Poly(ortho ester) (POE) nanoparticles were prepared by two different emulsification techniques: novel double emulsion solvent diffusion (DESD) and double emulsion solvent evaporation (DESE). We investigated the effect of sonication time/speed, type of organic phase solvents, polymer concentration, organic/aqueous phase ratio, surfactant type and concentration on the mean particle sizes of obtained POE nanoparticles by both DESD and DESE methods. Different organic solvents [ethyl acetate, methyl ethyl ketone, acetone, dichloromethane and chloroform] were used with several stabilizers [pluronic F 68, didodecyl dimethyl ammonium bromide, poly(vinyl alcohol), and sodium dodecyl sulfate]. The particle size of nanoparticles was observed by the dynamic light scattering method and transmission electron microscopy. We assumed that combining the double emulsion system with a partially water-soluble organic solvent, ethyl acetate, would result in better encapsulation efficiency of water-soluble drugs in nanoparticles and the utilization of both biocompatible surfactants and solvents. As a model drug, we used vancomycin hydrochloride, a hydrophilic low molecular weight glycopeptide antibiotic. The new nanoparticle preparation technique, DESD, resulted in improved formulation characteristics including smaller size, lower size distribution, higher encapsulation yield, and more biocompatible ingredients with unaltered bioactivity of vancomycin hydrochloride in comparison to classical DESE method used to load hydrophilic molecules. (C) 2013 Elsevier B.V. All rights reserved.