RESUMO
The intranasal administration of proteins using nanoparticles is a promising approach for several applications, especially for mucosal vaccines. Delivery of protein within the epithelial barrier is a key point to elicit an immune response and nano-carrier has to show no toxicity. The aim of this work was to elucidate the interactions of cationic porous nanoparticles loaded with protein delivery for antigen delivery in the nose. We investigated the loading, the cellular delivery and the epithelial transcytosis of proteins associated to these nanoparticles containing an anionic lipid in their core (NPL). NPL were highly endocytosed by airway epithelial cells and significantly improved the protein delivery into the cell. In vitro transcytosis studies showed that NPL did not modify the in vitro epithelial permeability suggesting no toxicity of these carriers. Moreover protein and NPL did not translocate the epithelial barrier. In vivo studies demonstrated that NPL prolonged the nasal residence time of the protein and no NPL were found beyond the epithelial barrier in vivo, precluding a negative side effect. All together these results establish the NPL as a bio-eliminable and optimal vaccine carrier.
Assuntos
Antígenos/administração & dosagem , Portadores de Fármacos/administração & dosagem , Nanopartículas/administração & dosagem , Mucosa Nasal/metabolismo , Ovalbumina/administração & dosagem , Administração Intranasal , Animais , Antígenos/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Endocitose , Epicloroidrina/administração & dosagem , Epicloroidrina/química , Compostos de Epóxi/química , Camundongos , Nanopartículas/química , Ovalbumina/química , Ovalbumina/farmacocinética , Permeabilidade/efeitos dos fármacos , Polissacarídeos/administração & dosagem , Polissacarídeos/química , Compostos de Amônio Quaternário/químicaRESUMO
A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Primers and probes were designed to detect specific sequences on the most conserved regions of the S segment. The limit of detection of the assay was 10 copies per reaction which is an improvement of 3 log(10)FFU/mL over the sensitivity of conventional RT-PCR. The specificity of the primers and probe was confirmed with the closely related Nairoviruses CCHFV and Hazara virus, and on the non-related viruses Coronavirus and Influenza A virus. This qRT-PCR assay was used to screen nucleic acids extracted from 498 ticks collected in the Republic of Chad. One sample was found positive suggesting that DUGV is present in this part of the world. The molecular assay developed in this study is sensitive, specific and rapid and can be used for research and epidemiological studies.