Insight into the mechanism and factors on encapsulating basic model protein, lysozyme, into heparin doped CaCO₃

Porous CaCO₃ microparticles are considered as one of the most popular and effective carriers for protein loading. Most of current studies have centered on elucidating particle formation and enhancing the protein loading efficiency, very few reports on the kinetics, driving forces and factors on protein loading process. Here, we took lysozyme as the basic model protein to investigate the kinetics, driving forces on protein loading and factors controlling loading efficiency into porous Hep/CaCO₃ microparticles by various techniques (protein quantification assay, QCM-D, SEM, BET, Zeta sizer, TGA, CLSM, CD spectrum, bioactive assay). As revealed, the adsorption process obeyed the pseudo second-order kinetics and Langmuir adsorption model. Doping heparin greatly influenced the detailed texture, pore size, surface area, and maximum loading capacity of lysozyme (LC_Lys,m). The dependence of LC_Lys,m on pH reflected the electrostatic interaction mainly contributed to lysozyme adsorption, especially below IEP of lysozyme. But the hydrophobic interaction also played the critical role on lysozyme adsorption at pH around IEP of lysozyme. Accompanying with pH change, the lysozyme orientation shifted from "side on" at lower pH to "end on" at pH around IEP. At proper concentration of NaCl (C_NaCl), the loaded lysozyme could be released from Hep/CaCO₃ microparticles, making them available for lysozyme reloading. Most importantly, such release-reloading cycle didn't disturb the bioactivity of released lysozyme and following reloading ability. We believe our work will contribute to understand protein adsorption behaviors, improve protein loading efficacy, biomaterials design, tissue engineering and disease treatment.