Dexamethasone conjugated polyethylenimine as a gene carrier to cardiomyocytes for anti-apoptotic gene therapy

초록

Dexamethasone is a potent glucocorticoid with anti-inflammatory effects. In addition, dexamethasone has a cyto-ptrotective effect of cardiomyocytes from apoptosis. To apply this effect to ischemic disease gene therapy, dexamethasone-conjugated polyethylenimine (PEI-Dexa) was evaluated as a gene carrier to cardiomyocytes. PEI-Dexa was synthesized with low molecular weight polyethylenimine (PEI2K, 2 kDa). PEI-Dexa may have a proton-buffering effect, due to the PEI segment. To confirm the buffering effect of PEI-Dexa, acid-base titration was performed. PEI-Dexa showed considerable buffering capacity, although the buffer capacity of PEI-Dexa was slightly lower than that of PEI. This may be due to protonation of the tertiary and secondary amine groups of PEI and PEI-Dexa. To evaluate the transfection efficiency, in vitro transfection assay was performed with PEI-Dexa. To optimize the transfection condition of PEI-Dexa, PEI-Dexa/plasmid DNA (pDNA) complexes were prepared at various weight ratios and transfected to H9C2 cells. The highest transfection efficiency of PEI-Dexa was obtained at an 8/1 weight ratio (PEI-Dexa/pDNA). PEI-Dexa had higher transfection efficiency than high molecular polyethylenimine (PEI25K, 25 kDa). This higher transfection efficiency may be due to efficient nuclear translocation of PEI-Dexa/pDNA complex, which is mediated by glucocorticoid receptor. To evaluate the cytotoxicity, the PEI-Dexa/pDNA complexes were transfected to H9C2 cells, and the cytotoxicity was evaluated by MTT assay. The cytotoxicity of PEI-Dexa was lower than that of PEI25K. PEI-Dexa reduced the caspase-3 activity compared to PEI or control groups, suggesting that PEI-Dexa has a cyto-protective effect against hypoxia-induced apoptosis of cardiomyocytes. Heme oxygenase-1 (HO-1) has an anti-apoptotic gene, and, in this study, HO-1 was used as a model gene. It was previously reported that over-expression of HO-1 in normal cells had deleterious effects such as tumor growth. Therefore, HO-1 Expression should be regulated for safe gene therapy. To limit HO-1 expression to hypoxic cells, hypoxia inducible expression vector, pEpo-SV-HO-1 was constructed and used for PEI-Dexa mediated gene delivery. In the transfection assay, PEI-Dexa delivered pEpo-SV-HO-1 into H9C2 cells effectively, and HO-1 expression was induced under hypoxia. In conclusion, PEI-Dexa in combination with pEpo-SV-HO-1 may be useful for development of ischemic disease gene therapy as a gene carrier and as an anti-apoptotic reagent.