1-2-dielaidoylphosphatidylethanolamine and fluorexon

Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.

Topics: Animals; Buffers; Cells, Cultured; Drug Delivery Systems; Flow Cytometry; Fluoresceins; Hydrogen-Ion Concentration; Liposomes; Phosphatidylethanolamines; Rats; Rhodamines

We have made a curious observation that the proteolytic enzyme, trypsin, induced a rapid and complete release of the contents of vesicles composed of dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA). Content release at 37 degrees C, monitored by the release of an entrapped fluorescence marker (calcein), was accompanied by an extensive vesicle aggregation. The lytic activity of trypsin on the vesicles depended on pH and liposome composition. The optimal pH for vesicle lysis was below pH 7.4, which was different from the optimal pH for catalytic activity of trypsin. The lytic activity of trypsin was specific for vesicles composed of DOPE and fatty acids such as OA and palmitoleic acid; vesicles composed of dioleoylphosphatidylcholine, N-methyl-DOPE, and OA, or DOPE combined with other negatively charged lipids such as phosphatidylserine and phosphatidic acid were not sensitive to trypsin. Inhibition of enzyme activity by trypsin inhibitors did not abolish the lytic activity, suggesting that the lytic activity of trypsin is not related to the catalytic activity. However, the lytic activity of trypsin on vesicles composed of DOPE and OA was inhibited in the presence of excess vesicles containing negative charges, or by a pretreatment of trypsin with acylating reagent to reduce the positive-charge content of trypsin. These data demonstrate that vesicle aggregation and lysis are the results of electrostatic interactions of positive charges on trypsin and negative charges on the vesicles. Phase separation and transition to nonbilayer phases of the vesicle lipids are likely involved.

Topics: Fluoresceins; Hydrogen-Ion Concentration; Liposomes; Oleic Acid; Oleic Acids; Phosphatidylethanolamines; Phospholipids; Surface Properties; Trypsin

The stability of two-component liposomes composed of the polymerizable 1,2-bis-[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphati dylcholine (SorbPC) and either a phosphatidylethanolamine (PE) or a phosphatidylcholine (PC) were examined via fluorescence leakage assays. Ultraviolet light exposure of SorbPC-containing liposomes forms poly-SorbPC, which phase separates from the remaining monomeric lipids. If the nonpolymerizable lipids are PE's, then the photoinduced polymerization destabilizes the liposome with loss of aqueous contents. The permeability of the control dioleoylPC/SorbPC membranes was not affected by photopolymerization of SorbPC. The photodestabilization of dioleoylPE/SorbPC (3:1) liposomes required the presence of oligolamellar liposomes. NMR spectroscopy of extended bilayers of dioleoylPE/SorbPC (3:1) showed that the photopolymerization lowers the temperature for the appearance of 31P NMR signals due to the formation of isotropically symmetric lipid structures. These observations suggest the following model for the photoinduced destabilization of liposomes composed of PE/SorbPC; photopolymerization induced phase separation with the formation of enriched domains of PE, which allows the close approach of apposed regions of enriched PE lamellae and permits the formation of an isotropically symmetric structure between the lamellae. The formation of such an interlamellar attachment (ILA) between the lamellae of an oligolamellar liposome provides a permeability pathway for the light-stimulated leakage of entrapped water-soluble reagents.

Topics: Drug Stability; Fluoresceins; Liposomes; Magnetic Resonance Spectroscopy; Models, Structural; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Photolysis; Structure-Activity Relationship; Ultraviolet Rays

Acid-sensitive liposomes composed of unsaturated phosphatidylethanolamine (PE) are efficient vehicles for cytoplasmic delivery of the target cells. We have recently shown that liposomes composed of dioleoyl-PE (DOPE) and dipalmitoyl-succinylglycerol (DPSG) retain the acid-sensitivity after exposure to human plasma. In the present work, we have extended these observations to investigate the role of ganglioside GM1 on the blood residence time of these liposomes. Small (d approximately 100 nm) unilamellar liposomes composed of DOPE and DPSG (4:1, molar ratio) became progressively less acid-sensitive when increasing amounts of GM1 were included in the lipid composition. However, partial sensitivity to acid (40-50% release of entrapped contents at pH 4) could be retained up to 5% GM1, even for liposomes which had been exposed to human plasma. Inclusion of GM1 in the lipid composition only slightly increased the release of entrapped contents in the presence of human plasma. The biodistribution of i.v. injected GM1-containing liposomes was studied by following the entrapped 125I-labeled tyraminylinulin marker in Balb/c mice. Inclusion of up to 5% GM1 showed a transient increase in the blood level and a concomitant decrease of liver and spleen uptake of liposomes. Thus, these liposomes are pH-sensitive, plasma-stable and show a relatively prolonged residence time in circulation. They are potentially significant drug carriers in vivo.

Topics: Animals; Drug Stability; Fluoresceins; G(M1) Ganglioside; Humans; Hydrogen-Ion Concentration; Inulin; Kinetics; Liposomes; Liver; Mice; Mice, Inbred BALB C; Phosphatidylethanolamines; Plasma; Spleen; Triglycerides; Tyramine

We have previously reported that small unilamellar liposomes (d less than or equal to 200 nm) composed of dioleoylphosphatidylethanolamine and oleic acid can be stabilized by incubating with normal human plasma (Liu and Huang, Biochemistry 1989, in press). The stabilized liposomes were very stable even under relatively harsh conditions such as extreme pH, high salt and trypsin treatment. Fluorescence depolarization of diphenylhexatriene showed that the stabilized liposome had a high microviscosity in the lipid core, which did not decrease even after the majority of proteins were removed by trypsin. These data suggest that plasma proteins inserted into the lipid bilayer are probably responsible for the stabilization activity. After i.v. injection into mouse, stabilized liposomes showed a relatively low affinity to liver and spleen as compared to a conventional liposome composition.

Topics: Animals; Blood Proteins; Drug Stability; Fluoresceins; Humans; Lipid Bilayers; Liposomes; Male; Mice; Mice, Inbred BALB C; Oleic Acid; Oleic Acids; Phosphatidylethanolamines; Plasma; Trypsin; Viscosity

Large unilamellar liposomes prepared by an octyl glucoside-dialysis method were examined for stability at 37 degrees C in the presence or absence of human plasma, using the release of the entrapped calcein as a fluorescence marker. The liposomes were acid-sensitive as they were composed of dioleoylphosphatidylethanolamine, oleic acid and cholesterol. The stability of the liposomes in the absence of plasma was significantly enhanced with increasing cholesterol content. However, the maximal calcein release at pH 5 decreased linearly with increasing cholesterol content of the liposome, indicating that cholesterol had reduced the acid-sensitivity of the liposomes. In the presence of human plasma, calcein release exhibited a biphasic behavior with a fast (plasma-sensitive) and a slow (plasma-resistant) component. Inclusion of cholesterol in the liposomes resulted in an increased proportion of the plasma-resistant component. Liposomes pretreated with human plasma, after removal of excess plasma and the released calcein by gel-filtration, showed a remarkable stability both in the presence and absence of human plasma. The acid-sensitivity of the plasma-treated liposomes with 40% cholesterol was the same as that of the untreated liposomes. These results are discussed in terms of the mechanism by which these liposomes deliver their contents to the cytoplasm of the cells via the endocytic pathway, a known biological activity of the type of liposome described here.

Topics: Blood; Chemical Phenomena; Chemistry, Physical; Cholesterol; Detergents; Dialysis; Fluoresceins; Glucosides; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Liposomes; Oleic Acid; Oleic Acids; Phosphatidylethanolamines

The importance of the protein-linked carbohydrates for the stabilization of dioleoylphosphatidylethanolamine (DOPE) bilayers has been investigated using glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, as a stabilizer. Two major types of glycophorin, differing in the sialic acid content, were used in the study. Type MM contains 19.2 +/- 2.5 sialic residues per molecule of glycophorin, and type NN contains 10.8 +/- 1.2. Type MM could stabilize DOPE bilayers at 0.5 mol%, whereas type NN was unable to do so even at 1 mol%. The importance of the sialic acid content to the stabilization activity of glycophorin was further confirmed by the observation that the neuraminidase-treated type MM showed a lower stabilization activity than the untreated type. Since type NN had no stabilizing activity, we attempted to couple a trisaccharide, NeuNAc----Gal----Glc, to type NN by reductive amination. 2.5 +/- 0.8 saccharide chains were added per molecule of type NN. The trisaccharide-attached type NN showed a greater stabilization activity than the parent type NN molecule, indicating again that the sialic acid content of the stabilizer molecule determines the stabilization activity. Addition of wheat-germ agglutinin (WGA), which binds to the sialic acid residues of a glycoprotein, to type MM-stabilized liposomes caused rapid aggregation and destabilization of liposomes, resulting in leakage of an entrapped marker, calcein. The aggregation increased with increasing amount of the lectin; however, the leakage rate was maximum at an optimum concentration of WGA. These results are discussed in terms of the role of sialic acid in the interfacial hydration and charge repulsion which determines the DOPE bilayer stability.

Topics: Carbohydrate Sequence; Chromatography, Affinity; Fluoresceins; Glycophorins; Humans; Indicators and Reagents; Lipid Bilayers; Molecular Sequence Data; Molecular Weight; Oligosaccharides; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Sialoglycoproteins

A novel type of liposome bilayer destabilization catalyzed by the enzyme, beta-galactosidase, is described. Unsaturated phosphatidylethanolamine (PE), an HII-phase-forming lipid, does not form stable liposomes at physiological temperature and pH. However, stable unilamellar liposomes can be prepared by mixing PE with a minimum of 5 mol% ganglioside GM1, a micellar-phase-forming lipid. Treatment of these GM1/PE liposomes with beta-galactosidase induces a rapid leakage (3-6 min) of the entrapped fluorescent dye, calcein. The studies indicate that liposome destabilization is the result of catalytic degradation of GM1, rather than a stoichiometric binding of GM1 by beta-galactosidase. Kinetic data indicate that the destabilization takes place via liposome collision. This simple, rapid method of liposome destabilization by beta-galactosidase will be useful in designing a liposome-based signal amplification mechanism for assays involving enzymes.

Topics: beta-Galactosidase; Drug Stability; Fluoresceins; Fluorescent Dyes; G(M1) Ganglioside; Galactosidases; Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers; Liposomes; Phosphatidylethanolamines; Temperature

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.

Topics: Erythrocytes; Fluoresceins; Fluorescent Dyes; Glycophorins; Humans; Hydrogen-Ion Concentration; Kinetics; Lipid Bilayers; Liposomes; Phosphatidylethanolamines; Sialoglycoproteins; Spectrometry, Fluorescence; Trypsin