title: Serum-resistant lipopolyplexes for gene delivery to liver tumour cells.
abstract: In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25kDa) or linear (22kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.
authors.affiliation: Department of Pharmacy and Pharmaceutical Technology, University of Navarra, Pamplona, Spain.
authors.forename: Leire
authors.initials: L
authors.lastname: García
authors.forename: Maria
authors.initials: M
authors.lastname: Buñuales
authors.forename: Nejat
authors.initials: N
authors.lastname: Düzgüneş
authors.forename: Conchita
authors.initials: C
authors.lastname: Tros de Ilarduya
mesh_terms: D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry; D004247:DNA / Q000008:administration & dosage* / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors; D004563:Electrochemistry; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D018014:Gene Transfer Techniques; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics; D008055:Lipids / Q000737:chemistry*; D008113:Liver Neoplasms / Q000235:genetics*; D010316:Particle Size; D011094:Polyethyleneimine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D044967:Serum / Q000737:chemistry*; D014162:Transfection
chemical_list: D002412:Cations; D005229:Fatty Acids, Monounsaturated; D008055:Lipids; D011108:Polymers; D000644:Quaternary Ammonium Compounds; D018664:Interleukin-12; D011094:Polyethyleneimine; D004247:DNA; D002784:Cholesterol; D003850:Deoxyribonuclease I; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane