The aim of this work was to develop a novel formulation method, termed modified-PGSS (modified-Particle from Gas Saturated Solution), for the encapsulation of protein into polymeric microparticles in CO2 medium. In this study, the isosorbide dimethyl ether (DMI), a non-toxic water-miscible solvent, was used for the formulation and lysozyme was chosen as a model protein for encapsulation into PLGA microparticles. Firstly, the mechanism of particle formation has been extensively studied and was discussed in detail. Phase behavior was investigated by measuring the solubility of CO2 in DMI and volumetric expansion of DMI saturated in CO2. Here, we demonstrated the consistency of the experimental values with the data obtained from the mathematical (such as the neural network) and thermodynamic (such as the Peng-Robinson equation of state) models. These models were built to develop predictive tools in the chosen experimental space for microparticles formulation. Furthermore, these microparticles were characterized in terms of size and zeta potential. The morphology and protein distribution within PLGA microparticles were determined using scanning electron microscopy and confocal microscopy respectively. High encapsulation efficiency (65%) was obtained as confirmed by lysozyme quantification using a specific bioassay (M. lysodeikticus). Moreover, the in vitro protein release profile from loaded microparticles was presented. In this study, we reported an innovative and green process for lysozyme encapsulation into PLGA microparticles. Thus, this process could be applied to the encapsulation of therapeutic proteins requiring protection and controlled release such as growth factors for regenerative medicine.
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