Centrifugation-based assay for examining nanoparticle–lipid membrane binding and disruption

Abstract

Centrifugation-based assays are commonly employed to study protein–membrane affinity or binding using lipid bilayer vesicles. An analogous assay has been developed to study nanoparticle–membrane interactions as a function of nanoparticle surface functionalization, membrane lipid composition, and monovalent salt concentration (NaCl). Anionic (carboxylic acid, Ag–COOH), cationic (amine, Ag–NH), and polyethylene glycol coated (Ag–PEG) silver nanoparticles (AgNPs) were examined based on their surface plasmon resonance (SPR), which was used to determine the degree of binding to anionic, cationic, and zwitterionic membrane vesicles by analyzing supernatant and sediment phases. SPR was also used to examine AgNP aggregation in solution and at membrane–water interfaces, and direct visualization of AgNP–membrane binding, vesicle aggregation, and vesicle disruption was achieved by cryogenic transmission electron microscopy (cryo-TEM). The extent of AgNP binding, based on AgNP + vesicle heteroaggregation, and vesicle disruption was dependent upon the degree of electrostatic attraction. Because of their biological and environmental relevance, Ag–PEG + anionic vesicles systems were examined in detail. Cryo-TEM image analysis was performed to determine apparent membrane–water partition coefficients and AgNP aggregation states (in solution and bound to membranes) as a function of NaCl concentration. Despite possessing a PEG coating and exhibiting a slight negative charge, Ag–PEG was able to bind to model anionic bacterial membranes either as individual AgNPs (low salt) or as AgNP aggregates (high salt). The centrifugation assay provides a rapid and straightforward way to screen nanoparticle–membrane interactions.