We previously developed an highly efficacious combination adjuvant comprised of innate defense regulator (IDR)-1002 peptide， poly(I:C) and polyphosphazene (TriAdj). Here we aimed to design and test the in vivo efficacy of a mucoadhesive nasal formulation of this adjuvant. To determine the physical properties of the formulation， the effect of addition of each individual component was characterised by gel electrophoresis and fluorescence quenching using rhodamine-poly(I:C). Cationic liposomes comprised of didodecyl dimethylammonium bromide (DDAB)， dioleoyl phosphatidylethanolamine (DOPE) (50:50 or 75:25?mol:mol) and DDAB， L-α-phosphatidylcholine (egg PC) and DOPE (40:50:10?mol:mol:mol) were prepared by t... More
We previously developed an highly efficacious combination adjuvant comprised of innate defense regulator (IDR)-1002 peptide， poly(I:C) and polyphosphazene (TriAdj). Here we aimed to design and test the in vivo efficacy of a mucoadhesive nasal formulation of this adjuvant. To determine the physical properties of the formulation， the effect of addition of each individual component was characterised by gel electrophoresis and fluorescence quenching using rhodamine-poly(I:C). Cationic liposomes comprised of didodecyl dimethylammonium bromide (DDAB)， dioleoyl phosphatidylethanolamine (DOPE) (50:50 or 75:25?mol:mol) and DDAB， L-α-phosphatidylcholine (egg PC) and DOPE (40:50:10?mol:mol:mol) were prepared by the thin-film extrusion method. The liposomes and TriAdj were combined by simple mixing. The formed complex (L-TriAdj) was characterized by dynamic light scattering， zeta potential， and mucin interactions. We found that IDR-1002 peptide， polyphosphazene and poly(I:C) self-assembled in solution forming an anionic complex. Exposure of RAW267.4 mouse macrophage cells to TriAdj alone vs. L-TriAdj indicated that DDAB/DOPE (50:50) and DDAB/EPC/cholesterol (40:50:10) complexation reduced TriAdj toxicity. Next， TriAdj-containing cationic liposomes were prepared at several molar ratios to determine optimal size， stability and desired positive charge. Transmission electron microscopy showed rearrangement of lipid structures on binding of liposomes to TriAdj and to mucin. Stable particles (<200?nm over 24?h) showed mucin binding of DDAB/DOPE?+?TriAdj was greater than DDAB/EPC/DOPE?+?TriAdj. To verify in vivo efficacy， mice were administered the DDAB/DOPE?+?TriAdj complex intranasally with ovalbumin as the antigen， and the immunogenic response was measured by ELISA (serum IgG1， IgG2a， IgA) and ELISpot assays (splenocyte IL-5， IFN-γ). Mice administered adjuvant showed a significantly greater immune response with L-TriAdj than TriAdj alone， with a dose-response proportionate to the triple adjuvant content， and an overall balanced Th1/Th2 immune response representing both systemic and mucosal immunity.
