Hydrophobic Ion Pairing Improves Protein Thermal Stability and Lipid Nanocarrier Integration through Controlled Surface Modification

Integrating therapeutic proteins into lipid-based nanocarriers remains challenging due to fundamental incompatibilities between hydrophilic protein surfaces and lipophilic carrier matrices. Here, we demonstrate that controlled hydrophobic ion pairing (HIP) using lysozyme-sodium dodecyl sulfate (SDS) at stoichiometric ratios simultaneously improves thermal stability during pharmaceutical processing and lipid compatibility for nanocarrier integration. Building on our previous molecular characterization of lysozyme-SDS complexes, we investigated their application in nanostructured lipid carrier (NLC) formulations for oral protein delivery. SDS complexation preserved lysozyme enzymatic activity at processing-relevant temperatures and increased apparent lipid miscibility by 50% in both solid and liquid pharmaceutical lipid excipients. This enhanced compatibility translated directly into superior NLC performance, with complexed lysozyme achieving a 4-fold higher encapsulation efficiency (79.6 ± 1.8% vs 18.4 ± 4.3% for native protein) with improved batch-to-batch reproducibility. In simulated gastrointestinal conditions, lysozyme-SDS NLCs demonstrated sustained intestinal release (87.2 ± 16.5% over 6 h) with 67.5 ± 7.5% enzymatic activity retained compared to only 15.2 ± 3.0% activity for native lysozyme formulations. Mechanistic analysis from differential scanning calorimetry and crystallinity measurements showed that SDS complexation induces a molten globule-like protein state that reduces lipid packing (56.8% vs 68.6% crystallinity for native lysozyme), enabling superior matrix integration while maintaining biological function. These findings establish that controlled surface modification through HIP provides a systematic approach to overcome protein–lipid incompatibilities, offering a generalizable framework for developing lipid-based protein therapeutics.