Catalytic Antibody Blunts Carfentanil-Induced Respiratory Depression

Carfentanil, the most potent of the fentanyl analogues, is at the forefront of synthetic opioid-related deaths, second to fentanyl. Moreover, the administration of the opioid receptor antagonist naloxone has proven inadequate for an increasing number of opioid-related conditions, often requiring higher/additional doses to be effective, as such interest in alternative strategies to combat more potent synthetic opioids has intensified. Increasing drug metabolism would be one strategy to detoxify carfentanil; however, carfentanil’s major metabolic pathways involve N-dealkylation or monohydroxylation, which do not lend themselves readily to exogenous enzyme addition. Herein, we report, to our knowledge, the first demonstration that carfentanil’s methyl ester when hydrolyzed to its acid was found to be 40,000 times less potent than carfentanil in activating the μ-opioid receptor. Physiological consequences of carfentanil and its acid were also examined through plethysmography, and carfentanil’s acid was found to be incapable of inducing respiratory depression. Based upon this information, a hapten was chemically synthesized and immunized, allowing the generation of antibodies that were screened for carfentanil ester hydrolysis. From the screening campaign, three antibodies were found to accelerate the hydrolysis of carfentanil’s methyl ester. From this series of catalytic antibodies, the most active underwent extensive kinetic analysis, allowing us to postulate its mechanism of hydrolysis against this synthetic opioid. In the context of potential clinical applications, the antibody, when passively administered, was able to reduce respiratory depression induced by carfentanil. The data presented supports further development of antibody catalysis as a biologic strategy to complement carfentanil overdose reversal

Reductively Responsive siRNA-Conjugated Hydrogel Nanoparticles for Gene Silencing

A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to target tissues and cells. Self-assembled lipid- and polymer-based systems have been most extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer therapies. Safety and compatibility of materials implemented in delivery systems must be ensured to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions, prepared via a particle molding process that is a unique off-shoot of soft lithography known as particle replication in nonwetting templates (PRINT), were explored in these studies as delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after systemic administration or during postfabrication processing for surface functionalization, a polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared. Triggered release of siRNA from the pro-drug hydrogels was observed under a reducing environment while cargo retention and integrity were maintained under physiological conditions. Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs, accompanied by minimal cell death. Further investigation into shape, size, and surface decoration of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies