title: Tetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemic, mucosal, and cellular immunostimulatory response following oral administration.
abstract: The present study reports the tetanus toxoid (TT)-loaded layer-by-layer nanoassemblies (layersomes) with enhanced protection, permeation, and presentation for comprehensive oral immunization. The stable and lyophilized TT-loaded layersomes were prepared by a thin-film hydration method followed by alternate layer-by-layer coating of an electrolyte. The developed system was assessed for in vitro stability of antigen and formulation, cellular uptake, ex vivo intestinal uptake, and immunostimulatory response using a suitable experimental protocol. Layersomes improved the stability in simulated biological media as well as protected the integrity/conformation and native 3D structure of TT as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorescence spectroscopy, respectively. The cell culture studies demonstrated a 3.8-fold higher permeation of layersomes in Caco-2 cells and an 8.5-fold higher uptake by antigen-presenting cells (RAW 264.7). The TT-loaded layersomes elicited a complete immunostimulatory profile consisting of higher systemic (serum IgG titer), mucosal (sIgA titer), and cellular (interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels) immune response after peroral administration in mice. The modified TT inhibition assay further confirmed the elicitation of complete protective levels of anti-TT antibody (>0.1 IU/mL) by layersomes. In conclusion, the proposed strategy is expected to contribute significantly in the field of stable liposome technology for mass immunization through the oral route.
authors.affiliation: Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Punjab, 160 062, India.
authors.forename: Harshad
authors.initials: H
authors.lastname: Harde
authors.forename: Ashish Kumar
authors.initials: AK
authors.lastname: Agrawal
authors.forename: Sanyog
authors.initials: S
authors.lastname: Jain
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D002460:Cell Line; D016503:Drug Delivery Systems; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood; D007371:Interferon-gamma / Q000276:immunology; D007376:Interleukin-2 / Q000276:immunology; D008297:Male; D008807:Mice, Inbred BALB C; D009092:Mucous Membrane / Q000276:immunology; D049329:Nanostructures / Q000008:administration & dosage*; D017207:Rats, Sprague-Dawley; D013745:Tetanus Toxoid / Q000008:administration & dosage*
chemical_list: D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007376:Interleukin-2; D013745:Tetanus Toxoid; D007371:Interferon-gamma