Physicochemical characterization and purification of cationic lipoplexes

Citation
Yh. Xu et al., Physicochemical characterization and purification of cationic lipoplexes, BIOPHYS J, 77(1), 1999, pp. 341-353
Citations number
33
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOPHYSICAL JOURNAL
ISSN journal
0006-3495 → ACNP
Volume
77
Issue
1
Year of publication
1999
Pages
341 - 353
Database
ISI
SICI code
0006-3495(199907)77:1<341:PCAPOC>2.0.ZU;2-#
Abstract
Cationic lipid-nucleic acid complexes (lipoplexes) consisting of dioleoyltr imethylammoniumpropane (DOTAP) liposomes and plasmid DNA were prepared at v arious charge ratios (cationic group to nucleotide phosphate), and the exce ss component was separated from the lipoplex. We measured the stoichiometry of the lipoplex, noted its colloidal properties, and observed its morpholo gy and structure by electron microscopy. The colloidal properties of the li poplexes were principally determined by the cationic lipid/DNA charge ratio and were independent of the lipid composition. In lipoplexes, the lipid me mbranes as observed in freeze-fracture electron microscopy were deformed in to high-radius-of-curvature features whose characteristics depended on the lipid composition. Lipoplexes prepared at a threefold or greater excess of either DOTAP or DNA could be resolved into complexes with a defined stoichi ometry and the excess component by sedimentation to equilibrium on sucrose gradients. The separated, positively charged complex retained high transfec tion activity and had reduced toxicity. The negatively charged lipoplex sho wed increased transfection activity compared to the starting mixture. In cr yoelectron micrographs the positively charged complex was spherical and con tained a condensed but indistinct interior structure. In contrast, the sepa rated negatively charged lipoplexes had a prominent internal 5.9 +/- 0.1-nm periodic feature with material projecting as spikes from the spherical str ucture into the solution. It is likely that these two lipoplexes represent structures with different lipid and DNA packing.