Therapeutic protein delivery in reverse micelles: influence of surfactant chemistry on payload stability

Reverse micelles (RM) have emerged as versatile supramolecular assemblies for the lipophilization of hydrophilic biomolecules, facilitating their incorporation into lipid-based drug delivery systems. However, the influence of surfactant chemistry on protein stability within RM remains poorly understood. In this study, a library of ten surfactants representing anionic, cationic, zwitterionic, and nonionic headgroup classes was systematically investigated to establish structure-stability relationships. Following determination of the reverse critical micelle concentration (rCMC), horseradish peroxidase (HRP), elastase (ELA), and papain (PAP) were encapsulated into RM by dry loading. Protein payload, partition behavior, and structural integrity were evaluated over 48 h, while an independent Zein fluorescence assay was employed to validate surfactant-induced protein denaturation. Although all surfactants formed stable RM and enabled successful protein encapsulation, pronounced differences in protein preservation were observed. Across all three proteins, a highly consistent denaturation ranking was established (DDAB > SLES > SOS > AOT > DEHP > So-Myr > SL > P2-OE > SMO > PG-3), demonstrating that surfactant chemistry is one significant determinant of protein stability within RM. This ranking was independently confirmed by the Zein assay and remained unchanged upon replacement of the continuous oil phase, indicating that protein denaturation originates primarily from surfactant-dependent interfacial interactions rather than the surrounding oil. These findings establish a systematic framework for the rational selection of surfactants in protein-compatible reverse micellar formulations and provide practical design principles for the development of lipid-based drug delivery systems for biologics.