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.The authors Ana I. Bourbon, Ana C. Pinheiro, Ricardo N. Pereira and Miguel A. Cerqueira are recipient of fellowships from the Fundacao para a Ciencia e Tecnologia, POPH-QREN and FSE (FCT, Portugal) through grants SFRH/BD/73178/2010, SFRH/BD/48120/2008, SFRH/BPD/81887/2011 and SFRH/BPD/72753/2010, respectively. Maria G. Carneiro-da-Cunha express is gratitude to the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) for research grant. The authors would like to acknowledge to Jorge Padrao from Centre of Biological Engineering, University of Minho and Andre Coelho from Federal University of Ceara for helping in electrophoresis measurements and to Rui Fernandes from IBMC, University of Porto for assistance in taking the TEM pictures. Also, the authors thank the FCT Strategic Projects PEst-OE/EQB/LA0023/2013 and PEst-C/EQB/LA0006/2013 (FCT and FEDER funds through COMPETE). The work also received financial support from the European Union (FEDER funds) under the framework of QREN (Programa Operacional Regional do Norte (ON.2 - O Novo Norte; FEDER) through projects NORTE-07-0124-FEDER-000028 and NORTE-07-0124-FEDER-000069
