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Biomedical subjects

D Papahadjopoulos

Publications and source records attributed to D Papahadjopoulos.

At least 109 records · Page 6Linked to original sources
PubMed1983

Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalization in coated vesicles.

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.

Ammonium Chloride↗
PubMed1983

Antibody-targeted liposomes: increase in specific toxicity of methotrexate-gamma-aspartate.

Liposomes conjugated with anti-H2Kk antibody associate with L929 murine fibroblasts in 6- to 20-fold greater amount than do nonspecific liposomes. The ability of methotrexate-gamma-aspartate to inhibit L929 growth is increased 10-fold when encapsulated in targeted liposomes but is decreased to 50% when encapsulated in liposomes with no specificity for the target cells. Ammonium chloride inhibits the effects of the encapsulated but not the free drug. Soluble antibody does not inhibit the efficacy of targeted vesicles, but empty targeted vesicles do inhibit the efficacy. The compound in both targeted and nontargeted vesicles has a minimal effect on BALB/c3T6 fibroblasts. These results demonstrate the potential of antibody-targeted liposomes and the importance of selecting liposome-dependent cytotoxic agents.

Animals↗
PubMed1982

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion.

We have examined the early events in Ca2+-induced fusion of large (0.2 microns diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.

Animals↗
PubMed1982

Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting.

Rabbit Fab' antibody fragments were covalently couple to preformed large unilamellar vesicles using a new sulfhydryl-reactive phospholipid derivative N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE). A highly efficient reaction between the sulfhydryl group on each Fab' fragment and the maleimide moiety of MPB-PE molecules incorporated at a low concentration in vesicle bilayers led to the formation of a highly stable Fab'-vesicle linkage. Coupling ratios in excess of 250 micrograms of Fab'/mumol of vesicle phospholipid were reproduciably obtained without vesicle aggregation. Bound Fab' fragments did not elute from vesicles in serum or in the presence of reducing agents (dithiothreitol or mercaptoethanol). Vesicles bearing Fab' fragments raised against specific human erythrocyte surface determinants bound selectively to human erythrocytes under physiological conditions (isotonic medium containing 50% human serum, pH 7.4) with minimal leakage of vesicle contents. Advantages of the present coupling method are discussed in relationship to our effects to optimize the properties of liposomes as a carrier system.

Animals↗
PubMed1982

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.

Animals↗
PubMed1982

Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.

The effect of synexin (an adrenal medullary protein) on the kinetics of Ca2+- and Mg2+-mediated membrane fusion was examined. Membrane fusion was studied by monitoring intermixing of the aqueous contents of phospholipid vesicles. Synexin facilitated Ca2+-mediated, but not Mg2+-mediated, fusion of phosphatidate/phosphatidylethanolamine (1:3) and phosphatidate/phosphatidylserine/phosphatidylethanolamine/cholesterol (1:2:3:2) vesicles. The threshold concentration of Ca2+ for fusion was decreased to approximately equal to 10 microM in the presence of synexin at 6 micrograms/ml and 1.5 mM Mg2+ in vesicle suspensions containing 50 microM lipid. This effect of synexin was drastically inhibited by including 25% phosphatidylcholine (mol/mol) in the vesicle membrane. It is proposed that the Ca2+-dependent lipid-specific enhancement of membrane fusion by synexin contributes to an increase in the sensitivity of specific intracellular membranes to Ca2+ with respect to fusion.

Animals↗
PubMed1982

Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: A sensitive assay for monitoring liposome-protoplast interactions.

Tobacco mosaic virus (TMV) RNA was encapsulated in large, unilamellar phospholipid vesicles (liposomes), and the encapsulated TMV RNA was shown to be infectious when incubated with tobacco protoplasts under appropriate conditions. Maximal virus production in protoplasts was observed after their incubation with TMV RNA entrapped in phosphatidylserine/cholesterol liposomes. Infection was dependent on the presence of polyalcohols in the incubation mixture. Other parameters, such as the extent of vesicle binding, the cell-induced leakage of vesicle contents, and the degree of liposome toxicity were shown to be important in determining the efficiency of infectivity. Liposome-mediated delivery offers an efficient and reproducible method for introducing RNA into plant protoplasts.

Journal Article↗
PubMed1982

Intermembrane contact affects calcium binding to phospholipid vesicles.

Binding of Ca2+ to liposomes composed of phosphatidylserine (PtdSer) was analyzed by potentiometric titrations. Ca2+ binding to large unilamellar PtdSer vesicles was saturable at a stoichiometry of 1:2 (Ca2+/PtdSer). At approximately 6 X 10(-4) M [Ca2+]free, the binding curve exhibited a discontinuity that can be attributed to the formation of a Ca2+/PtdSer complex with a higher affinity. When both Ca2+ and Mg2+ are present, depending on the relative concentrations, Mg2+ can either complete or can enhance Ca2+ binding. Concomitant to the enhanced binding, the vesicle suspension was found to aggregate, suggesting that close contact of membranes is a prerequisite for the abrupt change in affinity. This concept was tested by binding studies with liposomes of mixed composition. It was found that the incorporation of 50 mol% phosphatidylethanolamine (PtdEtn) into PtdSer liposomes produced a similar binding pattern to that of pure PtdSer with a saturable stoichiometry of 1:2 (Ca2+/PtdSer). However, incorporation of 50 mol% phosphatidylcholine (PtdCho) completely abolished the discontinuous shift in affinity and apparent saturation was reached at a stoichiometry of 1:4 (Ca2+/PtdSer). In addition, Ca2+ binding to PtdSer liposomes with 10 mol% galactosylcerebroside was not altered when compared to pure PtdSer, whereas 10 mol% of the glycolipid GL-4 abolished the increased binding. The results are closely correlated with recent findings on the role of the membrane composition in Ca2+-induced fusion of liposomes and argue in favor of a specific Ca2+/PtdSer complex (with 1:2 stoichiometry) forming only at points of close contact between membranes and serving as the trigger for membrane fusion.

Calcium↗
PubMed1981

Control of membrane fusion by phospholipid head groups. I. Phosphatidate/phosphatidylinositol specificity.

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca2+ or Mg2+ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90 degrees light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca2+ and Mg2+ at threshold concentration ranges of 0.03-0.1 mM (Ca2+) and 0.07-0.15 mM (Mg2+) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca2+ or Mg2+ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5-10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.

Calcium↗
PubMed1981

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↗
PubMed1981

Immunospecific targeting of liposomes to cells: a novel and efficient method for covalent attachment of Fab' fragments via disulfide bonds.

An efficient method for covalently cross-linking 50K Fab' antibody fragments to the surface of lipid vesicles is reported. Coupling up to 600 microgram of Fab'/mumol of phospholipid (about 6000 Fab' molecules per 0.2-micrometer vesicle) is achieved via a disulfide interchange reaction between the thiol group exposed on each Fab' fragment and a pyridyldithio-derivative of phosphatidylethanolamine present in low concentration in the membranes of preformed large unilamellar vesicles. The coupling reaction is efficient, proceeds rapidly under mild conditions, and yields well-defined products. Each vesicle-linked Fab' fragment retains its original antigenic specificity and full capacity to bind antigen. We have used Fab' fragments, coupled to vesicles by this method, to achieve immunospecific targeting of liposomes to cells in vitro. Vesicles bearing antihuman erythrocyte Fab' fragments bind quantitatively to human erythrocytes (at multiplicities up to 5000 0.2-micrometer vesicles per cell) while essentially no binding is observed to sheep or ox red blood cells. Vesicle-cell binding is stable over a pH range from 6 to 8 and is virtually unaffected by the presence of human serum (50%). Cell-bound vesicles retain their aqueous contents and can be eluted intact from cells by treatment with reducing agents (dithiothreitol or mercaptoethanol) at alkaline pH.

Animals↗
PubMed1981

Role of synexin in membrane fusion. Enhancement of calcium-dependent fusion of phospholipid vesicles.

Synexin, a soluble adrenal medullary and liver protein which causes calcium-dependent aggregation of isolated chromaffin granules, was isolated and purified according to published procedures. The effects of synexin on the kinetics of membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Synexin lowers the threshold of CA2+ concentration required for fusion of large unilamellar vesicles of phosphatidylserine and a mixture of phosphatidylserine with phosphatidylethanolamine. synexin also increases drastically the initial rate of fusion. the initial rate of fusion increases with the quantity of synexin present in the reaction mixture. In the presence of 1-2 mM Ca2+ and 50 microM phospholipid, synexin at 20 to 40 micrograms/ml increases the rate of fusion by two orders of magnitude. Mg2+ does not support synexin-induced fusion. With vesicles containing a mixture of phosphatidylserine with phosphatidylcholine, synexin enhances aggregation in the presence of CA2+, without promoting fusion. Synexin may play a role in exocytosis by promoting fusion of membranes containing specific phospholipids in the presence of Ca2+.

Adrenal Medulla↗
PubMed1981

Calcium- and magnesium-induced fusion of mixed phosphatidylserine/phosphatidylcholine vesicles: effect of ion binding.

The aggregation, leakage, and fusion of pure PS (phosphatidylserine) and mixed PS/PC (phosphatidylcholine) sonicated vesicles were studied by light scattering, the release of encapsulated carboxyfluorescein, and a new fusion assay which monitors the mixing of the internal compartments of fusing vesicles. On a time scale of 1 min the extent of fusion was considerably greater than leakage. The Ca2+ and Mg2+ concentrations required to induce fusion increased when the PS content of the vesicles was decreased, and/or when the NaCl concentration was increased. Calculations employing a modified Gouy-Chapman equation and experimentally determined intrinsic binding constants of Na+ and Ca2+ to PS were shown to predict correctly the amount of Ca2+ bound in mixed PS/PC vesicles. For vesicles composed of either pure PS or of mixtures with PC in 100 mM NaCl (4:1 and 2:1 PS/PC); the induction of fusion (on a time scale of minutes) occurred when the amount of Ca or Mg bound/PS molecule exceeded 0.35-0.39. The induction of fusion for both pure PS and PS/PC mixed vesicles (with PS exceeding 50%) can be explained by assuming that destabilization of these vesicles requires a critical binding ratio of divalent cations to PS.

Binding Sites↗
PubMed1981

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↗