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D Papahadjopoulos

Publications and source records attributed to D Papahadjopoulos.

At least 55 records · Page 3Linked to original sources
PubMed1992

Sterically stabilized liposomes. Reduction in electrophoretic mobility but not electrostatic surface potential.

The electrophoretic mobility of liposomes containing a negatively charged derivative of phosphatidylethanolamine with a large headgroup composed of the hydrophilic polymer polyethylene glycol (PEG-PE) was determined by Doppler electrophoretic light scattering. The results show that this method is improved by the use of measurements at multiple angles to eliminate artifacts and that very small mobilities can be measured. The electrophoretic mobility of liposomes with 5 to 10 mol% PEG-PE is approximately -0.5 mu ms-1/Vcm-1 regardless of PEG-PE content compared with approximately -2 mu ms-1/Vcm-1 for similar liposomes but containing 7.5% phosphatidylglycerol (PG) instead of PEG-PE. Measurements of surface potential by distribution of an anionic fluorescent probe show that the PEG-PE imparts a negative charge identical to that by PG, consistent with the expectation of similar locations of the ionized phosphate responsible for the charge. The reduced mobility imparted by the surface bound PEG is attributed to a mechanism similar to that described for colloidal steric stabilization: hydrodynamic drag moves the hydrodynamic plane of shear, or the hydrodynamic radius, away from the charge-bearing plane, that of the phosphate moities. An extended length of approximately 50 A for the 2,000 molecular weight PEG is estimated from the reduction in electrophoretic mobility.

Biophysical Phenomena↗
PubMed1991

Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy.

The results obtained in this study establish that liposome formulations incorporating a synthetic polyethylene glycol-derivatized phospholipid have a pronounced effect on liposome tissue distribution and can produce a large increase in the pharmacological efficacy of encapsulated antitumor drugs. This effect is substantially greater than that observed previously with conventional liposomes and is associated with a more than 5-fold prolongation of liposome circulation time in blood, a marked decrease in uptake by tissues such as liver and spleen, and a corresponding increased accumulation in implanted tumors. These and other properties described here have expanded considerably the prospects of liposomes as an effective carrier system for a variety of pharmacologically active macromolecules.

Animals↗
PubMed1991

Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times.

Therapeutic applications of intravenously injected liposomes have been limited by their rapid clearance from the bloodstream and their uptake by the macrophage cells of the liver and spleen (RES). Recently, however, liposomes which substantially evade the rapid uptake by the RES have been introduced. Since these liposomes exhibit dramatically different pharmacokinetics and biodistribution, new therapeutic opportunities have appeared. These include enhanced efficacy of antineoplastic agents against tumors, sites of inflammation, and targeting ligand-coupled liposomes to extravascular targets. Despite extensive experimental work, the mechanism underlying the ability of liposomes to avoid the rapid uptake by the RES is still not fully understood. Our approach is an alternative to seeking the answers in complex differential interactions of liposomes with various components of blood. We believe that the effect can be easily explained, at least in qualitative terms, by the fundamental principles of colloid stability. In this communication, we propose that steric stabilization of liposomes is responsible for their prolonged circulation times. We propose that stabilization results from local surface concentration of highly hydrated groups that sterically inhibit both electrostatic and hydrophobic interactions of a variety of blood components at the liposome surface.

Animals↗
PubMed1991

Increased binding of liposomes to cells by electric treatment.

The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.

Arylsulfonates↗
PubMed1991

Light microscopic localization of silver-enhanced liposome-entrapped colloidal gold in mouse tissues.

Silver-enhanced liposome-entrapped colloidal gold was developed for light microscopic localization of liposomes. Preparation of colloidal gold entrapped in liposomes was achieved by a modified method of Hong, et al. (1983) Biochim. Biophys. Acta 732, 320-323). In this report, a gold chloride/citrate solution of low pH (3.4) was used to inhibit the formation of gold granules during the liposome preparation. The diameter of most liposomes ranged from 80 to 100 nm. Following liposome preparation, the pH was adjusted to 6, and the temperature increased to 55 degrees C. The majority of the liposomes contained one to three gold particles. Liposomes were injected into mice via tail vein; 24 h later, tissues were collected. Sections were processed for silver enhancement of the gold particles and examined by light microscopy. Silver-enhanced gold particles were clearly observed in both liver and implanted tumor. Localization was confirmed by electron and fluorescence microscopy. Thus, we have shown that silver enhancement of colloidal gold liposomes is a direct and sensitive method for tracing the fate of liposomes in vivo, providing minimal background interference and a good definition of various cell types.

Animals↗
PubMed1991

Interactions of annexins with membrane phospholipids.

The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions.

Animals↗
PubMed1990

Effect of liposome composition and other factors on the targeting of liposomes to experimental tumors: biodistribution and imaging studies.

We have examined the distribution of radiolabeled liposomes in tumor-bearing mice after i.v. injection. Two mouse tumors (B16 melanoma, J6456 lymphoma) and a human tumor (LS174T colon carcinoma) inoculated i.m., s.c., or in the hind footpad were used in these studies. When various liposome compositions with a mean vesicle diameter of approximately 100 nm were compared using a radiolabel of gallium-67-deferoxamine, optimal tumor localization was obtained with liposomes containing a phosphatidylcholine of high phase-transition temperature and a small molar fraction of monosialoganglioside or hydrogenated phosphatidylinositol (HPI). At 24 h after injection, average values of tumor uptake higher than 10% of the injected dose per g and liver-to-tumor ratios close to 1 were reproducibly obtained. Increasing the molar fraction of HPI from 9% to 41% of the total phospholipid resulted in enhancement of liver uptake and decrease of tumor uptake. Methodological aspects that influence vesicle size appear to affect significantly liposome localization in the tumor. However, varying the phospholipid dose within a 10-fold range caused only minor changes in the percent of injected dose recovered in the tumor. A high uptake by tumors was also observed using other radiolabels [[3H]inulin and indium-111-labeled bleomycin (111In-Bleo)] in monosialoganglioside- and HPI-containing liposomes. In the case of 111In-Bleo, encapsulation in liposomes resulted in approximately 20- to 40-fold increase in tumor accumulation of the radiolabel at 24 h after injection. The marked localization of liposomes in the mouse footpad inoculated with tumor as opposed to the contralateral mock-injected footpad was also documented by imaging experiments with gallium-67-deferoxamine and 111In-Bleo-labeled liposomes. These results support the contention that some glycolipid-containing liposomes previously shown to have long circulating half-lives accumulate significantly in a variety of tumors and are promising tools for the delivery of anti-tumor agents.

Animals↗
PubMed1990

Endocytosis of liposomes by macrophages: binding, acidification and leakage of liposomes monitored by a new fluorescence assay.

The interaction of liposomes with macrophage cells was monitored by a new fluorescence method (Hong, K., Straubinger, R.M. and Papahadjopoulos, D., J. Cell Biol. 103 (1986) 56a) that allows for the simultaneous monitoring of binding, endocytosis, acidification and leakage. Profound differences in uptake, cell surface-induced leakage and leakage subsequent to endocytosis were measured in liposomes of varying composition. Pyranine (1-hydroxypyrene-3,6,8-trisulfonic acid, HPTS), a highly fluorescent, water-soluble, pH sensitive dye, was encapsulated at high concentration into the lumen of large unilamellar vesicles. HPTS exhibits two major fluorescence excitation maxima (403 and 450 nm) which have a complementary pH dependence in the range 5-9: the peak at 403 nm is maximal at low pH values while the peak at 450 nm is maximal at high pH values. The intra- and extracellular distribution of liposomes and their approximate pH was observed by fluorescence microscopy using appropriate excitation and barrier filters. The uptake of liposomal contents by cells and their subsequent exposure to acidified endosomes or secondary lysosomes was monitored by spectrofluorometry via alterations in the fluorescence excitation maxima. The concentration of dye associated with cells was determined by measuring fluorescence at a pH independent point (413 nm). The average pH of cell-associated dye was determined by normalizing peak fluorescence intensities (403 nm and 450 nm) to fluorescence at 413 nm and comparing these ratios to a standard curve. HPTS-containing liposomes bound to and were acidified by a cultured murine macrophage cell line (J774) with a t1/2 of 15-20 min. The acidification of liposomes exhibited biphasic kinetics and 50-80% of the liposomes reached an average pH lower than 6 within 2 h. A liposomal lipid marker exhibited a rate of uptake similar to HPTS, however the lipid component selectively accumulated in the cell; after an initial rapid release of liposome contents, 2.5-fold more lipid marker than liposomal contents remained associated with the cells after 5 h. Coating haptenated liposomes with antibody protected liposomes from the initial release. The leakage of liposomal contents was monitored by co-encapsulating HPTS and p-xylene-bis-pyridinium bromide, a fluorescence quencher, into liposomes. The time course of dilution of liposome contents, detected as an increase in HPTS fluorescence, was coincident with the acidification of HPTS. The rate and extent of uptake of neutral and negatively charged liposomes was similar; however, liposomes opsonized with antibody were incorporated at a higher rate (2.9-fold) and to a greater extent (3.4-fold).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Endocytosis and intracellular fate of liposomes using pyranine as a probe.

Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (lambda EX) at 350-415 nm but highly pH dependent with lambda EX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with lambda EX at 413 nm and low FEM with lambda EX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of approximately 6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with lambda EX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with lambda EX at 350-405 nm but decreased with lambda EX at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 degrees C. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic.

Animals↗
PubMed1990

Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes.

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.

Calcium↗
PubMed1990

Molecular mechanisms of calcium-induced membrane fusion.

We have reviewed studies on calcium-induced fusion of lipid bilayer membranes and the role of synexin and other calcium-binding proteins (annexins) in membrane fusion. We have also discussed the roles of other cations, lipid phase transitions, long chain fatty acids and other fusogenic molecules. Finally, we have presented a simple molecular model for the mechanism of lipid membrane fusion, consistent with the experimental evidence and incorporating various elements proposed previously.

Animals↗
PubMed1990

Liposomes designed to avoid the reticuloendothelial system.

Recent work has revealed some new and important characteristics of liposomes: Inclusion of certain glycolipids within liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol drastically prolongs the circulation time and reduces their uptake by liver and spleen. Concomitantly, their accumulation in several implanted tumors is substantially increased. These studies suggest that controlling the circulation time of liposomes and limiting their non-specific uptake by the Reticuloendothelial system (RES) opens up new opportunities for achieving specific targeting to tumors in vivo, with both diagnostic and therapeutic possibilities.

Animals↗
PubMed1989

Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times.

We have previously reported on liposome formulations with reduced uptake by the reticuloendothelial system, prolonged circulation time, and enhanced accumulation in transplanted tumors. One of these formulations, consisting of hydrogenated phosphatidylinositol (HPI), hydrogenated phosphatidylcholine (HPC), and cholesterol (Chol) (HPI-HPC-Chol), and a control formulation, consisting of phosphatidylglycerol (PG), phosphatidylcholine (PC), and Chol (PG-PC-Chol), were loaded with doxorubicin (DXR) and injected intravenously into BALB/c mice for pharmacokinetic studies. Although both formulations were similar in vesicle size, fraction of negatively charged lipid, and drug-to-lipid ratio, there were striking pharmacokinetic differences. DXR was cleared much faster in PG-containing liposomes than in HPI-containing liposomes. Liposome-associated drug was detectable in plasma up to 5 hours after injection in the case of PG-PC-Chol and as late as 72 hours after injection in the case of HPI-HPC-Chol. In agreement with the plasma clearance curves, peak drug concentrations in the liver were observed at 1/2, 5, and 24 hours after injection for free DXR, DXR in PG-PC-Chol, and DXR in HPI-HPC-Chol, respectively. Both types of liposomes reduced considerably the amount of drug accumulating in the heart compared with that accumulating after injection of free DXR.

Animals↗
PubMed1989

Role of ligand in antibody-directed endocytosis of liposomes by human T-leukemia cells.

The rate of uptake and intracellular processing of ligand-directed drug carriers may depend heavily on the endocytic pathway of the target antigen. We examined the role of the target antigen and type of antibody-liposome linkage in determining endocytosis of liposomes by three human T-cell leukemias, Jurkat, CEM, and Molt-4. Liposome-cell binding and internalization over time were studied using two independent assays for intracellular delivery of liposome contents: a new fluorescence assay using a pH-sensitive fluorescent dye; and a growth inhibition assay for delivery of cytotoxic drug, methotrexate-gamma-aspartate. Liposomes targeted against the transferrin receptor showed greater surface binding, internalization, and growth inhibition than liposomes targeted against the T-cell surface antigens, CD2, CD3, or CD5. Furthermore, liposomes made by conjugating the targeting antibody directly to the liposome surface were more efficiently internalized and retained than were liposomes linked to antibody-coated cells via Protein A. Selection of the type of antibody-liposome conjugate as well as the appropriate surface receptor to facilitate endocytosis is essential in antibody-directed drug treatment of cancer.

Antibodies, Monoclonal↗
PubMed1989

Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change.

The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Monolayer coupling in phosphatidylserine bilayers: distinct phase transitions induced by magnesium interacting with one or both monolayers.

We have investigated the thermotropic behavior of phospatidylserine bilayers interacting with Mg2+ either on one side or both sides, using differential scanning calorimetry. Large unilamellar vesicles (LUV) of phosphatidylserine exposed to Mg2+ on the external side only displayed an upward shift of the gel-liquid transition temperature (Tm) of about 6-8 degrees C relative to the Tm of LUV in Na+. Mg2+ was shown not to enter the vesicle interior, by means of fluorescence measurements on encapsulated 8-hydroxyquinoline-5-sulfonate. Multilamellar vesicles prepared in the presence of Mg2+, or vesicles prepared by Mg2+-induced fusion of small unilamellar vesicles, had Tm values that were shifted upward by about 16-17 C degrees. When the latter preparation was treated with EDTA to produce vesicles with Mg2+ inside and Na+ outside, the Tm was found to be shifted again by only 6-8 degrees C. These observations indicate that the monolayer interacting with Na+ fluidizes the monolayer interacting with Mg2+, and that the latter tends to solidify the former. The two monolayers thus appear to be coupled, possibly by hydrocarbon chain interdigitation.

Animals↗