13The aim of this work was to develop a novel formulation method, termed modified-PGSS (modified-14Particle from Gas Saturated Solution), for the encapsulation of protein into polymeric microparticles in 15 CO 2 media. In this study, the isosorbide dimethyl ether (DMI), a non-toxic water-miscible solvent, was 16 used for the formulation and lysozyme was chosen as a model protein for encapsulation into PLGA 17 microparticles. Firstly, the mechanism of particle formation has been extensively studied and was 18 discussed in detail. Phase behavior was investigated by measuring the solubility of CO 2 in DMI and 19 volumetric expansion of DMI saturated in CO 2 . Here, we demonstrated the consistency of the 20 experimental values with the data obtained from the mathematical (such as the neural network) and 21 thermodynamic (such the Peng-Robinson equation of state) models. These models were built to develop 22 predictive tools in the chosen experimental space for microparticles formulation. Furthermore, these 23 microparticles were characterized in terms of size and zeta potential. The morphology and protein 24 distribution within PLGA microparticles were determined using scanning electron microscopy and 25 confocal microscopy respectively. High encapsulation efficiency (65%) was obtained as confirmed by 26 lysozyme quantification using a specific bioassay (M. lysodeikticus). Moreover, the in vitro protein release 27 profile from loaded microparticles was presented. In this study, we reported an innovative and green 28 process for lysozyme encapsulation into PLGA microparticles. Thus, this process could be applied to the 29 encapsulation of therapeutic proteins requiring protection and controlled release such as growth factors for 30 regenerative medicine. 31 32
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