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As surfactants are capable of influencing the droplet formation, our study primarily aims the investigation of the effect of a nonionic surfactant e.g. Polysorbate 80 on the formation of microspheres on the course of vibrating nozzle method with coacervation. The experiments also concern the impact of the different process parameters (e.g. vibration frequency, feed rate and voltage) on the shape and size distribution of microspheres characterized by laser diffraction size determination completed with particle image analysis. The calcium-alginate microspheres were processed using freeze-drying to ensure solid state with better drug carrier capability.Addition of isomalt was advantageous in the formation of freeze-dried microspheres at low alginate concentration, which was explained by micro-CT analysis of the constructed particle structure. The internal three-dimensional network of calcium alginate demonstrated a more cancellous architecture ameliorating the roundness of microparticles.