beta-Galactosidase-induced destabilization of liposome composed of phosphatidylethanolamine and ganglioside GM1

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.     

Temperature-dependent aggregation of pH-sensitive phosphatidyl ethanolamine-oleic acid-cholesterol liposomes as measured by fluorescent spectroscopy.

pH-sensitive liposomes made of phosphatidyl ethanolamine-oleic acid-cholesterol (4:2:4 molar ratio) at neutral pH values aggregate at approximately 40 degrees C. The aggregation is accompanied by liposome destabilization and by the release of intraliposomal fluorescent marker (calcein). Both aggregation and calcein leakage start at the temperature corresponding to the lipid phase transition into hexagonal phase. In the system studied the phase transition temperature interval is within 45 to 55 degrees C as estimated with the use of the fluorescent probe 1,6-diphenylhexatriene. The presence of cell cultivation medium RPMI 1640 decreases liposome aggregation temperature. The addition of 10% serum to the system decreases the temperature at which the aggregation proceeds still further. The conclusion that serum-free media should be used for cell experiments involving pH-sensitive liposomes is made.     

Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic and ultrastructural studies of interactions of target-sensitive immunoliposomes with herpes simplex virus

The bilayer phase of dioleoylphosphatidylethanolamine (PE) can be stabilized with palmitoyl-IgG monoclonal antibody to the glycoprotein gD of the herpes simplex virus (HSV). Interactions of PE immunoliposomes with the target virions were characterized by analyzing the kinetics of lipid mixing, by liposomal content release, and by ultrastructural studies. As revealed by a resonance energy transfer assay, lipid mixing between PE immunoliposomes and virions was very rapid, with a second-order rate constant (kapp) of 0.173 (min)-1 (microgram/mL virus)-1. In comparison, content release from PE immunoliposomes was much slower and exhibited multiple-phase, mixed-order kinetics, indicating that liposome destabilization involved fusion of liposomes with HSV. The extent and the apparent rate of liposome destabilization were strongly dependent on liposome concentration. This was evident by the fact that only one to two liposomes were destabilized by each virus particle at low liposome concentration (0.1 microM). For higher liposome concentrations (1-10 microM), this value was 35-104. This finding implies that collision among the virus-bound liposomes is essential for the eventual collapse of PE immunoliposomes to form the hexagonal (HII) equilibrium phase which was observed using freeze-fracture electron microscopy. Studies employing soluble gD, immobilized on latex beads, indicated that a multivalent antigen source is essential for PE immunoliposome destabilization. Immediately after liposome-virus binding, fusion of liposome with the viral membrane then follows. Upon growth of the fusion complexes, which increase to 35-104 liposomes for each virus, an eventual collapse of the structure results, driving PE to its equilibrium structure of HII phase.     

Interactions of target-sensitive immunoliposomes with herpes simplex virus. The foundation of a sensitive immunoliposome assay for the virus

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.     

Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.     

脂质体通透性与脂多型性关系的研究

本文以TPE和TPE/DOPE(1:1.mol:mol)制成包裹荧光分子calcein的脂质体,通过测量荧光强度随扫描温度的变化,探讨了脂质体通透性与脂多型性之间的关系.结果表明,在不发生双层相(L)变成六角形Ⅱ相(H)相转变时,脂质体悬液的荧光强度不增加;当发生该转变时,脂质体悬液的荧光强度开始增加;完成该相转变后,脂质体悬液的荧光强度仍继续增加.据此,我们认为:脂质体的通透性与脂的多型性密切相关,当发生L→HⅡ相转变时,脂质体的通透性增加.由于荧光强度的变化对相变非常敏感,我们建议用测量脂质体荧光强度随温度的变化来监测脂质体稀悬液中脂的多型性.     

Destabilization of phosphatidylethanolamine-containing liposomes: hexagonal phase and asymmetric membranes

We have measured the temperature of the L alpha-HII phase transition, TH, for several types of phosphatidylethanolamine (PE), their binary mixtures, and several PE/cholesteryl hemisuccinate (CHEMS) mixtures. We have shown for liposomes composed of pure PE and in mixtures with CHEMS that there is an aggregation-mediated destabilization which is greatly enhanced at and above TH. We now ask the question: How well can a dioleoylphosphatidylethanolamine/CHEMS liposome, for example, destabilize TPE (transesterified from egg phosphatidylcholine)/CHEMS liposome and vice versa? We use Ca2+ and H+ to induce aggregation and to provide different values of TH: the TH of the PE/CHEMS mixture is much lower at low pH than with Ca2+. We find that if the temperature is above the TH of one lipid mixture, e.g., A, and below the TH of the other lipid mixture, e.g., B, then the destabilization sequence [measured by the fluorescent 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylylenebis(pyridinium bromide) leakage assay] is AA greater than AB much greater than BB. That is, the bilayer of the lipid A (which on its own would end up in the HII phase) destabilizes itself better than it destabilizes the bilayer of lipid B (which on its own would remain in the L alpha phase). The BB contact is the least unstable. From these experiments, we conclude that the enhanced destabilization of membranes provided by the polymorphism accessible to these lipids above TH is effective even if only one of the apposed outer monolayers is HII phase competent. The surprising result is that if the temperature is above the TH of both lipid mixtures, then the destabilization sequence is AB greater than AA, BB. That is, the mixed bilayers are destabilized more by contact than either of the pure pairs. We believe that this is due to specific differences in the kinetics of aggregation or close approach of the membranes. Similar results were obtained with pure PE liposomes induced to aggregate by Ca2+ at pH 9.5. We also found that the kinetics of low-pH-induced leakage from PE/CHEMS liposomes were initially faster when the CHEMS on both sides of the bilayer is fully protonated. However, in a citrate buffer, which cannot cross intact membranes, the leakage was eventually faster. Flip-flop of the protonated CHEMS to the inner monolayer can explain this observation.     

Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester polyethyleneglycol lipid

The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. This minimum-surface-shielding model describes the kinetics of the pH-triggered release of POD/PE liposomes. In the model, when acid-catalyzed hydrolysis lowers the mole percentage of POD on the liposome surface to a critical level, intervesicular lipid mixing is initiated, resulting in a burst of contents release. Two phases of content leakage are observed: a lag phase and a burst phase. During the lag phase, less than 20% of liposomal contents are released and the leakage begins to accelerate when approaching to the transition point. During the burst phase, the leakage rate is dependent on interbilayer contact. The burst phase occurs when the surface density of the PEG lipid is 2.3 +/- 0.6 mol%, regardless of the pH. Vesicles containing 4 mol% of a pH-insensitive PEG-lipid conjugate and 10% POD did not leak contents or collapse at any pH. These data are consistent with the stalk theory to describe the lamellar-to-inverted hexagonal phase transition and set a lower bound of approximately 16 PE lipids on the external monolayer as the contact site required for lipid mixing between two bilayers.     

pH-Sensitive Liposomes

Abstract

pH sensitive liposomes are lipid compositions that can be destabilized when the external pH is changed; usually from a neutral or slightly alkaline pH to an acidic pH. They are designed to circumvent delivery of liposome contents to the lysosomes of cells following internalization of the vesicle via the endocytic pathway. In the majority of compositions, a lipid containing a pH titratable group is mixed with phosphatidylethanolamine containing unsaturated acyl chains in a molar ratio (pH sensitive component/PE) of 1/4 or greater. There are five major groups of phosphatidylethanolamine containing pH-senstive lipid compositions. These can be classified by their acid-titratable component: phospholipids, acylated amino acids, fatty acids, cholesterol derivatives and miscellaneous double chain amphiphiles. The biophysical mechanism of action involves a transition of the lipids from the lamellar phase to the hexagonal phase. In cell culture, pH sensitive vesicles can increase the delivery of fluorescent markers, proteins, cytotoxic compounds, RNA and DNA into the cytoplasm. The mechanism of delivery is suggested to involve the destabilization of the liposome in the endosome as the pH is reduced from 7.4 to 5.0 and subsequent destabilization of, or fusion with, the endosomal membrane; some of the liposome contents are introduced into the cytoplasm. In most cases, the extent of liposome contents delivery into the cytoplasm is less than 1% of the amount that becomes cell associated. However further studies, with more reliable assays to differentiate cytoplasmic from lysosomal delivery, are required to place an exact value on this efficiency. The efficiency of pH sensitive liposomes in vivo is limited by stability of certain of the liposome compositions in serum and targeting to the appropriate cell. Cholesterol hemisuccinate is a particularly attractive component for in vivo use since it stabilizes the liposome when in serum at pH 7.4. The use of pH sensitive liposomes in drug delivery should continue to expand due to the increasing number of macromolecular therapeutic agents with intracellular targets.     

Temperature-sensitive poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate)-coated liposomes for triggered contents release

We prepared thermosensitive poly( N-(2-hydroxypropyl)methacrylamide mono/dilactate) (pHPMA mono/dilactate) polymer and studied temperature-triggered contents release from polymer-coated liposomes. HPMA mono/dilactate polymer was synthesized with a cholesterol anchor suitable for incorporation in the liposomal bilayers and with a cloud point (CP) temperature of the polymer slightly above normal body temperature (42 degrees C). Dynamic light scattering (DLS) measurements showed that whereas the size of noncoated liposomes remained stable upon raising the temperature from 25 to 46 degrees C, polymer-coated liposomes aggregated around 43 degrees C. Also, noncoated liposomes loaded with calcein showed hardly any leakage of the fluorescent marker when heated to 46 degrees C. However, polymer-coated liposomes showed a high degree of temperature-triggered calcein release above the CP of the polymer. Likely, liposome aggregation and bilayer destabilization are triggered because of the precipitation of the thermosensitive polymer above its CP onto the liposomal bilayers, followed by permeabilization of the liposomal membrane. This study demonstrates that liposomes surface-modified with HPMA mono/dilactate copolymer are attractive systems for achieving temperature-triggered contents release.     

Effects of the acyl chain composition of phosphatidylcholines on the stability of freeze-dried small liposomes in the presence of maltose.

The effects of the acyl chain composition of phosphatidylcholines (PCs) on the stability of small unilamellar vesicles during freeze-drying and rehydration in the presence of maltose were studied by monitoring the retention of a trapped marker, calcein, in the internal liposome compartment. In dipalmitoyl PC, beta-oleoyl-gamma-palmitoyl-PC and egg yolk PC liposomes, good or fair retentions (>50%) were observed in the presence of maltose, but maltose was ineffective in preserving retention in the dioleoyl PC (DOPC) liposomes (<10%). The extremely low retention in the DOPC liposome was ascribed to neither a formation of the inverted hexagonal phase of the liposomal membrane nor the fusion/aggregation of the liposomes in the drying-rehydration process. Differential scanning calorimetry measurements suggested that interactions of maltose with PC headgroups were essential to stabilizing the dry liposomes. These interactions were significant in the saturated or mixed chain liposomes but were markedly reduced in the DOPC liposomes.     

A novel pH-sensitive liposome formulation containing oleyl alcohol

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. First generation pH-sensitive liposomes, based on the cone-shaped lipid dioleoylphosphatidylethanolamine (DOPE), have been shown to lose fusogenicity in the presence of serum. Here, we report the design and evaluation of novel serum-resistant pH-sensitive liposome formulations that are based on the composition of egg phosphatidylcholine (PC), cholesteryl hemisuccinate (CHEMS), oleyl alcohol (OAlc), and Tween-80 (T-80). When loaded with the fluorescent probe calcein, these liposomes exhibited excellent stability at pH 7.4 and underwent rapid destabilization upon acidification as shown by calcein dequenching and particle size increase. Adjusting the mole percentages of T-80 and OAlc in the formulation could regulate the stability and pH-sensitive properties of these liposomes. Liposomes with a higher T-80 content exhibited greater stability but were less sensitive to acid-induced destabilization. Meanwhile, formulations with a higher OAlc content exhibited greater content release in response to low pH. The pH-triggered liposomal destabilization did not produce membrane fusion according to an octadecylrhodamine B chloride (R(18)) lipid-mixing assay. Compared to DOPE-based pH-sensitive liposomes, the above formulations showed much better retention of their pH-sensitive properties in the presence of 10% serum. These liposomes were then evaluated for intracellular delivery of entrapped cytosine-beta-D-arabinofuranoside (araC) in KB human oral cancer cells, which have elevated folate receptor (FR) expression. The FR, which is amplified in many types of human tumors, has been shown to mediate the internalization of folate-derivatized liposomes into an acidic intracellular compartment. FR-targeted OAlc-based pH-sensitive liposomes, entrapping 200 mM araC, showed approximately 17-times greater FR-dependent cytotoxicity in KB cells compared to araC delivered via FR-targeted non-pH-sensitive liposomes. These data indicated that pH-sensitive liposomes based on OAlc, combined with FR-mediated targeting, are promising delivery vehicles for membrane impermeable therapeutic agents.     

Sendai virus induced leakage of liposomes containing gangliosides

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.     

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.     

Serum-induced leakage of liposome contents

Efflux of contents from small unilamellar vesicles of various compositions, containing a highly quenched fluorescent compound (calcein, 175 mM) was determined as a function of temperature in the presence and absence of human serum. Efflux of calcein from the liposomes was monitored as an increase in fluorescence as calcein became dequenched upon release from the liposomes. The presence of serum significantly increased liposome leakage in all cases. Incorporation of increasing molar ratios of cholesterol into liposomes reduced leakage of calcein from liposomes incubated with buffer and with serum. Leakage was significantly faster from liposomes with an osmotic gradient across the membrane (higher inside) than from equiosmolar liposomes. The leakage of [¹⁴C]sucrose from egg lecithin liposomes at 37°C was also dramatically increased in the presence of serum.     

A novel pH-sensitive liposome formulation containing oleyl alcohol

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. First generation pH-sensitive liposomes, based on the cone-shaped lipid dioleoylphosphatidylethanolamine (DOPE), have been shown to lose fusogenicity in the presence of serum. Here, we report the design and evaluation of novel serum-resistant pH-sensitive liposome formulations that are based on the composition of egg phosphatidylcholine (PC), cholesteryl hemisuccinate (CHEMS), oleyl alcohol (OAlc), and Tween-80 (T-80). When loaded with the fluorescent probe calcein, these liposomes exhibited excellent stability at pH 7.4 and underwent rapid destabilization upon acidification as shown by calcein dequenching and particle size increase. Adjusting the mole percentages of T-80 and OAlc in the formulation could regulate the stability and pH-sensitive properties of these liposomes. Liposomes with a higher T-80 content exhibited greater stability but were less sensitive to acid-induced destabilization. Meanwhile, formulations with a higher OAlc content exhibited greater content release in response to low pH. The pH-triggered liposomal destabilization did not produce membrane fusion according to an octadecylrhodamine B chloride (R₁₈) lipid-mixing assay. Compared to DOPE-based pH-sensitive liposomes, the above formulations showed much better retention of their pH-sensitive properties in the presence of 10% serum. These liposomes were then evaluated for intracellular delivery of entrapped cytosine-β-d-arabinofuranoside (araC) in KB human oral cancer cells, which have elevated folate receptor (FR) expression. The FR, which is amplified in many types of human tumors, has been shown to mediate the internalization of folate-derivatized liposomes into an acidic intracellular compartment. FR-targeted OAlc-based pH-sensitive liposomes, entrapping 200 mM araC, showed ∼17-times greater FR-dependent cytotoxicity in KB cells compared to araC delivered via FR-targeted non-pH-sensitive liposomes. These data indicated that pH-sensitive liposomes based on OAlc, combined with FR-mediated targeting, are promising delivery vehicles for membrane impermeable therapeutic agents.     

Photoinduced destabilization of liposomes.

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.     

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.