Nanotechnology-based vaccine delivery systems have been developed to enhance the immunogenicity of the vaccine antigens by modulating antigen delivery to the residing immune cells in lymph node where antigen can properly be presented to the immune system. Nano-vector formulations of vaccines offer the advantages of co-delivery of antigen and immunomodulators. Toll like receptors (TLRs) agonists are promising vaccine adjuvants with the ability to enhance and modulate innate as well as adaptive immune responses in great potentials.The use of non-viral vectors as delivery carriers for adjuvants and vaccine antigens, enhance vaccine immungenecity which can lead to long lasting protective immunity. An effective way for co-delivery of antigens and TLRs agonist is to encapsulate both in one carrier to ensure that they both enter one cell at the same time. The aim of the present study was to design, synthesize and in vitro and in vivo characterization of a novel nano-adjuvant based vaccine delivery system based on single walled carbon nanotubes (SWNT) loaded with Polyethylenimine (PEI) and TLR4, TLR7/8 and TLR9 agonists which can deliver vaccine antigens entrapped in Poly Lactic-co-Glycolic Acid (PLGA). In this study, SWNT was covalently conjugated using amide chemistry to the surface of polyethylenimine (PEI) followed by addition of CpG oligodeoxynucleotides (CpG ODN) as TLR9 agonist, MPLA as TLR4 agonist and R848 as TLR7/8 agonist in order to produce a nanoparticulate vectors for the delivery of ovalbumin (OVA) protein encapsulated in PLGA (poly-lactic-co-glycolic acid). The resulting complexes was characterized in terms of size and zeta potential and the structures was analysed and confirmed by FT-IR spectroscopy, thermogravimetric analysis (TGA), atomic force microscopy (AFM) and transmission electron microscopy (TEM). FITC fluorescence staining was used to confirm the cellular uptakes of nano-vector complexes. Moreover, they were evaluated in terms of in vitro and in vivo cytotoxicity in C26 cells using an MTT assay and in mouse model using histopathology and LD50 measurement. In vivo immunological properties of nano-vaccine compounds were evaluated in a prime-boost experiment in mouse model and several immunological parameters including different antibody isotypes (IgG1, IgG2a), pro and anti-inflammatory cytokines ( IL-1β, IL-10) and Th1/Th2/Treg cytokine profiles (IFN-, IL-4, IL-10) were measured using ELISA and ELISPOT assay. The vaccine delivery potentials of the resulting complexes have shown promising results which will be discussed in more detail during the symposium.

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