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Recombinant bacterial cocaine esterase (CocE) represents a potential protein therapeutic for cocaine use disorder treatment. Unfortunately, the native enzyme was highly unstable and the corresponding mutagenized derivatives, RBP-8000 and E196-301, although improving in vitro thermo-stability and in vivo half-life, were a partial solution to the problem. For cocaine use disorder treatment, an efficient cocaine-metabolizing enzyme with a longer residence time in circulation would be needed. We investigated in vitro the possibility of developing red blood cells (RBCs) loaded with RBP-8000 and E196-301 as a biocompatible system to metabolize cocaine for a longer period of time. RBP 8000 stability within human RBCs is limited (approximately 50% residual activity after 1 h at 37°C) and not different as for the free enzyme, while both free and encapsulated E196-301 showed a greater thermo-stability. By reducing cellular glutathione content during the loading procedure, in order to preserve the disulfide bonds opportunely created to stabilize the enzyme dimer structure, it was possible to produce an encapsulated protein maintaining 100% stability at least after 4 h at 37°C. Moreover, E196-301-loaded RBCs were efficiently able to degrade cocaine in a time- and concentration-dependent manner. The same stability results were obtained when murine RBCs were used paving the way to preclinical investigations. Thus, our in vitro data show that E196-301-loaded RBCs could act as efficient bioreactors in degrading cocaine to non-toxic metabolites to be possibly considered in substance-use disorder treatments. This approach should now be investigated in a preclinical model of cocaine use disorder to evaluate if further protein modifications are needed to further improve long term enzyme stability.

Red blood cells (RBC) are actually exploited as innovative drug delivery systems with unconventional and convenient properties. Because of a long in vivo survival and a non-random removal from circulation, RBC can be loaded with drugs and/or contrasting agents without affecting these properties and maintaining the original immune competence. However, native or drug-loaded RBC, can be modified decorating the membrane with peptides, antibodies or small chemical entities so favoring the targeting of the processed RBC to specific cells or organs. Convenient modifications have been exploited to induce immune tolerance or immunogenicity, to deliver antibodies capable of targeting other cells, and to deliver a number of constructs that can recognize circulating pathogens or toxins. The methods used to induce membrane processing useful for biomedical applications include the use of crosslinking agents and bifunctional antibodies, biotinylation and membrane insertion. Another approach includes the expression of engineered membrane proteins upon ex vivo transfection of immature erythroid precursors with lentiviral vectors, followed by in vitro expansion and differentiation into mature erythrocytes before administration to a patient in need. Several applications have now reached the clinic and a couple of companies that take advantage from these properties of RBC are already in Phase 3 with selected applications. The peculiar properties of the RBC and the active research in this field by a number of qualified investigators, have opened new exciting perspectives on the use of RBC as carriers of drugs or as cellular therapeutics.