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, an d the effects of the bulk mass ratio of protein to polymer (r) on the forma tion of polymer-protein conjugates have been studied. HPLC, electrophoresis , viscosimetry and fluorescence spectroscopy suggest that the mode of coval ent binding of BSA to poly(NIPAAm-AA) depends upon the weight concentration ratio (r) of BSA to poly(NIPAAm-AA). At r less than or equal to 1, free po ly(NIPAAm-AA) molecules coexist with conjugate, and when r reaches I the am ount of free polymer is too small to be observed. It is shown that dependin g on the ratio r, two types of conjugate particles were formed: at r < 1, t he protein molecules in the structure of conjugate particles are densely co vered as a shell by polymer chain and practically "fenced off" from water e nvironment; at r > I the conjugate-forming particles possess more friable s tructures in which protein molecules are practically exposed to the solvent . The complex formation involving electrostatic interactions between BSA an d carbodiimide activated polymer are proposed as the driving force for the covalent binding of BSA to polymer macromolecules. The coil-globule transit ion of macromolecules in low and thermally induced precipitation in more co ncentrated solutions of bioconjugates was observed. The immunogenic propert ies of covalent conjugates of CP-BSA were investigated and the temperature- modulated solubility-immunogenicity alterations was analyzed. A single immu nization of mice with conjugates at the thermally precipitating concentrati on without an adjuvant evoked increased specific immune response to BSA, wh ich practically did not depend on the initial conjugation ratio of componen ts. Such a modulated system is attractive for application as a novel immuno genic system in vaccine technology. (C) 2001 Elsevier Science Ltd. All righ ts reserved.