Enhanced plasmid DNA transfection with lysosomotropic agents in cultured fibroblasts.
Transfer of plasmid DNA into mammalian cells has posed major challenges for gene therapy. Most non-viral vectors are known to internalize in the cells by endocytosis. Therefore, low transfection efficiency of non-viral vectors may be due to intracellular degradation of input DNA in the endosomes and/or lysosomes. DNA degradation can be inhibited either by inactivating the lysosomal enzymes or obliterating endosome fusion to lysosomes using lysosomotropic agents. We report here the effects of individual lysosomotropic agents such as chloroquine, polyvinylpyrolidone (PVP) and sucrose on beta-gal expression in cultured fibroblasts COS, 293 and CHO. Cell viability was influenced by type, exposure time and concentration of lysosomotropic agents. Exposure to chloroquine at high concentration (1000 microM) or more than 4 h at any concentration (10-1000 microM) caused extensive cell death, however, cytotoxicity due to sucrose (5-500 mM) and PVP (0.01-1 mg/ml) was minimal in the cell lines tested. All the agents utilized in this study enhanced the gene expression and the transfection efficiency followed the order of sucrose>chloroquine>PVP at the concentrations used in all cell lines. Results suggest that lysosomotropic agents can enhance transfection efficiency but the degree of transgene expression may be cell- and agent-specific. Of the agents studied, sucrose appears to be an attractive agent in improving gene expression without toxic effect in the cultured fibroblasts. Thus, it can be used as an excipient in the formulation of new gene delivery systems.
Ciftci
K
K
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, USA. kciftci@astro.ocis.temple.edu
Levy
R J
RJ
D000818:Animals; D018501:Antirheumatic Agents / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts; D015316:Genetic Therapy; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D010592:Pharmaceutic Aids / Q000494:pharmacology; D010957:Plasmids; D011205:Povidone / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018501:Antirheumatic Agents; D010592:Pharmaceutic Aids; D013395:Sucrose; D002738:Chloroquine; D004247:DNA; D001616:beta-Galactosidase; D011205:Povidone