Real-time observation of lipoplex formation and interaction with anionic bilayer vesicles.
A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.
Pantazatos
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SP
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston IL 60208, USA.
MacDonald
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RC
D000327:Adsorption; D000838:Anions; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry* / Q000648:ultrastructure*; D004058:Diffusion; D008051:Lipid Bilayers / Q000138:chemical synthesis / Q000737:chemistry; D008081:Liposomes / Q000138:chemical synthesis / Q000737:chemistry*; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence / Q000379:methods*; D008968:Molecular Conformation; D009038:Motion; D010713:Phosphatidylcholines / Q000138:chemical synthesis / Q000737:chemistry*; D010743:Phospholipids / Q000138:chemical synthesis / Q000737:chemistry; D013997:Time Factors; D014162:Transfection / Q000379:methods; D014741:Video Recording / Q000379:methods*
D000838:Anions; D004279:DNA, Viral; D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D010743:Phospholipids