Formation of Giant Unilamellar Vesicles

Three compounds, MOH (8, R'CO = Oleoyl), DOSH (10, R'CO = R'CO = Oleoyl), and DOPH (11) were studied with respect to the conditions allowing for Giant Unilamellar Vesicle (GUV) formation. The goal was to find a lipid forming GUVs at the pH needed for the ligation experiments (pH 7, HEPES). Vesicle formation was induced by the swelling of a evaporated lipid film in the respective buffer, eventually followed by sonification, freeze-drying, and alternatively by electroformation. GUVs were observed, both by light microscopy, and by confocal laser microscopy after staining the membranes with suitable fluorescent dyes. In all cases, GUV-formation required the presence of cholesterol as a co-lipid, of which 5-15% were found to be optimal for DOSH (10) and MOH (8). Vesicles from DOPH required alkaline buffers (pH 8-8.2) and coexisted with oil droplets under the conditions studied (40-50% cholererol). In the cases of DOSH and MOH, GUVs were obtained at neutral to slightly acidic pHs under conditions compatible with ligation experiments.

Vesicle filling was demonstrated by freeze-drying a fluorescence-labeled oligonucleotide with DOSH, cholesterol, and HEPES buffer, rehydration with a small amount of water, and 10-fold dilution of the vesicle population (Figure 13).

The preparation of giant vesicles (GVs) from the synthesized catalipids MOH 8a (R' in 8 = oleyl) and 10 (R , R'in 10 = oleyl) was optimized using the swelling or electroformation method. Large GVs of high unilamellarity are accessible and therefore suitable for further experiments (Figure 14).

MOH-GV-encapsulation of the reaction component Cy3TTCCGp was possible und could be observed under the fluorescence microscope (Figure 15).

Experiments showed that in the presence of EDC the component Cy3TTCCGp is enriched on the surface of MOH-GVs - whether this is due to the formation of a phosphorimidazolide or the unspecific absorption on the positively charged surface of the GV- needs further control experiments.



Figure 13  TAMRA-CGT ATG AAC GTC ATC in GUVs from DOSH (10), cholesterol in 4 mM HEPES, pH 7. Top: light microscopy. Bottom: fluorescence microscopy.



Figure 14  GVs of MOH (8a) formed in water using the swelling method (bright image)


Figure 15   Encapsulation of ss-DNA Cy3TTCCGp into a GV (MOH 8a); left bright image, right fluorescence image (filter 540nm)