DNA-induced endocytosis upon local microinjection to giant unilamellar cationic vesicles

Abstract

We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 microns in diameter, made of phosphatidyl-cholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. "Short" DNAs (oligonucleotide 21 b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (Cendo) of D-erythrosphingosine (Sph+) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below Cendo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. Cendo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph+ GUVs and about 20 mol% for SOPC/Sph+ or eggPC/Sph+ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes.