Evaluating the Consequences of Adsorption-Induced Structural Perturbations of Proteins

Abstract

Maintaining the stability of a biologically-active therapeutic agent throughout its lifecycle is a critical parameter in successful pharmaceutical formulation. Interfacial effects such as surface adsorption, which can occur during drug storage and delivery, may culminate in various forms of instabilities: protein unfolding, activity loss and population of non-native, aggregate-prone states. A central paradigm that underpins our understanding of proteins with solid surfaces is that protein adsorption leads to changes in secondary structure. Bound proteins tend to denature and these non-native, adsorbed structures are likely stabilized by loss of alpha-helices and concomitant formation of intermolecular beta-sheets. This research seeks to critically assess the impact this behavior has on protein desorption, where irreversible conformational change may lead to aggregation or other forms of instability. We employ a robust study design to examine the kinetics of adsorption, desorption, and structural transitions of lysozyme on fumed silica nanoparticles as a function of surface coverage. We use circular dichroism (CD) spectroscopy to monitor structural transitions on the surface, in situ. The results show that despite significant adsorption-induced structural loss, surprisingly, adsorption is reversible, and protein desorption is predictable in a coverage-dependent manner. We find evidence of a two-state model, involving exchange between a native-like dissolved and highly perturbed adsorbed state. Since the in situ circular dichroism (CD) derived secondary structure of the adsorbed proteins are essentially unaffected by changes in surface coverage, these results are not consistent with previous claims that surface-induced denaturation is coverage dependent. Inspired by results from homopolymer adsorption experiments, we speculate that more local descriptors, such as the number of amino acids per chain that are physically adsorbed on the surface, likely control the desorption process.