School of Ophthalmology and Optometry, Eye Hospital, School of Biomedical Engineering, Wenzhou Medical University, Wenzhou, Zhejiang Province, 325035, PR China; Wenzhou Institute of Biomaterials and Engineering, CNITECH, CAS, Wenzhou, Zhejiang Province, 325001, PR China; Engineering Research Center of Clinical Functional Materials and Diagnosis&Treatment Devices of Zhejiang Province, Wenzhou Institute of Biomaterials and Engineering, CAS, Wenzhou, Zhejiang Province, 325001, PR China.
Wenzhou Vocational and Scientific College, Wenzhou, Zhejiang Province, 325006, PR China.
Colloids Surf B Biointerfaces. 2019 Mar 1;175:184-194. doi: 10.1016/j.colsurfb.2018.11.079. Epub 2018 Nov 29.
Porous CaCO 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/CaCO 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). The dependence of LC 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), the loaded lysozyme could be released from Hep/CaCO 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.
多孔 CaCO3 微粒被认为是蛋白质负载的最受欢迎和最有效的载体之一。目前的大多数研究都集中在阐明颗粒形成和提高蛋白质负载效率上，很少有关于蛋白质负载过程动力学、驱动力和影响因素的报道。在这里，我们以溶菌酶为基本模型蛋白，通过各种技术（蛋白质定量分析、QCM-D、SEM、BET、Zeta 粒径仪、TGA、CLSM、CD 光谱、生物活性测定）研究了动力学、驱动力和控制载药效率的因素，将其负载到多孔 Hep/CaCO3 微粒中。结果表明，吸附过程符合准二级动力学和 Langmuir 吸附模型。肝素的掺杂极大地影响了溶菌酶的详细结构、孔径、比表面积和最大载药量（LC）。LC 对 pH 的依赖性反映了静电相互作用主要贡献于溶菌酶的吸附，特别是在溶菌酶等电点（IEP）以下。但在溶菌酶 IEP 左右的 pH 值下，疏水力也对溶菌酶的吸附起着关键作用。伴随着 pH 值的变化，溶菌酶的取向从较低 pH 值时的“侧挂”转变为 IEP 左右时的“端挂”。在适当浓度的 NaCl（C）存在下，负载的溶菌酶可以从 Hep/CaCO3 微粒中释放出来，使其能够进行溶菌酶的再负载。最重要的是，这种释放-再负载的循环不会干扰释放的溶菌酶的生物活性和随后的再负载能力。我们相信我们的工作将有助于理解蛋白质吸附行为，提高蛋白质负载效率，生物材料设计，组织工程和疾病治疗。
