Development of cocaine esterase W/O/W nanoemulsions by a novel low-temperature double emulsification approach for cocaine abuse treatment.

Cocaine esterase is highly effective against cocaine but has a short in vivo half-life and is sensitive to temperature, limiting its clinical use. Encapsulation of cocaine esterase in water-in-oil-in-water (W/O/W) nanoemulsions offers a promising approach to improve its stability and therapeutic effectiveness. In this study, we developed a novel low-temperature double emulsification method to encapsulate the cocaine esterase mutant (E196-301) into W/O/W nanoemulsions (designated as CocE NEs). The oil phase, containing phospholipids, was cooled to room temperature under high-speed stirring before the enzyme-containing aqueous phase was added. This process prevented the precipitation of soybean phospholipids and preserved over 90 % of E196-301's enzymatic activity. Notably, the temperature stability of E196-301 has been greatly improved, E196-301 within the inner aqueous phase maintained over 90 % of its activity under temperature variations between 37 °C and 40 °C. Importantly, the in vivo half-life of E196-301 increased from 16.26 ± 1.94 min to 57.25 ± 14.71 min (3 mg/kg, i.v.). Pharmacodynamic studies revealed CocE NEs (3 mg/kg, i.v.) significantly reduced cocaine-induced locomotor sensitization in mice within 45 min, by attenuating cocaine-induced (25 mg/kg, i.p.) dopamine signaling in the brain. CocE NEs represent a promising candidate for the sustained prevention and treatment of cocaine abuse.