RESUMO
Multifunctional nanocomplexes (NCs) consisting of urocanic acid-modified galactosylated trimethyl chitosan (UA-GT) conjugates as polymeric vectors, poly(allylamine hydrochloride)-citraconic anhydride (PAH-Cit) as charge-reversible crosslinkers, and vascular endothelial growth factor (VEGF) siRNA as therapeutic genes, were rationally designed to simultaneously overcome the extracellular, cellular, and intracellular barriers for siRNA delivery. The strong physical stability of UA-GT/PAH-Cit/siRNA NCs (UA-GT NCs) at pH 7.4 and 6.5 endowed protection from massive dilution, competitive ions, and ubiquitous nucleases in the blood and tumorous microenvironment. Their internalization into hepato-carcinoma cells was facilitated through the recognition of galactose receptors, followed by effective escape from endosomes/lysosomes owing to the strong buffering capacity of imidazole residues. At the meantime, the endosomal/lysosomal acidity triggered the charge reversal of PAH-Cit in UA-GT NCs, thus evoking their structural disassembly and subsequently accelerated release of siRNA in the cytosol. As a result, robust in vivo performance in terms of both gene silencing and tumor inhibition was achieved by UA-GT NCs at a low siRNA dose. Moreover, neither histological nor hematological toxicity was detected following repeated intravenous administration. Therefore, UA-GT NCs potentially served as an efficient and safe candidate in the treatment of hepatocellular carcinoma through knocking down the overall barriers for siRNA delivery.
Assuntos
Antineoplásicos/farmacologia , Técnicas de Transferência de Genes , Nanopartículas/química , RNA Interferente Pequeno/metabolismo , Animais , Soluções Tampão , Linhagem Celular Tumoral , Quitosana/síntese química , Quitosana/química , Anidridos Citracônicos/síntese química , Anidridos Citracônicos/química , Endocitose , Endossomos/metabolismo , Feminino , Galactose/química , Inativação Gênica/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/toxicidade , Tamanho da Partícula , Poliaminas/síntese química , Poliaminas/química , Espectroscopia de Prótons por Ressonância Magnética , Eletricidade Estática , Ácido Urocânico/síntese química , Ácido Urocânico/química , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
A new nonviral gene vector--urocanic acid-coupled chitosan (UAC) was prepared by the reaction of the activated urocanic acid (UA) with the amine group on the chitosan (CTS). The structure of UAC was confirmed with FT-IR, 1H NMR and element analysis. The influencing factors of substitution values were studied by orthogonal test, and the substitution values of UAC increased with the prolongation of activating time of UA and the increasing ratio of UA to CTS. The condensation ability and the resistance to DNase I of UAC/pDNA were evaluated by agarose gel electrophoresis, and UAC showed good condensation ability with pDNA, well protecting pDNA from the degradation by DNase I. The particle size and zeta potential were evaluated by zetasizer, and the results showed that the UAC/pDNA complex was well stable and could easily enter into cells. The transfection studies were performed with HepG2 cells in vitro. It showed that the in vitro transfection of UAC/pDNA was efficient in HepG2 cells and could express more green fluorescent proteins than that of CTS. So the UAC is easy to prepare and a promising non-viral gene vector.
Assuntos
Quitosana/administração & dosagem , Terapia Genética/métodos , Vetores Genéticos , Ácido Urocânico/administração & dosagem , Quitosana/síntese química , Quitosana/metabolismo , DNA/genética , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Sistemas de Liberação de Medicamentos , Células Hep G2 , Humanos , Tamanho da Partícula , Plasmídeos , Transfecção , Ácido Urocânico/síntese química , Ácido Urocânico/metabolismoRESUMO
Ethyl, n-dodecyl, and n-hexadecyl esters of urocanic acid (4-imidazoleacrylic acid) were prepared from 4-imidazolecarboxaldehyde in satisfactory yields via the Wittig reaction.
