PLASMIDEOS COM POTENCIAL BIOTECNOLOGICO: AVALIAÇÃO DA ESTABILIDADE FISICO-QUIMICA E PRODUÇÃO DE NANOPARTÍCULAS FUNCIONALIZADAS COM POLIETILENOIMINA
Plasmid; Super-curled; Stability; Nucleases; Nano particle; Polyethyleneimine
The plasmids (pDNAs) have been standing out regarding to its application at biotechnology, mostly in DNA vaccination and genic therapy. pDNAs are DNA molecules with extra chromosomal double bands, presenting circular shape and have different isoforms. The Food and Drug Administration recommends that during pDNAs utilization on immunization protocols, they must be at least 80% on its super-curled isoform, once the studies assign that super-curled isoforms have a larger access to the interior of cells. Super-curled isoform may be altered by chemical, physical and biological factors. Owing to its importance at pDNAs structure stability at biological function, this study aims to evaluate the pDNAs stability used at biotechnology, ands tests a nano formulation with polyethyleneimine (PEI) associated to pDNAs, in order to promote a stability and bioavailability increasing of pDNAs. We evaluate the temperature effect at incubation, plasmid size and nucleases concentrations under super-curled isoform conservation. Also, it was evaluated pDNAs stability using functionalized nano particles with PEI through time variables, nucleases concentration and comparing to non functionalized nano particles formulated pDNAs. A kinetics protocol for plasmids enzymatic degradation was optimized. The higher the temperature, higher the plasmid size and the lower conservation of super-curled isoforms occur. The preparation of functionalized nano particles with PEI associated to pDNAs showed itself a promising alternative to protection against enzymatic degradation, however it requires more in vitro and in vivo studies to verifying security factors and genic transference rate.