Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The aqueous-core enclosed in lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water mimic, at least in theory, the environment within viable cells, thus being suitable for housing hydrophilic protein entities such as bioactive proteins, peptides and bacteriophage particles. This study reports a complete physicochemical characterization of optimized biomimetic aqueous-core lipid nanoballoons housing hydrophilic (BSA) protein entities, evolved from a statistical 2(3)x3(1) factorial design study (three variables at two levels and one variable at three levels) that was the subject of the first paper of a series of three, aiming at complete stabilization of the three-dimensional structure of protein entities attempted via housing the said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion. The statistical factorial design followed led to the production of an optimum W/O/W multiple emulsion possessing quite homogeneous particles with an average hydrodynamic size of (186.2 +/- 2.6) nm and average Zeta potential of (-36.5 +/- 0.9) mV, and exhibiting a polydispersity index of 0.206 +/- 0.014. Additionally, the results obtained for the diffusion coefficient of the lipid nanoballoons integrating the optimized W/O/W multiple emulsion were comparable and of the same order of magnitude (10(-12) m(2)s(-1)) as those published by other authors since, typically, diffusion coefficients for molecules range from 10(-10) to 10(-7) m(2)s(-1), but diffusion coefficients for nanoparticles are typically of the order of magnitude of 10(-12) m(2)s(-1).421219902000CNPq, National Council for Scientific and Technological Development - Brazil [306113/2014-7, 309598/2014-1]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)