Physicochemical stability, antioxidant and ACE-inhibitory activities of cryoprotected liposomes encapsulating stone fish-derived peptides

This study investigates the physicochemical stability, antioxidant activity, and ACE-inhibitory effects of cryoprotected liposomes encapsulating bifunctional peptides derived from stone fish, with unencapsulated peptides serving as a positive control. Microcapsular liposomal suspensions were prepared using the thin lipid film hydration method and classified into an empty liposomes (F1) , peptide-loaded liposomes without cryoprotectants (F2), and peptide-loaded liposomes with sucrose (F3) or trehalose (F4) as cryoprotectants. The suspension formulations were evaluated for particle size (PS), zeta potential (ZP), encapsulation efficiency (EE), and bioactivity before and after storage at 4 °C and 25 °C for eight weeks. Liposomal formulations ( F3 and F4 ) showed significantly higher ( p < 0.05) physicochemical stability , with smaller PS at 4 °C (14.49±0.16 µm for F3, 15.48±0.26 µm for F4) and 25 °C (26.69±0.18 µm for F3, 23.90±0.19 µm for F4) , while F2 exhibited aggregation with PS of 28.20±0.12 µm at 4 °C, 56.61±0.40 µm at 25 °C . Zeta potential was higher in F3 (-42.00±4.96 mV) and F4 (-39.42±3.12 mV) than in F2 (-32.45±2.21 mV) at 25 °C post-storage, indicating greater electrostatic stability. Encapsulation efficiency was significantly higher ( p < 0.05) in F3 (70.57±2.69 % at 4 °C, 46.65±3.44 % at 25 °C) and F4 (65.69±2.00 % at 4 °C, 59.55±2.37 % at 25 °C) compared to F2 (44.89±2.55 % at 4 °C, 35.10±2.77 % at 25 °C) post-storage. Bioactivity retention was significantly higher ( p < 0.05) in F3 and F4, with enhanced DPPH * scavenging , Fe²⁺-chelating, and ACE-inhibitory activities at both 4 °C and 25 °C compared to F2. Structural analyses of the lyophilized liposomes using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) confirmed the formation of stable peptide-phospholipid complexes, with higher thermal stability in F3 and F4, as indicated by a higher thermal temperature of 36.62 °C and 59.58 °C, compared to 20.13 °C in F2.These findings demonstrate the critical role of liposomal encapsulation and cryoprotection in preserving bioactive peptides, indicating their potential for functional foods and therapeutic applications targeting oxidative stress and hypertension.