College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada.
College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada.
Vaccine. 2019 Mar 7;37(11):1503-1515. doi: 10.1016/j.vaccine.2019.01.058. Epub 2019 Feb 7.
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.
我们之前开发了一种高效的组合佐剂，由先天防御调节剂（IDR）-1002 肽、聚肌苷酸（poly(I:C)）和聚磷腈（TriAdj）组成。在这里，我们旨在设计和测试该佐剂的粘膜粘附性鼻用制剂的体内疗效。为了确定制剂的物理性质，通过凝胶电泳和使用罗丹明-poly(I:C)的荧光猝灭来表征添加每种单独成分的效果。由二癸基二甲基溴化铵（DDAB）、二油酰基磷脂酰乙醇胺（DOPE）（50:50 或 75:25 mol:mol）和 DDAB、L-α-磷脂酰胆碱（蛋黄 PC）和 DOPE（40:50:10 mol:mol:mol）组成的阳离子脂质体通过薄膜挤出法制备。通过简单混合将脂质体和 TriAdj 组合。形成的复合物（L-TriAdj）通过动态光散射、Zeta 电位和粘蛋白相互作用进行表征。我们发现 IDR-1002 肽、聚磷腈和 poly(I:C)在溶液中自组装形成阴离子复合物。单独暴露于 TriAdj 的 RAW267.4 小鼠巨噬细胞与 L-TriAdj 相比表明，DDAB/DOPE（50:50）和 DDAB/EPC/胆固醇（40:50:10）复合物降低了 TriAdj 的毒性。接下来，制备了含有 TriAdj 的阳离子脂质体，以确定最佳尺寸、稳定性和所需正电荷的几个摩尔比。透射电子显微镜显示，脂质体与 TriAdj 和粘蛋白结合后，脂质结构发生重排。稳定的颗粒（24 小时内<200nm）显示 DDAB/DOPE+TriAdj 的粘蛋白结合大于 DDAB/EPC/DOPE+TriAdj。为了验证体内疗效，用卵清蛋白作为抗原经鼻腔给予 DDAB/DOPE+TriAdj 复合物，并通过 ELISA（血清 IgG1、IgG2a、IgA）和 ELISpot 测定（脾细胞 IL-5、IFN-γ）测量免疫应答。与单独使用 TriAdj 相比，给予佐剂的小鼠表现出显著更强的免疫反应，其剂量反应与三佐剂含量成比例，并且表现出全身性和粘膜性免疫的整体平衡 Th1/Th2 免疫反应。
