Undergraduate Research | George R. Brown School of Engineering

Undergraduate Research | George R. Brown School of Engineering | Rice University

Undergraduate Research in Engineering at Rice

David Tran, Senior Bioengineering student
Fusogenic Potential of Liposomes to Outer Hair CellsandFormation and Stability of Cationic Vesicles in Asymmetric Ionic Environments
with Dr. Robert Raphael
My research project involved investigating the interaction of liposomal membranes with cochlear outer hair cells. Preliminary studies by Dr. Robert Raphael revealed that lipids containing phosphatidylcholine (PC) did not adhere to outer hair cells while those containing PC, phosphatidylserine (PS), and phosphatidylethanolamine (PE) did adhere. The project confirmed and extended these studies with the introduction of a cationic liposome 1,2-Diodeoyl-3(trimethylammonium) propane (DOTAP). I investigated the fusogenic potential of different liposome compositions through micropipette aspiration of vesicles attached to cochlear outer hair cells and the transfer of fluorescently labeled liposomes to cochlear outer hair cells. In addition, I investigated the formation and stability of cationic vesicles in asymmetric ionic environments.
Fusion is a biochemical and mechanical phenomenon that is responsible for cellular processes such as exocytosis, endocytosis, fertilization, and mitosis. The mechanics of fusion are still not completely understood. Previous studies have shown that fusion is promoted through instabilities and wedges in vesicles through the formation of high curvature structures (Hui et al., 1989). PE is a liposome known to form the hexagonal II phase where polar head groups are on the inside and hydrophobic hydrocarbon tails are on the outside rendering vesicles closely packed; as a result, they have a tendency to form surfaces with large radii of curvature (Yeagle, 79). Charged vesicles have been shown to favor fusion and will also be explored (Hui et al., 1997).
Phospholipids were purchased from Avanti Polar Lipids, Inc. Phospholipid vesicles were formed using a modification of the electroformation technique (Angelova et al., 1992) where a lipid mixture was coated on the surface of electrodes and subjected to an electric field oscillating at 10 Hz. Dehydrated lipids swell under the AC electric field while they are rehydrated and bud off to form vesicles. The formation of vesicles was viewed under an inverted light microscope and Hoffman Modulation Contrast optics. Phospholipid vesicles formed and detached from the electrode with time. Modification included the increase in voltage potential and timing.
Stearoyl-oleoyl-phosphotidylcholine (SOPC) was chosen as the base lipid because phosphatidylcholine is found and known to behave like natural cell membranes. Different concentrations of charged lipids were added to see their affects on fusion, encapsulation, and vesicle mechanical properties. Palmitoyl-oleoyl-phosphotidyl-serine (POPS) is desired for its anionic charge. 1,2-Diodeoyl-3(trimethylammonium) propane (DOTAP) is desired for its cationic charged. 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) was added to investigate the relationship between its fusogenic and encapsulation potential with vesicle mechanical properties.
Micropippette aspiration was utilized to study the fusogenic potential of liposomes to outer hair cells. Micropipette aspiration is a technique to measure the elastic moduli in model systems. Vesicles are aspirated into micropipettes at known values of pressure which correlates with the stress imposed on the vesicle. In the case of the fusion experiments, micropipette aspiration was used as a tool to determine the force required to remove SOPC (100), POPS:DLPE:SOPC (20:40:40), and DOTAP:DLPE:SOPC (20:40:40) vesicles attached to outer hair cells. Pipette and aspiration chamber were filled with filtered HBS. Vesicles, isolated outer hair cells, and 2.5 um microspheres were pipetted into the chamber. Vesicles were brought into contact with outer hairs cells via micropipette for 15 to 30 minutes. Pressure was then increased until vesicles detached from the outer hair cells. As before, SOPC (100) vesicles did not adhere to outer hair cells; little to no pressure was required to detach the vesicles. POPS:DLPE:SOPC (20:40:40) vesicles adhered to outer hair cells; a pressure between 300 to 1000 N/m2 was required to detach the vesicles. Due to the instability of DOTAP:DLPE:SOPC (20:40:40) vesicles in asymmetric environments, no successful data was collected.
The fusogenic potential of liposomes to outer hair cells was also studied through a fluorescent transfer study. SOPC (100), POPS:DLPE:SOPC (20:40:40), and DOTAP:DLPE:SOPC (20:40:40) vesicles made with 1% Rhd-DOPE were incubated with outer hair cells in a Petri dish in HBS. The dishes were covered with aluminum foil and stored in 4C refrigerator. Over a period of 5 days, outer hair cells were viewed under a Zeiss Axiovert 200M Microscope at 500nm and pictures were taken with a Zeiss AxioCam MRm camera to see whether there was a transfer of fluorescent liposomes. Cells in SOPC (100) vesicles exhibited no significant excitation over a period of five days. Cells in POPS:DLPE:SOPC (20:40:40) vesicles exhibited fluorescent specs after 15 hours and increased in days 2 and 5. Cells in DOTAP:DLPE:SOPC (20:40:40) vesicles exhibited fluorescence days 1 and 2. A Deiters cell was also seen to exhibited intense fluorescence after 1 day. These show that both cationic and anionic charges appear to increase liposomal transfer.
The ability to perform meaningful experiments on giant unilamellar vesicles (GUVs) has been limited by the difficulty in forming these vesicles in physiological saline. We have successfully formed GUVs containing the cationic lipid dioleoyl-1,2-diacyl-3-trimethylammonium-propane (DOTAP) in HEPES-buffered saline solution (HBS) by modifying the electroformation technique. The formation of these positively charged liposomes was also achieved in a sucrose solution containing trace amounts of bovine serum albumin (BSA). In contrast to the formation of pure phosphatidylcholine (PC) membranes, vesicles containing DOTAP could not be formed in pure sucrose solution. Interestingly, when DOTAP vesicles were formed in BSA and then exposed to HBS, the vesicles were observed to jitter and then burst. Some vesicles were also observed to bud off and then fuse back together. We postulate that these effects were due to differential surface tension arising from the asymmetric ionic composition of the external solutions. This hypothesis is supported by the observation that when PC vesicles were formed in BSA and then exposed to HBS, they were stable. Moreover, vesicles were stable in symmetric ionic environments. The instability of membranes in asymmetric ionic environments illustrates the inherent coupling between membrane surface charge and mechanics that may produce electromechanical effects.
During the course, I have learned the arts of micropipette fabrication, vesicle formation, cochlear outer hair cell isolation, and micropipette aspiration. Eventually, I hope to transfer fluorescent molecules such as DNA from the vesicles to the cells. I also wish to further perfect and characterize the stability and formation of lipid vesicles in physiological saline.
References
Angelova, M. J., S. Soleau, Ph. Melaeard, J.F. Faucon and P. Bothorel. 1992. Preparation of giant vesicles by AC electric fields. Kinetics and applications. Progr. Colloid. Polym. Sci. 89: 127-131.
Hui, S. W., Langner, M., Zhao, Y., Ross, P. Hurley, E. and K. Chan. 1997. The role of helper lipids in cationic liposome-mediated gene transfer. Biophysical Journal. 71:590-599.
Hui, S. W. and A. Sen. 1989. Effects of lipid packing on polymorphic phase behavior and membrane properties. Proc. Natl. Acad. Sci. USA. 86:5825-5829.
Hui, S. W., T. P. Steward, and L. T. Boni. 1981. Membrane fusion through point defects in bilayers. Science. 212:921-923.
Yeagle, Philip L. The Membranes of Cells. Academic Press, Inc., San Diego, California. 1993.

Department of Bioengineering
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