RESUMEN
OBJECTIVE: To construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. METHODS: The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. RESULTS: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. CONCLUSION: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.
Asunto(s)
Técnicas de Transferencia de Gen , Neoplasias Hepáticas/terapia , Péptidos/farmacología , Polietileneimina/farmacología , Neoplasias de la Próstata/terapia , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Animales , Sitios de Unión , Carcinoma/terapia , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Vectores Genéticos/síntesis química , Vectores Genéticos/química , Vectores Genéticos/farmacología , Humanos , Técnicas In Vitro , Ligandos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Tamaño de la Partícula , Péptidos/química , Péptidos/metabolismo , Polietileneimina/química , Polietileneimina/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Relación Estructura-Actividad , Propiedades de Superficie , Transfección , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To investigate the influencing factors of polyethylenimine (PEI) in gene transfer in vitro. METHODS: Cytotoxic effects of PEI on in vitro cultured NIH 3T3 cells were quantified by MTT assay. The interaction between PEI and DNA at different charge ratios was analyzed by agarose gel electrophoresis retardation assay. The expression of gene transfer was monitored in Cos-7 cells using pEGFP and pSV beta plasmids as the reporter gene systems. Influences of chloroquine, albumin, serum, salt ion strength, and Mg(2+) ion and other factors on PEI/DNA transfer efficiency were evaluated. RESULT: The survival rate of NIH3T3 cells at 6 mg/L of PEI was 64.2% and at 7 mg/L of PEI was 54.4%. Gel electrophoresis retardation assays showed that PEI completely retarded DNA migration at 3.0 PEI nitrogen per DNA phosphate. Chloroquine enhanced the transfection efficiency of PEI. Albumin and serum in the culture medium decreased the transfection efficiency. HBS(HEPES buffered solution) or 150 mmol/L NaCl as the dilution solution of PEI/DNA was superior over 278 mmol/L glucose solution in the transfection efficiency. Mg(2+) in the dilution solution decreased the transfer efficiency of PEI/DNA. CONCLUSION: PEI is efficient gene transfer agent of eukaryotes in vitro, and can be possibly used in vivo.