Hydroxytyrosol‑Loaded Zein Nanoparticles: A Versatile Formulation for Pharmaceutical and Nutraceutical Applications

Hydroxytyrosol (Htyr) is one of the most bioactive phenolic compounds derived from olive by‑products and is widely recognized for its antioxidant, anti‑inflammatory and protective biological activities. Despite its relevance, its use in functional foods or nutraceutical formulations is hindered by poor stability, rapid metabolism and limited absorption. To address these issues, an eco‑friendly nanoformulation was developed using zein (2 mg/mL), a GRAS corn protein, stabilized with sodium deoxycholate (SD, 1.25% w/v) and loaded with Htyr (0.2–2.0 mg/mL). Empty nanoparticles showed a mean diameter of approximately 100 nm, low polydispersity and a negative zeta potential (~ –30 mV). The addition of Htyr at 1.6 mg/mL produced nanosystems with an entrapment efficiency of ~60% (≈1.0 mg/mL retained) and a loading capacity of ~25%. FT‑IR spectroscopy indicated that hydrogen bonding and electrostatic interactions were the main stabilizing forces within the formulation. The nanoparticles preserved their structural integrity after pasteurization (65 °C, 30 min), heating up to 95 °C and exposure to ionic strengths up to 150 mM NaCl. Simulated gastrointestinal assays confirmed effective protection of Htyr in gastric fluid (~7% release at pH 1.2 with pepsin) and a controlled release profile under intestinal conditions (~40% at pH 6.8 with pancreatin). In CaCo‑2 cells treated with H₂O₂, Htyr‑loaded nanoparticles enhanced cell viability up to 80% (at 20 µM), outperforming free Htyr (73%), likely due to synergistic radical‑scavenging interactions between the bioactive compound and the zein matrix. Bioactivity preservation was further supported by the ORAC assay.  Overall, this study highlights the potential of zein nanoparticles as a versatile formulation able of improving the stability and functional performance of Htyr. The combination of natural materials, robustness under processing conditions and preservation of bioactivity supports their application in next‑generation pharmaceuticals, nutraceuticals and fortified foods enriched with phenolic antioxidants.