The experimental model of lysozyme sustained release based on poly(3-hydroxybutyrate)-poly(ethylene glycol)/hydroxyapatite microparticles

Development of experimental models of proteins sustained release is a promising trend in modern biochemistry and biopharmacology. Application of proteins sustained release systems based on biopolymer microparticles allow to model prolonged protein action in vitro and in vivo. Moreover, the use of these devices in biopharmacology can eliminate the most of the drawbacks of traditional medicines: high toxicity, infectivity, substance instability, inconvenience of administration etc. For development of proteins sustained release system the biodegradable polymer poly(3-hydroxybutyrate) (PHB) and its copolymers, obtained b iotechnologically i n our l aboratory with Azotobacter chroococcum strain 7B, w ith n anohydroxyapatite w ere u sed. H ere we i nvestigated microparticles loaded with composite of nanohydroxyapatite and model protein lysozyme t hat w ere pr oduced by modificated t wo-step e mulsification s olid/oil/water (S/O/W) technique. For microparticles production we used a novel PHB c opolymer pol y(3-hydroxybutyrate)-poly(ethylene glycol) ( PEG-PHB) with m olecular weight of 250 000, as w ell as PHB w ith m olecular w eight of 250000 for comparison. We have chosen lysozyme as model protein with positive net charge and enzymatic activity. A lysozyme sustained release from biopolymer microparticles was carried out in vitro in a phosphate buffer (pH 7,4) at 37°C. We showed that protein release from the microparticles occurs via two processes: diffusion and degradation of microparticles. The release kinetics of the protein was connected with polymer molecular weight and hydrophobicity. Therefore, to improve the performance of lysozyme sustained release we used a more hydrophilic P HB-PEG. A s a r esult, t he r elease k inetics o f P HB-PEG microparticles was s ignificantly more l inear t han t hat o f PHB m icroparticles. The e fficiency o f pr otein e ncapsulating t o PHB-PEG m icroparticles w as al so better. However, the encapsulated protein can lose i ts native structure and enzymatic activity during its release from polymer microparticles. To verify lysozyme stability we examined the lysozyme enzymatic activity. The obtained data demonstrated that the lysozyme do not lose their integrity and enzymatic activity for 14 days sustained release from microparticles. Thus, the developed technique f or pr otein e ncapsulation in PHB-PEG microparticles can p roduce t he experimental model of protein sustained release.