Improving lipoplex-mediated gene transfer into C6 glioma cells and primary neurons

Abstract

The development of methodologies for gene transfer into the central nervous system is crucial for gene therapy of neurological disorders. In this study, different cationic liposome formulations were used to transfer DNA into C6 glioma cells and primary hippocampal and cortical neurons by varying the nature of the helper lipid (DOPE, Chol) or a mixture of DOPE and cholesterol (Chol) associated to DOTAP. In addition, the effect of the lipid/DNA (+/À) charge ratio, the association of the ligand transferrin to the lipoplexes, and the stage of differentiation of the primary cells on the levels of transfection activity, transfection efficiency, and duration of gene expression were evaluated. Mechanistic studies were also performed to investigate the route of delivery of the complexes into neurons. Our results indicate that DOTAP:Chol (1:1 mol ratio) was the best formulation to transfer a reporter gene into C6 glioma cells, primary hippocampal neurons, and primary cortical neurons. The use of transferrin-associated lipoplexes resulted in a significant enhancement of transfection activity, as compared to plain lipoplexes, which can be partially attributed to the promotion of their internalization mediated by transferrin. While for hippocampal neurons the levels of luciferase gene expression are very low, for primary cortical neurons the levels of transgene expression are high and relatively stable, although only 4% of the cells has been transfected. The stage of cell differentiation revealed to be critical to the levels of gene expression. Consistent with previous findings on the mechanisms of cell internalization, the experiments with inhibitors of the endocytotic pathway clearly indicate that transferrin-associated lipoplexes are internalized into primary neurons by endocytosis. Promising results were obtained in terms of the levels and duration of gene expression, particularly in cortical neurons when transfected with the Tfassociated lipoplexes, this finding suggesting the usefulness of these lipid-based carriers to deliver genes within the CNS. D 2004 Elsevier Inc. All rights reserved.

Transferrin lipoplex-mediated suicide gene therapy of oral squamous cell carcinoma in an immunocompetent murine model and mechanisms involved in the antitumoral response

“…In this regard, we and others have demonstrated that association of human transferrin to cationic liposome/DNA complexes enhances transfection in a large variety of cells, including dividing 18,19,27 and non-dividing cells. [28][29][30] As demonstrated in our recent work, 27 Tf-lipoplexes containing 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/cholesterol mediated the highest levels of transfection in oral cancer cells in culture, HSC-3 and SCC-7, when prepared at the 3/2 lipid/DNA ( ± ) charge ratio. We have also reported that delivery of HSV-tkexpressing plasmid to these cells mediated by Tflipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity.…”

Tf-lipoplex-mediated NGF gene transfer to the CNS: neuronal protection and recovery in an excitotoxic model of brain injury

“…We have previously demonstrated that Tf is highly effective in promoting lipoplexmediated transfection of a large variety of cells, [34][35][36][37] including both cortical and hippocampal neuronal primary cultures. 38 Therefore, in this work, we evaluated whether this strategy would also result in an enhancement of in vivo rat brain transfection. For this purpose, lipoplexes prepared upon association of the pSIN-PGKnls-LacZ-WHV plasmid to 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP): cholesterol (Chol) liposomes, at the 8/1 lipid/DNA (+/À) charge ratio, in the presence or absence of Tf, were stereotactically injected in the striatum of Wistar rats.…”

“…This partial neutralization of the surface positive charges promotes particle aggregation, which results in lipid structures of larger size. 56 Although the existence of such changes may explain the higher in vitro transfection efficiency of the 2/1 and 4/1 (+/À) Tf-lipoplexes, 38 for which the high sedimentation capability overcomes the decrease in electrostatic interactions with the plasma membrane, the same is not true for in vivo gene transfer. The need for high lipid and DNA concentrations, in order to prepare a small volume of lipoplexes for stereotactic administration, favours the formation of very large aggregates, which may hamper diffusion through the brain parenchyma and cellular internalization by endocytosis.…”

Enhancement of p53 gene transfer efficiency in hepatic tumor mediated by transferrin receptor through trans-arterial delivery

“…[21][22][23] The ideal gene transduction vector should be safe, specific, with high transfection efficiency and convenient to operate. 2,3 Viral vectors generally facilitate highly efficient transfer and expression of foreign genes, but attempts to modify specificity to their target cells have proven difficult.…”