Efficient Cocaine Degradation by Cocaine Esterase-Loaded Red Blood Cells.

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.

Melatonin protects hippocampal HT22 cells from the effects of serum deprivation specifically targeting mitochondria.

Neurons contain a high number of mitochondria, these neuronal cells produce elevated levels of oxidative stress and live for a long time without proliferation; therefore, mitochondrial homeostasis is crucial to their health. Investigations have recently focused on mitochondrial dynamics revealing the ability of these organelles to change their distribution and morphology. It is known that mitochondrial fission is necessary for the transmission of mitochondria to daughter cells during mitosis and mitochondrial fragmentation has been used as an indicator of cell death and mitochondrial dysfunction. Oxidative stress is a trigger able to induce changes in the mitochondrial network. The aim of the present study was to determine the effects of melatonin on the mitochondrial network in HT22 serum-deprived cells. Our results showed that serum deprivation increased reactive oxygen species (ROS) content, promoted the activation of plasma membrane voltage-dependent anion channels (VDACs) and affected the expression of pDRP1 and DRP1 fission proteins. Moreover, parallel increases in apoptotic and autophagic features were found. Damaged and dysfunctional mitochondria are deleterious to the cell; hence, the degradation of such mitochondria through mitophagy is crucial to cell survival. Our results suggest that melatonin supplementation reduces cell death and restores mitochondrial function through the regulation of autophagy.

A new therapy prevents intellectual disability in mouse with phenylketonuria.

Untreated phenylketonuria (PKU) results in severe neurodevelopmental disorders, which can be partially prevented by an early and rigorous limitation of phenylalanine (Phe) intake. Enzyme substitution therapy with recombinant Anabaena variabilis Phe Ammonia Lyase (rAvPAL) proved to be effective in reducing blood Phe levels in preclinical and clinical studies of adults with PKU. Aims of present study were: a) to gather proofs of clinical efficacy of rAvPAL treatment in preventing neurological impairment in an early treated murine model of PKU; b) to test the advantages of an alternative delivering system for rAvPAL such as autologous erythrocytes. BTBR-Pahenu2-/- mice were treated from 15 to 64 post-natal days with weekly infusions of erythrocytes loaded with rAvPAL. Behavioral, neurochemical, and brain histological markers denoting untreated PKU were examined in early treated adult mice in comparison with untreated and wild type animals. rAvPAL therapy normalized blood and brain Phe; prevented cognitive developmental failure, brain depletion of serotonin, dendritic spine abnormalities, and myelin basic protein reduction. No adverse events or inactivating immune reaction were observed. In conclusion present study testifies the clinical efficacy of rAvPAL treatment in a preclinical model of PKU and the advantages of erythrocytes as carrier of the enzyme in term of frequency of the administrations and prevention of immunological reactions.

Reengineering red blood cells for cellular therapeutics and diagnostics.

Recently optimized technologies that permit the reversible opening of nanopores across the red blood cell membrane, give the extraordinary opportunity for reengineering human erythrocytes to be used in different biomedical applications, both for therapeutic and diagnostic purposes. Engineered erythrocytes have been exploited as a system for the controlled release of drugs in circulation upon encapsulation of prodrugs or small molecules; as bioreactors when they are endowed of recombinant enzymes able to catalyze the conversion of toxic metabolite into inert products; as drug targeting system for the delivery of compounds to the reticuloendothelial system inducing proper senescent signals on the drug-loaded erythrocyte membrane; as carrier of contrasting agents for diagnostic procedures. Preclinical development of these different applications has taken advantage from the use of proper animal models whose erythrocytes can be reengineered as the human ones or the encapsulation procedures can be adapted on the basis of their specific erythrocyte biological features. Successful results, obtained both in vitro and in preclinical studies, have prompted several clinicians to start pilot clinical investigations in different conditions and some new companies to start the industrialization of selected loading technologies and to initiate clinical development programs. This short review summarizes the key features that, to the best of our knowledge, have been crucial to advance the products toward regulatory clinical approval making reengineering of erythrocytes a modality to treat patients with limited or absent therapeutic options. WIREs Nanomed Nanobiotechnol 2017, 9:e1454. doi: 10.1002/wnan.1454 For further resources related to this article, please visit the WIREs website.

Engineering erythrocytes for the modulation of drugs' and contrasting agents' pharmacokinetics and biodistribution.

Pharmacokinetics, biodistribution, and biological activity are key parameters that determine the success or failure of therapeutics. Many developments intended to improve their in vivo performance, aim at modulating concentration, biodistribution, and targeting to tissues, cells or subcellular compartments. Erythrocyte-based drug delivery systems are especially efficient in maintaining active drugs in circulation, in releasing them for several weeks or in targeting drugs to selected cells. Erythrocytes can also be easily processed to entrap the desired pharmaceutical ingredients before re-infusion into the same or matched donors. These carriers are totally biocompatible, have a large capacity and could accommodate traditional chemical entities (glucocorticoids, immunossuppresants, etc.), biologics (proteins) and/or contrasting agents (dyes, nanoparticles). Carrier erythrocytes have been evaluated in thousands of infusions in humans proving treatment safety and efficacy, hence gaining interest in the management of complex pathologies (particularly in chronic treatments and when side-effects become serious issues) and in new diagnostic approaches.