AM1-Stabilized PEG-Modified Nanoemulsions for Interfacial Engineering and Biomolecule Conjugation

Nanoemulsions (NEs) are promising platforms for biomolecule delivery but are limited by poor colloidal stability and insufficient surface control under physiological conditions. This study aimed to engineer peptide-stabilized, tailorable nanoemulsions (TNEs) with improved stability using systematic polyethylene glycol (PEG) interfacial modification. Oil-in-water NEs were stabilized using the AM1 peptide and modified with 2 kDa PEG in linear, arm, and hybrid configurations. Physicochemical characterization included interfacial tension measurements, dynamic light scattering, and zeta potential analysis. Stability was evaluated under ionic, chelating, serum, and cell-culture conditions and during storage at 4 and 25 °C. Apparent dye retention was assessed using the hydrophobic fluorescent dye DiI. Surface functionalization was examined via the adsorption of CpG-ODN1826, and cellular interactions were studied using peripheral blood mononuclear cells. AM1 reduced squalene-water interfacial tension from 26.05 to 12.04 mN/m, generating positively charged droplets of approximately 168 nm that destabilized in phosphate-buffered saline and EDTA. PEGylation yielded droplets of approximately 179-183 nm with reduced surface charge and improved stability. The hybrid P200-A-P200-L formulation remained stable for 28 days across all tested media. Arm and hybrid PEGylated TNEs showed 96-99% apparent dye retention over 5 days, whereas unmodified AM1-TNEs exhibited a rapid decrease in DiI fluorescence signal and pronounced swelling. CpG adsorption increased droplet size and reversed surface charge, consistent with effective oligonucleotide functionalization. In cellular assays, arm and hybrid PEG coatings reduced nonspecific uptake and enhanced cellular association after CpG functionalization. Peptide-stabilized nanoemulsions with tuned PEG architectures achieve enhanced stability, apparent cargo retention, and selective cellular interactions for drug and nucleic acid delivery.