Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro

Background: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam(3)Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam(3)Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-alpha secretions (ELISA). Results: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam(3)Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor alpha demonstrated clearly that Pam(3)Cys functionalisation elicited an overall higher immune response with the ranking of Pam(3)Cys chitosan DNA NP > chitosan DNA NP = DNA unloaded chitosan NP > control (culture medium). Conclusions: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam(3)Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF alpha levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).