Biophysical characterization of anionic lipoplexes.
Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca2+ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca2+-DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca2+-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca2+ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca2+ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.
Patil
Siddhesh D
SD
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA.
Rhodes
David G
DG
Burgess
Diane J
DJ
D000838:Anions; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D000838:Anions; D008081:Liposomes; D010714:Phosphatidylethanolamines; D010715:Phosphatidylglycerols; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D004247:DNA; D004996:Ethidium; D002118:Calcium