, RNA interference, EPR effect, nanomedicine, melanoma