Water-soluble covalent conjugates of bovine serum albumin with anionic poly(N-isopropyl-acrylamide) and their immunogenicity

We have conjugated bovine serum albumin (BSA) to poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAAm-AA)) by using water-soluble carbodiimide, and the effects of the bulk mass ratio of protein to polymer (r) on the formation of polymer–protein conjugates have been studied. HPLC, electrophoresis, viscosimetry and fluorescence spectroscopy suggest that the mode of covalent binding of BSA to poly(NIPAAm-AA) depends upon the weight concentration ratio (r) of BSA to poly(NIPAAm-AA). At r 1, free poly(NIPAAm-AA) molecules coexist with conjugate, and when r reaches 1 the amount of free polymer is too small to be observed. It is shown that depending on the ratio r, two types of conjugate particles were formed: at r<1, the protein molecules in the structure of conjugate particles are densely covered as a shell by polymer chain and practically “fenced off” from water environment; at r>1 the conjugate-forming particles possess more friable structures in which protein molecules are practically exposed to the solvent. The complex formation involving electrostatic interactions between BSA and carbodiimide activated polymer are proposed as the driving force for the covalent binding of BSA to polymer macromolecules. The coil-globule transition of macromolecules in low and thermally induced precipitation in more concentrated solutions of bioconjugates was observed. The immunogenic properties of covalent conjugates of CP–BSA were investigated and the temperature-modulated solubility–immunogenicity alterations was analyzed. A single immunization of mice with conjugates at the thermally precipitating concentration without an adjuvant evoked increased specific immune response to BSA, which practically did not depend on the initial conjugation ratio of components. Such a modulated system is attractive for application as a novel immunogenic system in vaccine technology.