ABSTRACT
Encapsulation of cells in biocompatible polymer matrices represents a powerful tool for cell-based therapies and therapeutic delivery systems. This technology has successfully been used to deliver pancreatic islets to humans for the treatment of Type 1 diabetes. However, the clinical impact of this technology may be improved by reducing the inflammatory response brought on after implantation of capsules in vivo. Within this study a biocompatible polymeric delivery system combining alginate and photo-crosslinked methacrylated glycol chitosan (MGC) was developed. This approach involved encapsulating cells in calcium-alginate beads, coating with MGC and photo-polymerizing using UVA in the presence of photo-initiator (VA-086), resulting in the formation of capsules â¼600 µm in size. Crosslinking of the MGC outer wall allowed control over capsule swelling and improved the capsules overall properties. Capsule characterization demonstrated the stabilizing influence of polymerization and fluorescence imaging showed that the distribution of glycol chitosan is dependent on molecular weight. Good islet viability and insulin release was demonstrated in vitro over the course of a month, and in vivo transplantation of the capsules demonstrated good biocompatibility, particularly when compared with standard alginate/poly-l-ornithine/alginate capsules.
Subject(s)
Alginates/chemistry , Biocompatible Materials/chemistry , Chitosan/analogs & derivatives , Drug Compounding/methods , Graft Rejection/prevention & control , Islets of Langerhans Transplantation/methods , Islets of Langerhans , Methacrylates/chemistry , Alginates/isolation & purification , Animals , Capsules , Carbohydrate Conformation , Cells, Cultured , Chitosan/chemistry , Chitosan/immunology , Chitosan/isolation & purification , Chitosan/radiation effects , Female , Foreign-Body Reaction/prevention & control , Glucuronic Acid/chemistry , Glucuronic Acid/immunology , Glucuronic Acid/isolation & purification , Hexuronic Acids/chemistry , Hexuronic Acids/immunology , Hexuronic Acids/isolation & purification , Hydrogels , Hydrophobic and Hydrophilic Interactions , Islets of Langerhans/metabolism , Limulus Test , Male , Materials Testing , Methacrylates/isolation & purification , Methacrylates/radiation effects , Mice , Microspheres , Molecular Structure , Peptides , Peritoneal Cavity , Permeability , Polymerization/radiation effects , Sus scrofa , Swine , Transplantation, Heterologous , Ultraviolet RaysABSTRACT
Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.
