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Trilateral '3P' Mechanics of Stabilized Layersomes Technology for Efficient Oral Immunization.

The main objective of the present work is to investigate the potential of polyelectrolyte stabilized layersomes as vaccine adjuvants for oral immunization. Herein, bovine serum albumin (BSA) loaded vesicular nanoformulations were prepared via thin film hydration method. The effect of various explanatory variables on responses were examined by a 6-factor, 3-level Box-Behnken design using JMP statistical software. An improved stability of vesicular nanoformulations in simulated biological media revealed that alternate layer-by-layer coating using polyacrylic acid and polyallylamine hydrochloride improved the robustness of vesicular structure. The formulation was lyophilized to improve long-term storage stability using trehalose as cryoprotectant. As evident from in process stability analysis, the chemicals, process and lyophilization conditions had no detrimental effect on the conformational stability and integrity of antigen. Mathematical modelling and DDsolver analysis confirmed that the release profile of vesicular nanoformulations altered from Baker-Lonsdale to Higuchi fashion, following integration of layer-by-layer coating. Confocal microscopy and spectrofluorometric quantification demonstrated a selective, dose and time dependent accumulation of nanoformulations in antigen presenting cells. As evident from in vivo immunization study, layersomes produced higher humoral (mean IgG in serum), mucosal (mean sIgA titre in biological fluids) and cellular immune response (IL-2 and IFN-y level) compared to free antigen. The observed effects might be attributed to improved protection, permeation and presentation of antigen using layersomes. In conclusion, polyelectrolyte stabilized vesicular nanoformulations contribute promising toolbox for mass immunization using human/veterinary vaccine or other protein/peptides delivery especially by oral route.