Micropipette aspiration studies of lipid membrane fusion mechanisms and polymersome adhesion energies
Micropipette aspiration was used to study the interactions of a fusion peptide with lipid vesicles, fusion between lipid vesicles, and the adhesion energy between polymer vesicles. First, the activity of a novel trimeric fusogenic peptide (ccX31) designed by W. Lau et al. (Lau, Ege et al. in press) was compared to the monomeric X31 fusion peptide from influenza virus. Measurements of 99.5:0.5::SOPC:SOPS lipid giant unilamellar vesicles (GUV) demonstrate that oligomerization of fusogenic peptides promotes membrane fusion by facilitating membrane destabilization. The ccX31 peptide caused lipid mixing and contents leakage at pH 7 and pH 5, whereas X31 only destabilized the vesicles at pH 5. The ccX31 showed a high level of activity at low concentration (∼1μM), while X31 required as much as a 10X higher concentration to achieve a similar level of destabilization. Also, ccX31 and X31 destabilize GUVs by different mechanisms. In contrast to X31, ccX31 did not cause the formation of stable pores, and membrane failure was characterized by sudden lysis after only a small amount of peptide had adsorbed. Second, we have for the first time directly observed fusion between two GUVs using optical microscopy and micropipette aspiration. Notably, this fusion occurred without a trigger such as PEG or a mediating protein. The composition of GUVs observed to fuse were 65:35::eggPC:DOPE (4 out of 7 trials) and 65:35::SOPC:DOPE (only 1 of 12 trials). Finally, the water permeability and energy of adhesion were measured for polymersome GUVs. The water permeability of OE-7 polymersomes was Pf = 2.5 +/− 1.2μm/s, one to two orders of magnitude smaller than liposome membranes (Pf = 25 to 150μm/s). The osmotic depletion-driven adhesion of OB-2, OB-9 and 90:10::OB2:OB9 polymersomes was seen to be a strong function of the concentration of 10kDa or 37.5kDa dextran in solution, similar to the effect seen with SOPC vesicles. The value of adhesion energy for OB-9 in 37.5kDa dextran was wa = 0.0077 +/− 0.0006 mJ/m2 at &phis;p = 0.015, increasing up to wa = 0.11 +/− 0.010 mJ/m2 at &phis;p = 0.09. Values of w a were similar for OB2 polymersomes in 37.5kDa and in 10kDa dextran solutions, as well as for 90:10::OB2:OB9 vesicles in 37.5kDa dextran solutions. In the absence of dextran, there was no measurable adhesion between any of the polymersomes studied.
Ege, David Stewart, "Micropipette aspiration studies of lipid membrane fusion mechanisms and polymersome adhesion energies" (2003). Dissertations available from ProQuest. AAI3109174.