Hydrophobic ion pairing as a rational surface engineering strategy for enhanced protein integration within lipid-based nanocarriers

Abstract

Hydrophobic ion pairing is an established solubility engineering strategy that uses surfactants to modulate drug lipophilicity and facilitate encapsulation in lipid-based nanocarriers. Although promising for enhancing lipophilicity, its influence on protein stability, activity, and interactions with lipids remains unclear at the molecular level. In this study, lysozyme was used as a model protein, and ion paired with two surfactants, sodium dodecyl sulphate (SDS) and dioctyl sodium sulfosuccinate (DOSS). The impact of surfactant complexation on lysozyme solubility, activity, and structure was investigated at various charge ratios. An up-to-2-fold increase in lysozyme catalytic activity was observed alongside enhanced lipophilicity upon complexation with SDS at sub-stoichiometric ratios, suggesting that favourable conformational changes, driven by partial protein unfolding, led to higher substrate accessibility. To understand the mechanism of protein release for downstream formulation design, an ion exchange chromatography method was developed and validated using the ÄKTA start fast protein liquid chromatography system for accurate quantification of lysozyme–surfactant complexes dissociation as a function of pH, ionic strength, and simulated intestinal fluid components, with lysozyme–SDS complex showing greater susceptibility to dissociation. The next step involved the incorporation of the lysozyme–SDS complex into nanostructured lipid carriers (NLCs). By complexing lysozyme with SDS at stoichiometric ratio, an improved thermal stability at pharmaceutical processing temperature and a 2-fold increase of apparent lipid solubility in both solid and liquid lipid excipients were observed. The enhanced lipid compatibility translated directly into superior NLC performance, with the lysozyme–SDS complex exhibiting a 4-fold increase in encapsulation efficiency compared to native lysozyme. Under enzyme-free simulated gastrointestinal conditions, lysozyme–SDS NLCs exhibited sustained intestinal release and retained enzymatic activity. These findings highlight the potential of controlled surface modification to resolve protein–lipid incompatibilities while preserving biological function, establishing a versatile framework for the delivery of therapeutic proteins. Thesis is embargoed until 31 July 2027.