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 to 14.4 nm and 0.4 to 440 kPa, respectively. AFM images and profiles, as well as LTSEM showed different morphologies depending on the microsphere composition. Furthermore, microbeads prepared by gelation of alginate with Ca2+ and Ba2+, exhibited a much smoother surface compared to microspheres obtained after a second reaction of the calcium-alginate beads with poly(methylene-co-guanidine) hydrochloride (PMCG). However, a similar smooth surface to that of calcium beads was obtained when reacting the beads with poly(L-lysine) (PLL). Comparing barium- and calcium-alginate beads, the first showed the higher average surface roughness. Furthermore, for the PMCG-containing capsules a decrease in surface average roughness by, approximately, one half was obtained by coating with SA or SCS. The microsphere type studied could be classified into four groups, according to the differences in the Young's modulus values. The highest surface compressibility corresponded to calcium-alginate beads and PLL containing-microspheres. Barium beads follow in the subsequent groups, with YM values increasing with the SA molar mass. The PMCG-containing microspheres exhibited the highest surface stiffness, being 405 times higher than the value determined for the calcium-beads. Surface stiffness further increased when coating with SA or SCS was applied. In addition to local surface analysis, microsphere network parameters such as mechanical stability (compression work (CW)) and permeability were studied. Results front compression experiments on barium-beads showed a stronger influence of SA concentration on network mechanical resistance compared to the effect that this change generated on the local surface elastic modulus. SA molar mass, however, is highly influencing both, bulk and surface compressibility. The type of divalent cation affected surface and bulk mechanical properties. Specifically, barium-alginate beads with stiffer surfaces exhibited more compressible gels, while calcium-alginate beads showed a relatively high resistance to compression possessing the lowest local surface elastic modulus. In addition, different profiles of compression force curves, obtained for the beads, could be correlated to different homogeneity of bead structure. The multicomponent capsule, prepared by subsequent reaction of calcium-alginate beads with PMCG, showed much higher values of CW compared to the beads. Coating with SCS did not have a major effect on bulk stiffness, while the coating with low-G-low-molar mass SA decreased considerably the bulk resistance to compression. Furthermore, calcium-alginate beads were more permeable to pullulan standards than barium-alginate beads. PMCG-containing microspheres non-coated and those coated with SCS showed a significantly lower pullulan ingress compared to the beads, both microcapsules exhibited similar permeability to pullulan molecules. The third part of the thesis summarizes in vitro and in vivo experiments on small animals, including the optimization of the encapsulation of small amounts of islets of Langerhans. The good in vitro insulin response to glucose of the encapsulated islets demonstrated a minor influence of the encapsulation process on islet viability and function. The practical experience on islets encapsulation showed that a strictly controlled quality of the islet suspension is determinant for suitable encapsulation. The in vivo experiments revealed the best biocompatibility for barium-M-alginate beads if implanted into the portal vein of the liver. Finally, the combination of highly purified materials with short-term inmunosuppression was identified as a suitable approach to reduce cellular overgrowth on rnicrobeads injected intraportally. The results of the thesis are significant for the goal directed design of immununoprotection and in vivo delivery systems composed of natural and/or synthetic polyelectrolytes.

Wandrey, Christine
Lausanne, EPFL

Note: The status of this file is: EPFL only

 Record created 2005-03-16, last modified 2018-01-27

Rate this document:

Rate this document:
(Not yet reviewed)