title: Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.
abstract: 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 Pam3Cys (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. Pam3Cys 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-α 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 Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys 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, Pam3Cys 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-α 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).
authors: School of Pharmaceutical Sciences Geneva-Lausanne (EPGL), University of Geneva, University of Lausanne, Geneva, Switzerland.
authors: Simon
authors: S
authors: Heuking
authors: Barbara
authors: B
authors: Rothen-Rutishauser
authors: David Olivier
authors: DO
authors: Raemy
authors: Peter
authors: P
authors: Gehr
authors: Gerrit
authors: G
authors: Borchard
mesh_terms: D001980:Bronchi / Q000378:metabolism*; D048271:Chitosan / Q000494:pharmacology; D018920:Coculture Techniques; D004247:DNA / Q000378:metabolism*; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007109:Immunity / Q000187:drug effects; D016209:Interleukin-8 / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D008970:Molecular Weight; D009000:Monocytes / Q000166:cytology; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D010587:Phagocytosis / Q000187:drug effects; D010957:Plasmids / Q000378:metabolism*; D051194:Toll-Like Receptor 1 / Q000819:agonists* / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000819:agonists* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016209:Interleukin-8; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D004247:DNA; D048271:Chitosan
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