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Factors affecting DNA binding and stability of association to cationic liposomes.
Even-Chen, Simcha; Cohen, Rivka; Barenholz, Yechezkel.
Afiliação
  • Even-Chen S; Laboratory of Membrane and Liposome Research, Department of Biochemistry, IMRIC, The Hebrew University, Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel.
Chem Phys Lipids ; 165(4): 414-23, 2012 May.
Article em En | MEDLINE | ID: mdl-22715503
ABSTRACT
Lipoplexes are complexes formed between cationic liposomes (L(+)) and polyanionic nucleic acids (P(-)). They are commonly used in vitro and in vivo as a nucleic acid delivery system. Our study aims are to investigate how DOTAP-based cationic liposomes, which vary in their helper lipid (cholesterol or DOPE) and in media of different ionic strengths affect the degree, mode of association and degree of condensation of pDNA. This was determined by ultracentrifugation and gel electrophoresis, methods based on different physical principles. In addition, the degree of pDNA condensation was also determined using the ethidium bromide (EtBr) intercalation assay. The results suggest that for cationic lipid compositions (DOTAP/DOPE and DOTAP/cholesterol), 1.5 M NaCl, but not 0.15 M NaCl, both prevent lipoplex formation and/or induce partial dissociation between lipid and DNA of preformed lipoplexes. The higher the salt concentration the greater is the similarity of DNA condensation (monitored by EtBr intercalation) between lipoplex DNA and free DNA. As determined by ultracentrifugation and agarose gel electrophoresis, 30-90% of the DNA is uncondensed. SDS below its critical micellar concentration (CMC) induced "de-condensation" of DNA without its physical release (assessed by ultracentrifugation) for both DOTAP/DOPE and DOTAP/cholesterol lipoplexes. As was assessed by agarose gel electrophoresis SDS induced release of 50-60% of DNA from the DOTAP/cholesterol lipoplex but not from the DOTAP/DOPE lipoplex. This study shows that there are conditions under which DNA is still physically associated with the cationic lipids but undergoes unwinding to become less condensed. We also proved that the helper lipid affects level and strength of the L(+) and DNA(-) electrostatic association; these interactions are weaker for DOTAP/cholesterol than for DOTAP/DOPE, despite the fact that the positive charge and surface pH of DOTAP/cholesterol and DOTAP/DOPE are similar.
Assuntos
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Base de dados: MEDLINE Assunto principal: Plasmídeos / DNA / Ácidos Graxos Monoinsaturados / Compostos de Amônio Quaternário / Lipossomos Idioma: En Ano de publicação: 2012 Tipo de documento: Article
Buscar no Google
Base de dados: MEDLINE Assunto principal: Plasmídeos / DNA / Ácidos Graxos Monoinsaturados / Compostos de Amônio Quaternário / Lipossomos Idioma: En Ano de publicação: 2012 Tipo de documento: Article