Development and characterization of lactoferrin-GMP nanohydrogels: Evaluation of pH, ionic strength and temperature effect

• Lactoferrin and glycomacropeptide were used to produce stable nanohydrogels.
• Interactions between proteins proved be extremely relevant in design of nanohydrogels.
• Increasing gelation temperature led to a faster and more extensive aggregation of the mixture.
• Interactions between proteins depend not only upon the protein concentration but also upon their ratio of mixture.
• Effect of environmental conditions were evaluated on Lf-GMP nanohydrogels stability.

Lactoferrin (Lf) and glycomacropeptide (GMP), two bioactive proteins from milk, were used to produce nanohydrogels by electrostatic interaction and thermal gelation. Parameters such as protein concentration, molar ratio, pH, temperature and heating time were evaluated in order to develop the nanohydrogels, which were characterized in terms of morphology (Transmission Electron Microscopy – TEM, Dynamic Light Scattering – DLS, Atomic Force Microscopy – AFM and Confocal Laser Scanning Microscopy – CLSM) and stability. The aggregation of the mixture between Lf and GMP increased with the increase of temperature, resulting in particles with lower hydrodynamic diameter and polydispersity index (PdI). Nanohydrogels were obtained from the mixture of Lf and GMP solutions (0.02% (w/w) in molar ratio 1:7) at pH 5.0, and subsequently stirred and heated at 80 °C, during 20 min. Results showed that nanohydrogels have a spherical shape with a hydrodynamic diameter around 170 nm, a PdI of 0.1 and a swelling ratio of 30. The minimum size of nanohydrogels depends on protein concentration and molar ratio. Decreasing the protein concentrations and increasing the content of GMP molecules in solution, the hydrodynamic diameter and PdI of nanohydrogels decreased. The electrical charge values of nanohydrogels at different pH values suggest that Lf molecules are in the surface and GMP molecules are mostly in the core of the structure (confirmed by confocal microscopy). Also, it was observed that nanohydrogels' hydrodynamic diameter and PdI, after formation, are influenced when submitted at different conditions of pH, ionic strength and temperature. Lf-GMP nanohydrogels showed properties that indicate that they are a promising delivery system for food and pharmaceutical applications.

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