Development of zein-based nanoparticles for oral co-delivery of insulin and liraglutide and preliminary in vivo evaluation in Danio rerio.

Oral delivery of peptide-based therapeutics remains a major challenge due to enzymatic degradation and poor intestinal permeability. This study reports the development of hybrid zein-Eudragit RS100-chitosan nanoparticles for the co-delivery of insulin and liraglutide (Z-ERS-CS/I-LIRA), aiming to protect both peptides and support their oral administration. The nanoparticles were prepared by nanoprecipitation and exhibited suitable physicochemical properties, including nanoscale size (~230 nm), low polydispersity (PDI < 0.1), and positive zeta potential (~ + 46 mV). Structural and thermal analyses (FTIR, XRD, TGA, and DSC) revealed the formation of a predominantly amorphous and molecularly dispersed system, indicating strong interactions among the polymeric components and effective incorporation of both peptides. The formulation remained stable under refrigerated conditions, showed pronounced mucoadhesive behavior after interaction with mucin, and displayed a biphasic release profile in PBS (pH 7.4), with cumulative release values of approximately 87% for insulin and 58% for liraglutide after 48 h. Under simulated gastrointestinal conditions, minimal peptide release (<15%) was observed, suggesting protection against harsh gastric and intestinal environments. In adult zebrafish, unloaded nanoparticles showed no mortality or significant toxicological alterations, supporting preliminary biocompatibility. In a glucose-induced hyperglycemia model, Z-ERS-CS/I-LIRA reduced blood glucose to levels comparable to healthy controls, preserving antidiabetic activity after encapsulation. However, hemorrhagic and pancreatic alterations in liraglutide-containing groups indicate that further liraglutide dose optimization is required to minimize adverse effects. Overall, Z-ERS-CS/I-LIRA combine peptide protection, mucoadhesion, prolonged release, preliminary in vivo biocompatibility, and antidiabetic activity, representing a promising platform for oral peptide-based combination therapy.