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Biomedical subjects

D Papahadjopoulos

Publications and source records attributed to D Papahadjopoulos.

At least 127 records · Page 7Linked to original sources
PubMed1981

Covalent attachment of immunoglobulins to liposomes via glycosphingolipids.

We describe a method for covalent binding of proteins to large unilamellar liposomes which involves the periodate oxidation of glycosphingolipids in the vesicle membrane. Proteins such as IgG and F(ab')2 may then be attached to the aldehyde groups on the glycolipid by Schiff-base formation at pH 9.5 and reduction with NaBH4, or by reductive amination with NaBH3CN at pH 8.4. Exposure of the vesicles to periodate, protein coupling and separation from unbound protein by a novel method of flotation is discontinuous dextran gradients does not release the vesicle contents when performed at pH 8.4. Studies on the oxidation of neutral glycolipid-containing vesicles, and on the oxidation of encapsulated glycerol 1-phosphate show that periodate influx into neutral vesicles during a 4 h exposure is appreciable at pH 5.5 but not at pH 8.4. Under optimal conditions, approx. 20% of the protein may be coupled to vesicles, and a ratio of 100--200 microgram of protein/mumol of lipid is readily achieved. This method will be of great importance for the antibody-mediated targeting of vesicles to cells.

Animals↗
PubMed1980

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↗
PubMed1980

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↗
PubMed1980

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↗
PubMed1980

Preparation of unilamellar liposomes of intermediate size (0.1-0.2 mumol) by a combination of reverse phase evaporation and extrusion through polycarbonate membranes.

Liposomes can be prepared by a combination of reverse phase evaporation and sequential extrusion through polycarbonate membranes. The vesicles have diameters in the range 0.05-0.5 micron and are mostly unilamellar as indicated by electon microscopy, capture volume, and availability of reactive groups to periodate oxidation. Sequential extrusion leads to a decrease in the encapsulation efficiency by 2-4-fold, depending upon the lipid composition. The inclusion of cholesterol at a 1 : 1 molar ratio of cholesterol-to-phospholipid increases both the mean size and the size heterogeneity of the liposomes as measured by negative-stain electron microscopy. The mean size of vesicles with an equal molar ratio of cholesterol-to-phospholipid after extrusion through a 0.1 micron membrane is 0.140 micron. Vesicles composed of phosphatidylglycerol/phosphatidylcholine (1 : 4) have a mean size of 0.08 micron after extrusion through a 0.1 micron membrane. The intermediate-size (0.1-0.2 micron) vesicles formed by this process have an aqueous space-to-lipid ratio of 3 : 5 and capture between 12 and 25% of the aqueous phase. The procedure is relatively simple, rapid, and yields almost quantitative recovery of vesicles that encapsulate a large percentage of the total aqueous space.

Carbonates↗
PubMed1980

Fluorescence studies on the mechanism of liposome-cell interactions in vitro.

Sonicated unilamellar liposomes containing fluorescent lipid analogs or biotinyl phosphatidylethanolamine as a ligand for fluorescein avidin have been used to study the mechanism of interaction of phospholipid vesicles with eucaryotic cells. Microscopy revealed that after short incubations the fluorescence was associated with the cell surface in a punctate as opposed to a uniform staining pattern. Fluid vesicles, regardless of charge, were found to associate with cells to the same degree. Solid neutral and negatively charged vesicles associated to a 3-fold greater extent, while solid positively charged vesicles associated to a 10-fold greater extent than fluid vesicles. Fluorescence recovery after photobleaching, a technique used to measure the lateral mobility of cell surface components, was used to measure the lateral mobility of the associated fluorescence probes. No recovery was observed, implying that greater than 90% of the fluorescent lipid analogs are not free to diffuse over distances of the order of 1 micrometer. When these analogs were introduced into the cell membrane by an ethanol-injection technique, rapid and full recovery after photobleaching was observed. This can be accounted for by a lateral diffusion coefficient characteristic of phospholipids in model and biomembranes. The image and photobleaching results suggest that the majority of liposomes that become cell-associated under the conditions used here are adsorbed on the surface. The consequences of this binding for liposome-mediated delivery of molecules into the cytoplasm or plasma membrane of the cell are discussed.

Animals↗
PubMed1979

Ca2+-induced fusion of phospholipid vesicles monitored by mixing of aqueous contents.

Ca2+ has a central role in various cellular phenomena involving membrane fusion. However, little is known about the mechanisms involved. Model membrane systems such as phospholipid vesicles have been used extensively to study the mechanism of membrane fusion at the molecular level. For example, phosphatidylserine (PS) vesicles have been shown to undergo massive aggregation and structural rearrangements on additon of Ca2+, with eventual formation of large cochleate structures. Although these structures do not retain appreciable internal volume, their formation has been proposed to result from fusion of the initial vesicles. The significance of the PS--Ca2+ system as a model for biological membrane fusion has been questioned recently by Ginsberg. Based on the observation that divalent cations induce the release of contents from PS vesicles but fail to bring about the uptake of a marker from the medium, he proposes that the vesicles are ruptured completely during interaction with divalent cations and reassemble subsequently to form large non-vesicular structures. The present study demonstrates that the question raised by Ginsberg is not particularly relevant to the phenomenon concerned, and that his experimental observations do not allow the exclusive conclusion that Ca2+ induces lysis of PS vesicles rather than fusion.

Calcium↗
PubMed1979

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 micron membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 micron while maintaining an acceptable level of encapsulation of the aqueous phase.

Carbonates↗
PubMed1979

Complex phase mixing of phosphatidylcholine and phosphatidylserine in multilamellar membrane vesicles.

The phase mixing in dipalmitoyl phosphatidylcholine and bovine brain phosphatidylserine mixtures suspended in aqueous salt solutions was studied by differential scanning calorimetry, freeze-fracture electron microscopy and X-ray and electron diffraction. The pure dipalmitoyl phosphatidylcholine has two well-defined solidus phases P beta' and L beta' and a liquidus phase L alpha while the pure phosphatidylserine has a broad transition from L beta to L alpha. The mixture exhibits some dipalmitoyl phosphatidylcholine characteristics up to 30% phosphatidylserine when pure P beta' phase no longer exists. Phase mixing is observed at all compositions. An addition of 3% phosphatidylserine is found to be sufficient to destroy the sharp L beta'--P beta' transition observed in pure dipalmitoyl phosphatidylcholine. The transition between L beta' and P beta' in these mixtures is displaced to lower temperatures and becomes gradual as depicted by all three techniques, but freeze-fracture gives more definite information of the continuous transition. The most striking observation on the morphology of the mixed membranes (3--30% phosphatidylserine in dipalmitoyl phosphatidylcholine) is the presence of banded patterns (P beta') at temperatures well below the main transition peak as detected by differential scanning calorimetry.

Calorimetry, Differential Scanning↗
PubMed1979

The use of aqueous space markers to determine the mechanism of interaction between phospholipid vesicles and cells.

A method has recently been introduced that quantitates the extent of phospholipid vesicle-cell interactions by following the amount of a vesicle-entrapped water-soluble fluorescent probe, carboxyfluorescein (CF) that becomes cell associated (Weinstein, J.N., Yoshikami, S., Henkart, P., Blumenthal, R. and Hagins, W.A. (1977) Science 195, 489--492). We have characterized some of the properties of this probe in sonicated phospholipid vesicles. The CF undergoes a pH-dependent quenching as previously reported and both a pH- and temperature-dependent efflux from vesicles. Decreasing the pH from 7.4 to 5.0 results in almost a 100-fold increase in CF efflux from the vesicles. The simultaneous measurement of cell-associated tritiated lipid and CF fluorescence reveals a discrepancy between the two markers with the tritiated phospholipid becoming associated to 5--10-fold greater extent than the CF. In the presence of cells the leakage of CF from vesicles increases from 1.5- to 10-fold depending on the vesicle composition. This data suggests that interpretations of cell-vesicle interactions followed by the CF technique or other aqueous space markers should be done with caution. However, in experiments where the leakage of CF from vesicles can be controlled, the technique can provide useful information.

Animals↗
PubMed1979

Studies on the mechanism of membrane fusion: evidence for an intermembrane Ca2+-phospholipid complex, synergism with Mg2+, and inhibition by spectrin.

The interaction of Ca2+ and Mg2+ with phosphatidylserine (PS) vesicles in 0.1 M NaCl aqueous solution was studied by equilibrium dialysis binding, X-ray diffraction, batch microcalorimetry, kinetics of cation-induced vesicle aggregation, release of vesicle contents, and fusion. Addition of either cation causes aggregation of PS vesicles and produces complexes with similar stoichiometry (1:2 cation/PS) at saturating concentrations, although the details of the interactions and the resulting complexes are quite different. Addition of Ca2+ to PS vesicles at T greater than or equal to 25 degrees C induces the formation of an "anhydrous" complex of closely apposed membranes with highly ordered crystalline acyl chains and a very high transition temperature (Tc greater than 100 degrees C). The formation of this complex is accompanied by a release of heat (5.5 kcal/mol), rapid release of vesicle contents, and fusion of the vesicles into larger membranous structures. By contrast, addition of Mg2+ produces a complex with PS which is much more hydrated, has no crystallization of the acyl chains at T greater than or equal to 20 degrees C, and has comparatively little fusion. Studies with both Ca2+ and Mg2+ added simultaneously indicate that there is a synergistic effect between the two cations, which results in an enhancement of the ability of Ca2+ to form its specific complex with PS at lower concentrations. The presence of the erythrocyte protein "spectrin" inhibits this synergism and interferes with the formation of the specific PS/Ca complex. It also inhibits the fusion of PS vesicles. It is proposed that the unique PS/Ca complex, which involves close apposition of vesicle membranes, is an intermembrane "trans" complex. We further propose that such a complex is a key step for the resultant phase transition and fusion of PS vesicles. By contrast, the PS/Mg complex is proposed to be a "cis" complex with respect to each membrane. The results are discussed in terms of the mechanism of membrane fusion.

Calcium↗
PubMed1979

Specificity of Na+ binding to phosphatidylserine vesicles from a 23Na NMR relaxation rate study.

23Na NMR relaxation rate measurements show that Na+ binds specifically to phosphatidylserine vesicles and is displaced partially from the binding site by K+ and Ca2+ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na+ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na+-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na+) fall in the range 0.4--1.2 M-1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na+ at 0.1 M Na+ concentration.

Calcium↗
PubMed1979

Interaction of mitochondrial malate dehydrogenase monomer with phospholipid vesicles.

The association between bovine and porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) and phospholipid vesicles was investigated. At concentrations at which malate dehydrogenase exists as a dimer, entrapment within the aqueous compartment but not binding of the 14C-labelled enzyme was observed. The dissociated enzyme was labile to moderate heat and to p-chloromercuribenzoate, but in both cases inactivation was decreased by incubation with suspensions of charged phospholipid vesicles. This suggested an interaction between enzyme subunits and phospholipid, and this was confirmed by direct binding measurements and by studies that followed changes in the fluorescein-labelled enzyme. The circular-dichroism spectra of the enzyme indicated a high alpha-helix content, and suggested that a small conformational change occurred when the enzyme dissociated. Fluorescence data also suggested less-rigid molecules after dissociation. A possible mechanism, based on the flexibility of enzyme monomer and its interaction with phospholipids, by which mitochondrial matrix enzymes are specifically localized in cells, is discussed.

Animals↗
PubMed1979

The introduction of poliovirus RNA into cells via lipid vesicles (liposomes).

Large unilamellar vesicles (LUV) composed of phosphatidylserine are capable of encapsulating poliovirus ribonucleic acid (RNA) and delivering it efficiently to cells in an infectious form. The biological activity of vesicle-entrapped poliovirus RNA was 1-2 x 10(4) plaque forming units/nanogram (pfu/ng) and appeared to be enhanced by ribonuclease treatment of the vesicle preparations (infectivity = 1-2 x 10(5) pfu/ng). Vesicle-mediated RNA infection produced equivalent titers in primate and nonprimate cells. Moreover, the data strongly suggest that the ratio of molecules per infectious unit is close to one when the RNA is properly delivered to the cell. A comparative study of LUV and multilamellar vesicles (MLV) indicates that LUV deliver their contents to the cell cytoplasm much more efficiently than MLV. LUV-entrapped poliovirus RNA produced infectious titer 10-100 fold higher than comparable RNA preparations delivered to cells by other techniques.

Animals↗
PubMed1979

Liquid diffraction analysis of the model membrane system. Egg lecithin + myelin protein (N-2).

The scattering from the model membrane system, egg lecithin + myelin protein (N-2), has been analyzed by liquid diffraction methods. It is found that by manipulating the protein-lipid ratio, the scattering domains of the protein and lipid can be identified. The multilayer contribution can also be identified by its position and concentration behavior in both the intensity pattern and its Fourier transform. When the multilayer and protein components are subtracted, the phospholipid scattering and an interaction term are left: these two can be resolved by a reasonable assessment of their relative magnitude in the boundary region where they overlap. The interaction term can then be used to determine the most probably position of the protein in the membrane. The deconvoluted protein and lipid transforms can then be combined in the proper way to obtain the electron density profiles. The resolution of the interaction term is not yet complete, but a method for accomplishing this is discussed.

Egg Yolk↗
PubMed1979

A model for the study of the oral administration of peptide hormones.

The intragastric administration of lysine vasopressin (LVP) to rats is used as a model to study the biological activity of orally administered peptide hormones. Using a modification of the antidiuretic assay of Sawyer, LVP given by stomach tube caused a significant antidiuresis that was dose dependent in doses of 300 to 2000 mU. The simultaneous administration of the protease inhibitor, Trasylol, increased the antidiuretic effect of LVP. The synthetic peptide (1-deamino, 4 valine)-8-D-arginine-vasopressin also caused a dose-dependent prolonged and significant antidiuresis. No pressor effect was observed after intragastric administration of LVP in doses up to 40 U/rat. We are now using this model to test other procedures for enhancing the activity of lysine vasopressin administered in the gastrointestinal tract such as encapsulation into liposomes. The information gained with vasopressin will then be applied to insulin with the ultimate goal of making oral administration practical.

Administration, Oral↗