Polyelectrolyte-based immunprotection systems : polymer purification and surface analysis

The research of this doctoral thesis was dedicated to polyelectrolytes, specifically to hydrogel materials, which result from coacervation and polyelectrolyte complex formation and have a potential as immunoprotection systems. In particular, microspheres obtained by electrostatic interaction of alginate, as main polyanionic component. with multivalent cations such as Ca2+ and Ba2+, and polycations were investigated. The research has primarily focused on polymer purification and microsphere surface analysis. Complementary in vitro and in vivo studies were performed. In a first part, a purification procedure was developed and optimized. The relatively simple and economic procedure is suitable to reduce endotoxins (ET), proteins and polyphenols in polyanions such as sodium alginate (SA) and sodium cellulose sulfate (SCSI). No major influence of the purification procedure on the structural and macromolecular characteristics of the polymers was detected. The procedure was optimized with regards to the total yield and reproducibility, considering the differences in composition and macromolecular characteristics of the raw materials. Optimum process conditions were identified for two types of sodium alginate and sodium cellulose sulfate. The factors having an impact on the yield are the polymer concentration, the volume filtered in one step, the non-solvent to solvent ratio as well as the number of precipitation steps. The purity of the final product is determined by the initial concentration of contaminants in the polymer, the concentration of the aqueous polymer solution, and the number of precipitation steps. The purification process could be reproduced in the gram scale with endotoxin contents, as low as 50 EU/g alginate, which arc far below the FDA threshold. The experimental results of the research in this first part of the thesis provided the basis for a protocol to routinely purify polyanions. Several batches of pure and well-characterized alginate have been produced for application research following this protocol. Detailed complex stability studies revealed ET complexation by the cationic components involved in microsphere preparation. It was proven that endotoxin forms stable complexes with polycations. This represents additional security in terms of biocompatibility but does not exclude the need of polymer purification for applications in immunoprotection systems. In the second part of the thesis, surface and network properties of hydrogel microspheres were studied and correlated with composition and preparation conditions. Nine different types of microspheres were investigated. Surface analysis was assessed by atomic force microscopy (AFM) and low temperature scanning electron microscopy (LTSEM). Sample preparation for both techniques required special instrumentation and manipulation. Surface imaging and mechanical response to indentation revealed different average surface roughness and Youngs' moduli for all hydrogel types ranging from 0.9