Molecular insights into the gastrointestinal fate of multicomponent liposomes within a WPI–chitosan nanocarrier

• Nutraceuticals and biopolymers critically modulate liposome bilayer properties. • pH and enzymes drive distinct structural changes during gastrointestinal transit. • Controlled, location-specific release of liposomes occurs in the GIT. • Mucoadhesion shifts from strong (stomach) to weak (intestine), aiding uptake. • Bile salts transform liposomes into nano-bilosomes, enhancing bioavailability. Liposomes have emerged as attractive delivery systems due to their ability to co-encapsulate synergistic nutraceutical combinations. This study investigates a whey protein isolate (WPI)–chitosan electrostatic nanocomplex designed to enhance the stability and control the gastrointestinal bioaccessibility of phosphatidylcholine (PC) liposomes. These liposomes are co-loaded with a combination of nutraceuticals: long-chain omega-3 polyunsaturated fatty acids from fish oil, β-carotene, and eugenol (a key component of clove essential oil). The loading efficiency of the nutraceutical combination into PC liposomes and the encapsulation efficiency of the liposomes within the WPI-chitosan matrix were calculated spectrophotometrically, yielding values of 98 ± 3% and 83 ± 6%, respectively. Transmission electron microscopy showed the nanoscale size and spherical shape of both the co-loaded liposomes and their biopolymer-encapsulated forms. Multi-mode laser light scattering revealed key physicochemical parameters (weight-average molar mass ( M w ), radius of gyration ( R G ), hydrodynamic radius ( R h ), ζ-potential, architecture (ρ), density, second virial coefficient) of both the binary (WPI-liposome) and ternary (WPI-liposome-chitosan) complexes including ζ-potential and R h of the liposomes. Electron paramagnetic resonance spectroscopy revealed key intermolecular interactions governing the structural state of the lipid bilayer in both free and biopolymer-encapsulated liposomes. The structural evolution of the ternary complex and released liposomes was subsequently monitored under simulated gastrointestinal conditions. The high particle density of the ternary complex (6.3 mg/cm³) ensured protection of the liposomes and facilitated a two-stage release: gastric retention (16% bioaccessibility) followed by intestinal liberation (84% bioaccessibility), governed by mucoadhesion and bile-salt-mediated disruption. This design ensures targeted delivery to the intestinal site of absorption.