Characterization of chemically defined poly-N-isopropylacrylamide based copolymeric adjuvants.

PNiPAAm is a thermo-responsive polymer with an adjuvant activity. To identify the minimal chemical structure present within PNiPAAm responsible for its adjuvant property, three different constituent polymers with specific functional groups were synthesized through free radical reaction and tested their adjuvant potential along with PNiPAAm. Among them, polymer with isopropyl attached to an amide showed maximal adjuvant activity in rodents followed by polymer with amide or ketone functional groups. However, secondary amine containing polymer did not show any adjuvant activity. In addition, to improve the adjuvant properties of PNiPAAm, we incorporated an affinity ligand, boronate. At first, we synthesized and characterized the dual responsive copolymers PNiPAAm-co-VPBA and PNiPAAm-co-VPBA-co-DMAEMA. Biocompatibility of these copolymers was confirmed both in vitro and in vivo. Mice injected with these copolymers mixed with collagen (CII) developed significant levels of anti-CII antibodies comprising of all the major IgG subclasses and an increased T cell activation. At the injection site, massive infiltration of immune cells was observed. However, only PNiPAAm-co-VPBA-co-DMAEMA-CII induced arthritis in mice after injection of 0.5M fructose confirming the importance of effective release of CII from the polymer for its adjuvant activity. Thus, a fine balance of hydrophobicity and hydrophilicity promotes adjuvant properties and continuous release of antigen, in this case CII, from polymer is essential for its adjuvant activity.