Gamma-oryzanol Stabilized Nanoliposomes for Enhanced Delivery of Hempseed Protein Hydrolysate: Physicochemical Characterization and Antioxidant Potential for Nutraceutical Applications

Background: Hempseed protein hydrolysate (HPH) exhibits potent antioxidant activity but suffers from physicochemical instability and low bioavailability, limiting its pharmaceutical and nutraceutical applications. Sterol stabilizers can liposomal membrane integrity, yet comparative studies on their effects in HPH-loaded nanoliposomes remain limited. Objective: To develop and characterize HPH-loaded nanoliposomes (HPH-NLs) stabilized with cholesterol (CHO), curcumin (CURC), and γ-oryzanol (GO), and to evaluate their physicochemical properties, encapsulation efficiency, structural characteristics, and antioxidant activity. Materials and Methods: HPH was produced from hemp protein concentrate via pepsin-catalyzed enzymatic hydrolysis. Nanoliposomes were prepared using the thin-film hydration-sonication method. Formulations were characterized for particle size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE%), and morphology (SEM). Structural interactions were analyzed by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Antioxidant activity was monitored over 30-day storage. Results: Enzymatic hydrolysis significantly enhanced HPH antioxidant activity compared to non-hydrolyzed protein (DPPH: 42.3% vs. 35.8%; ABTS⁺: 41.1% vs. 39.1%). All formulations exhibited nanoscale dimensions (<110 nm). GO-stabilized nanoliposomes (GO-NL) demonstrated superior characteristics: smallest particle size (70 ± 5 nm), lowest PDI (0.183), highest EE% (92 ± 2.1%), and greatest retention after 30 days (89.5%). FTIR confirmed HPH encapsulation through amide I/II bands. DSC revealed that GO eliminated the cooperative gel-to-liquid crystalline phase transition, indicating enhanced membrane stability. During storage, GO-NL maintained 90.5% of DPPH scavenging activity, significantly outperforming CURC-NL and CHO-NL (P ≤ 0.05). Conclusions: GO functions as a dual-action stabilizer, simultaneously enhancing membrane integrity and providing antioxidant protection. GO-stabilized HPH nanoliposomes demonstrate enhanced physicochemical stability and antioxidant retention under in vitro storage conditions. These findings support further investigation of this system for potential nutraceutical applications, pending evaluation of release kinetics, gastrointestinal stability, and cellular uptake.