Covalent Attachment of Superoxide Dismutase to Phospholipid Membranes: A Nanoarchitectonics Strategy for Preparing Liposome–Enzyme Complexes

Superoxide dismutase (SOD) is a biologically important antioxidant enzyme and various strategies have been employed to immobilize SOD at material interfaces. However, there has been limited exploration of SOD attachment to lipid membranes despite emerging interest in SOD-liposome systems. Addressing this gap would improve physicochemical understanding of how attachment chemistry affects SOD-membrane interactions and could guide practical applications. Herein, we explored two strategies for attaching SOD to supported lipid bilayer (SLB) platforms in phosphate-buffered saline (PBS) conditions by comparing covalent and noncovalent approaches based on maleimide–thiol chemistry and electrostatic-mediated adsorption, respectively. Quartz crystal microbalance-dissipation (QCM-D) experiments revealed stable SOD attachment to SLBs via the covalent strategy, whereas there was minimal noncovalent adsorption on positively charged SLBs despite the net negative charge of SOD. These results led us to further investigate the covalent strategy by preparing SOD-liposome complexes. Dynamic light scattering (DLS) and electrophoretic mobility measurements identified a suitable range of enzyme-to-lipid ratios where colloidal stability was maintained. At higher ratios, the onset of apparent liposome aggregation coincided with decreased zeta potential magnitude. Importantly, liposome-conjugated SOD retained enzymatic activity, demonstrating that maleimide–thiol chemistry is an effective strategy to covalently conjugate SOD to lipid membrane interfaces and prepare colloidally stable liposome-enzyme complexes that exhibit antioxidant activity. Based on these findings, we discuss how charge screening likely aids the covalent strategy while attenuating noncovalent adsorption, highlighting the interplay between chemical conjugation and interfacial forces to modulate SOD attachment to lipid membranes.