RESUMO
A promising method that offers both time- and site-specific delivery of macromolecules is photochemical internalization technology (PCI). Here, we have characterized various polyamidoamine (PAMAM) carriers [generation (G) 0-7], for light-directed delivery of nucleic acids in vitro by the use of PCI technology. A number of parameters for optimal delivery of nucleic acids into human cancer cells, that is, various light-doses, carrier-doses, and small interfering RNA (siRNA)/messenger RNA (mRNA) doses were investigated for either up- or down-regulation of enhanced green fluorescent protein (EGFP) gene expression. In summary, our results showed in an osteosarcoma cell line (OHS) [EGFP] model system the possibility for efficient light-directed siRNA silencing (>80% silencing) when using PAMAM G3 to G7 as carriers. Surprisingly, no EGFP mRNA up-regulation was detected either with or without PCI after EGFP mRNA/PAMAM (G0-G7) transfection in standard OHS cells. We have here identified properties for PAMAM formulations enabling light-directed siRNA delivery with the aim of developing a site-specific strategy for delivery of nucleic acids in vivo.
Assuntos
Processos Fotoquímicos , Poliaminas/administração & dosagem , RNA Mensageiro/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Luz , Osteossarcoma/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/química , Poliaminas/química , RNA Mensageiro/química , RNA Interferente Pequeno/química , Regulação para CimaRESUMO
A promising strategy for increased intracellular delivery of nucleic acids with the benefit for targeting is photochemical internalization (PCI). PCI relies on the use of a photosensitizing compound that photochemically destroys membranes in the endocytic pathway after illumination, resulting in cytosolic transfer of endosomal content. PCI technology combined with biodegradable polyamino acid carriers and nucleic acids delivers effective targeting and improved biosafety. In an in vitro model system, we have evaluated various poly-l-lysine (PLL), poly-l-histidine (PLH), and poly-l-arginine (PLA) formulations for light-directed small interference RNA (siRNA) gene silencing and messenger RNA (mRNA) delivery. We find that PLA formulations are suitable as siRNA and mRNA carriers in a strictly light-directed manner.
Assuntos
Melanoma , Terapia de Alvo Molecular , Peptídeos , RNA Interferente Pequeno/administração & dosagem , Arginina/administração & dosagem , Arginina/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endossomos/química , Endossomos/metabolismo , Terapia Genética , Humanos , Luz , Lisina/administração & dosagem , Lisina/química , Melanoma/tratamento farmacológico , Melanoma/genética , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/química , Peptídeos/administração & dosagem , Peptídeos/química , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/química , RNA Interferente Pequeno/química , TransfecçãoRESUMO
Among non-viral cationic polymers, biodegradable chitosan has during the last decade become an attractive carrier for small interference RNA (siRNA) delivery. Currently, degradation of macromolecules in the lysosomes is assumed to be a major barrier for effective siRNA transfection. Hence, transfection protocols are focused toward endosomal release mechanisms. In this work, we have tested 3 novel chitosan polymers and their siRNA delivery properties in vitro. To obtain efficient gene silencing of our model gene, S100A4, various transfection parameters were investigated, such as pH, nitrogen/phosphate ratio, photochemical internalization (PCI), media for complex formation, and cell lines. Our results showed that 2 linear chitosan polymers demonstrated excellent siRNA gene silencing, better than Lipofectamine 2000. The silencing effect was achieved without PCI treatment, under physiological pH, and with no observable reduction in cell viability.