Atomic force microscopy imaging of DNA-cationic liposome complexes optimised for gene transfection into neuronal cells

Background: Cationic liposomes represent an important gene delivery system due to their low immunogenicity, but are relatively inefficient, with optimisation of DNA-liposome complexes (lipoplexes) for transfection necessary for each cell type of interest. There have been few studies examining optimisation in neuronal cell types or determining how the structure of lipoplexes affects transfection efficiency. Methods: Four commercially available cationic liposome formulations were used to optimise transfection efficiency in neuronal cells. The DNA to liposome ratio and the amount of DNA used in transfections were varied. Transfection efficiency was determined by the percentage of cells positive for the β-galactosidase reporter gene product. The structure of lipoplexes was studied using atomic force microscopy. Lipoplexes were characterised further using dynamic light scattering to determine size and fluorescence techniques to show DNA compaction. Results: Optimal transfection conditions were found to differ between immortalised cell lines and primary cells. High transfection efficiencies in immortalised cell lines were achieved predominantly with multivalent cationic liposomes while primary neuronal cells showed optimal transfection efficiency with monovalent cationic liposomes. The structure of lipoplexes was observed with atomic force microscopy and showed globular complexes for multivalent cationic liposomes, while monovalent liposomes gave less compact structures. In support of this finding, high levels of DNA compaction with multivalent liposomes were observed using fluorescence quenching measurements for all DNA to liposome ratios tested. One monovalent liposome showed increasing levels of compaction with increasing liposome amount. Dynamic light scattering showed little change in complex size when the different lipoplexes were studied. Conclusions: Optimisation of transfection efficiency was different for cell lines and primary neurons. Immortalised cells showed optimal transfection with multivalent liposomes while primary neurons showed optimal transfection with monovalent liposomes. The charge ratio of the monovalent liposome was below one, suggesting a different mechanism of lipoplex binding and uptake in primary neurons. The structure of lipoplexes, as determined with atomic force microscopy, showed more compact structures for multivalent liposomes than monovalent liposomes. This finding was supported by DNA compaction studies

Non-viral suicide gene therapy in cervical, oral and pharyngeal carcinoma cells with CMV- and EEV-plasmids

Background: Cervical cancer is the third most common cause of cancer in women. The 5-year survival rate in oropharyngeal squamous cell carcinomas is approximately 50% and this rate has not improved in recent decades. These cancers are accessible to direct intervention. We examined the ability of a highly efficient non-viral vector, TransfeX (ATCC, Manassas, VA, USA), to deliver the suicide gene HSV-tk to cervical, oral and pharyngeal cancer cells and to induce cytotoxicity following the administration of the prodrug, ganciclovir. Methods: HeLa cervical carcinoma, HSC-3 and H357 oral squamous cell carcinoma and FaDu pharyngeal carcinoma cells were transfected with cytomegalovirus (CMV)- or enhanced episomal vector (EEV)-driven HSV-tk plasmids and treated with ganciclovir for 24–120 h. Cell viability was assessed by Alamar blue. Results: The viability of HeLa cells was reduced to only 30–40%, despite the very high levels of transgene expression. By contrast, the viability of HSC-3 cells was reduced to 10%, although transgene expression was 18-fold lower than that in HeLa cells. An approximately five-fold higher transgene expression was obtained with the EEV-plasmid than from the CMV-plasmid. Nevertheless, HeLa cell viability after suicide gene + ganciclovir treatment was reduced by only 35% compared to 70% with the CMV-plasmid. For HSC-3 cells, the reduction was 40% for the EEV- and 80% for the CMV-plasmid. The lower efficiency of transfection with the EEV-plasmid may explain the lower cytotoxicity. Conclusions: TransfeX-mediated gene delivery to cervical, pharyngeal and oral cancer cells may be used for suicide gene therapy. The levels of transgene expression, however, do not translate directly to cytotoxicity