In the present study we found that the polyethylene glycol (PEG)-induced membrane fusion involved primarily the disruption of lipid bilayers, and no active pore was found for membrane proteins. We propose to verify the importance of membrane lipids in PEG-induced fusion by studying vesicles and cells containing different lipids, and having different protein/lipid ratios. Vesicles reconstituted with erythrocyte membrane proteins, and exogenous lipids of different surface charge, headgroup hydration, bilayer stability, acyl chain length and saturation will be used. The lipids in human and avian erythrocyte and human lymphocyte membranes will be modified by lipid exchange techniques. The fusion efficiency of these samples will be analyzed in terms of known lipid properties. To access the unique characteristics of PEG, the interaction between PEG, phospholipids and the surface layer of water molecules will be studied by NMR, FTIR and radioactive binding assays. Peg-induced changes of membrane potential will be measured by fluorescence methods. Membrane fusion induced by electric pulses is said to involve the breakdown and the structural disruption of two tightly opposed bilayers. The postulated pore formation and reseal sequence will be investigated by time-resolved freeze fracture electron microscopy. The factors related to PEG-induced fusion, namely, surface charges, bilayer defects, susceptibility to form intramembranous particle-denuded areas and swelling will be tested for their effects on the dielectrophoresis and the reversible electrical breakdown processes. The results of this study are expected to lead to a more rational design of fusion protocols, and to contribute to the general understanding of membrane fusion mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030969-05
Application #
3278863
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1982-07-01
Project End
1990-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Roswell Park Cancer Institute Corp
Department
Type
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14263
Hui, Sek-Wen (2002) The application of electroporation to transfect hematopoietic cells and to deliver drugs and vaccines transcutaneously for cancer treatment. Technol Cancer Res Treat 1:373-84
Chandaroy, Parthapratim; Sen, Arindam; Alexandridis, Paschalis et al. (2002) Utilizing temperature-sensitive association of Pluronic F-127 with lipid bilayers to control liposome-cell adhesion. Biochim Biophys Acta 1559:32-42
Chandaroy, P; Sen, A; Hui, S W (2001) Temperature-controlled content release from liposomes encapsulating Pluronic F127. J Control Release 76:27-37
Langner, M; Hui, S (2000) Effect of free fatty acids on the permeability of 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer at the main phase transition. Biochim Biophys Acta 1463:439-47
Wells, J M; Li, L H; Sen, A et al. (2000) Electroporation-enhanced gene delivery in mammary tumors. Gene Ther 7:541-7
Li, L H; Ross, P; Hui, S W (1999) Improving electrotransfection efficiency by post-pulse centrifugation. Gene Ther 6:364-72
Ross, P C; Hui, S W (1999) Polyethylene glycol enhances lipoplex-cell association and lipofection. Biochim Biophys Acta 1421:273-83
Li, L H; Sen, A; Murphy, S P et al. (1999) Apoptosis induced by DNA uptake limits transfection efficiency. Exp Cell Res 253:541-50
Xu, Y; Hui, S W; Frederik, P et al. (1999) Physicochemical characterization and purification of cationic lipoplexes. Biophys J 77:341-53
Ross, P C; Hui, S W (1999) Lipoplex size is a major determinant of in vitro lipofection efficiency. Gene Ther 6:651-9

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