Expertise: implications for radiological education.
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Clinical Competence↗
Biomedical subjects
R Fraley
Publications and source records attributed to R Fraley.
Physicochemical characterization of large unilamellar phospholipid vesicles prepared by reverse-phase evaporation.
Properties of large unilamellar vesicles (LUV), composed of phosphatidylcholine and prepared by reverse-phase evaporation and subsequent extrusion through Unipore polycarbonate membranes, have been investigated and compared with those of small unilamellar vesicles (SUV) and of multilamellar vesicles (MLV). The unilamellar nature of the LUV is shown by 1H-NMR using Pr3+ as a shift reagent. The gel to liquid-crystalline phase transition of LUV composed of dipalmitoylphosphatidylcholine (DPPC) monitored by differential scanning calorimetry, fluorescence polarization of diphenylhexatriene and 90 degrees light scattering, occurs at a slight lower temperature (40.8 degrees C) than that of MLV (42 degrees C) and is broadened by about 50%. The phase transition of SUV is shifted to considerably lower temperatures (mid-point, 38 degrees C) and extends over a wide temperature range. In LUV a well-defined pretransition is not observed. The permeability of LUV (DPPC) monitored by leakage of carboxyfluorescein, increases sharply at the phase transition temperature, and the extent of release is greater than that from MLV. Leakage from SUV occurs in a wide temperature range. Freeze-fracture electron microscopy of LUV (DPPC) reveals vesicles of 0.1-0.2 micron diameter with mostly smooth fracture faces. At temperatures below the phase transition, the larger vesicles in the population have angled faces, as do extruded MLV. A banded pattern, seen in MLV at temperatures between the pretransition and the main transition, is not observed in the smaller LUV, although the larger vesicles reveal a dimpled appearance.
Animals↗
Liposomes: the development of a new carrier system for introducing nucleic acid into plant and animal cells.
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Animals↗
Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions.
Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.
Animals↗
Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles.
We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 micrometer diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca2+ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca2+ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca2+ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg2+ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg2+ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca2+ and also enhanced the rate and extent of fusion. Mg2+ had a synergistic effect on Ca2+-induced fusion of phosphatidylserine/phosphatidylethanolamine vesicles. We suggest that the role of phospholipids in membrane fusion is related to their ability to form dehydrated intermembrane complexes with divalent cations.
Calcium↗
Studies on the mechanism of membrane fusion: kinetics of calcium ion induced fusion of phosphatidylserine vesicles followed by a new assay for mixing of aqueous vesicle contents.
We describe an assay for following the mixing of aqueous contents during fusion of phospholipid vesicles. Terbium is encapsulated as the Tb(citrate)3(6-) chelation complex in one population of vesicles, dipicolinic acid (DPA) in another. Vesicle fusion results in the formation of the fluorescent Tb(DPA)3(3-) chelation complex. The presence of EDTA (0.1 mM) and Ca2+ (greater than 1 mM) prevents the formation of the Tb/DPA complex in the external medium. We have studied the Ca2+-induced fusion of small or large unilamellar vesicles (SUV or LUV, respectively) composed of phosphatidylserine (PS). In addition, vesicle aggregation was monitored by light scattering, and release of vesicle contents was followed by carboxyfluorescein (CF) fluorescence enhancement. The addition of Ca2+ induced an immediate enhancement in Tb fluorescence with both SUV and LUV, which occurs on the same time scale as aggregation but much faster than the release of CF. The release of contents from LUV occurs with a considerable delay. It is estimated that the initial fusion of SUV is accompanied by 10% leakage of the internal volume per fusion event; in contrast, fusion of LUV is essentially nonleaky. Massive release of vesicle contents appears to be a secondary phenomenon related to the collapse of fused vesicles. The initial rate and the extent of Tb fluorescence enhancement are markedly dependent on the Ca2+ concentration. Threshold Ca2+ concentrations are 1.2 and 2.4 mM for SUV nd LUV, respectively. At saturating Ca2+ concentrations (greater than 10 mM), the rate of fusion of LUV is slightly lower than that of SUV at the same vesicle concentration. At any Ca2+ concentration, the rates of both SUV and LUV fusion are consistent with vesicle aggregation being rate limiting. When measured at a subsaturating Ca2+ concentration, fusion is essentially second order over a wide range of relatively low vesicle concentrations, whereas at higher vesicle concentrations the order is decreased. This suggests that at high vesicle concentrations (and at relatively low Ca2+ concentrations) aggregation may proceed faster than fusion.
Calcium↗
Studies on the mechanism of membrane fusion: role of phosphate in promoting calcium ion induced fusion of phospholipid vesicles.
The role of phosphate in enhancing the calcium-induced fusion of phosphatidylserine (PS) vesicles has been examined by using the new fluorescent (terbium/dipicolinic acid) assay described by Wilschut et al. (1980) [Wilschut, J., Düzgüneş, N., Fraley, R., & Papahajopoulos, D. (1980) Biochemistry (preceding paper in this issue)]. In the presence of physiological levels of phosphate, the calcium concentration required for fusion of PS vesicles was lowered significantly (3-4-fold), and the rate of vesicle fusion was increased dramatically (up to 1000-fold). The fusion of PS vesicles by calcium and phosphate is shown to be specific and critically dependent on temperature, pH, ion concentrations, and the composition of the calcium phosphate crystalline phase present during the incubation. The results indicate that a significant enhancement in vesicle fusion occurs only when calcium phosphate precipitation is initiated in the presence of PS vesicles, suggesting that crystal nucleation on the vesicle surface is a prerequisite for fusion. Calcium and phosphate were shown to promote phospholipid phase separations and vesicle fusion under conditions (e.g., mixtures of PS and phosphatidylcholine) in which calcium alone is ineffective, indicating that formation of PS calcium phosphate complexes may facilitate the molecular segregation of PS into distinct domains. These experiments underline the important role that phosphate may play in calcium-mediated fusion phenomena in biological membranes.
Calcium↗
Introduction of liposome-encapsulated SV40 DNA into cells.
DNA, isolated from Simian virus 40 (SV40), has been encapsulated in large (0.4-micrometer diameter) unilamellar phospholipid vesicles. The procedure used for liposome preparation encapsulated the SV40 DNA at high efficiency (30 to 50% entrapment) and did not alter the physical or biological properties of the DNA molecules. The biological activity of the liposome-entrapped viral DNA was determined by plaque assays on a permissive monkey cell line. The infectivity of liposome-entrapped SV40 DNA was enhanced at least 100-fold over that of free naked DNA. Importantly, the infectivity of vesicle-entrapped DNA was resistant to DNase digestion, dependent on the amount of DNA encapsulated per vesicle and on the vesicle lipid composition. Liposomes composed of phosphatidylserine were the most efficient for delivery of DNA to cells (1.8 x 10(3) plaque-forming units/micrograms of DNA). Following the incubation of DNA-containing liposomes with cells, their infectivity could be enhanced an additional 10- to 200-fold by exposing the cells to high concentrations of polyethylene glycol or glycerol. Under these conditions the infectivity of liposome-encapsulated SV40 DNA (3 x 10(5) plaque-forming units/microgram) was comparable with values reported using the calcium phosphate method. In addition to providing a sensitive assay for monitoring and optimizing the delivery of vesicle contents to cells, the liposome-mediated delivery of nucleic acids may have potential for increasing the efficiency of DNA delivery to cells and for extending the number of cell types which can be transformed or transfected.
Animals↗