pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 degrees C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.     

pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 °C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.     

Monomers of the Neurospora plasma membrane H+-ATPase catalyze efficient proton translocation

Liposomes prepared by sonication of asolectin were fractionated by glycerol density gradient centrifugation, and the small liposomes contained in the upper region of the gradients were used for reconstitution of purified, radiolabeled Neurospora plasma membrane H+-ATPase molecules by our previously published procedures. The reconstituted liposomes were then subjected to two additional rounds of glycerol density gradient centrifugation, which separate the H+-ATPase-bearing proteoliposomes from ATPase-free liposomes by virtue of their greater density. The isolated H+-ATPase-bearing proteoliposomes in two such preparations exhibited a specific H+-ATPase activity of about 11 mumol of Pi liberated/mg of protein/min, which was approximately doubled in the presence of nigericin plus K+, indicating that a large percentage of the H+-ATPase molecules in both preparations were capable of generating a transmembrane protonic potential difference sufficient to impede further proton translocation. Importantly, quantitation of the number of 105,000-dalton ATPase monomers and liposomes in the same preparations by radioactivity determination and counting of negatively stained images in the electron microscope indicated ATPase monomer to liposome ratios of 0.97 and 1.06. Because every liposome in the preparations must have had at least one ATPase monomer, these ratios indicate that very few of the liposomes had more than one, and simple calculations show that the great majority of active ATPase molecules in the preparations must have been present as proton-translocating monomers. The results thus clearly demonstrate that 105,000-dalton monomers of the Neurospora plasma membrane H+-ATPase can catalyze efficient ATP hydrolysis-driven proton translocation.     

钙库调控的钙通道Orai1体外研究方法的建立

目的:建立钙通道Orai1的体外研究方法。方法:利用脂质体重组技术,将体外纯化的Orai1蛋白重组到脂质体膜上,利用蔗糖密度梯度离心来检测其重组效率及Orai1蛋白在脂质体膜上的结构,并利用钙染料Fura-2检测脂质体内钙离子的释放。结果:成功制备了脂质体及体外纯化了GST-Orai1融合蛋白,蔗糖密度梯度离心结果证明GST-Orai1蛋白成功重组到脂质体上,以及Orai1蛋白以多聚体的形式定位在脂质体膜上。钙离子释放实验证明脂质体内钙离子包装完好,可用于后续Orai1钙通道的功能研究。结论:利用脂质体重组技术建立了一种新的Orai1的研究方法,能够更直接有效地研究其功能及其活化机制。     

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.     

Density gradient separation of carborane-containing liposome from low density lipoprotein and detection by inductively coupled plasma spectrometry

Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for analyzing the new cholesterol-based compounds (BCH, BCH-Da, BCH-Db and BCH-Dc) in liposomal formulations. Not only the boron compounds but also the phospholipid compositions of the liposome formulation were quantitatively analyzed. Reasonable limit of detection for boron (0.5?µg/ml) and phosphorous (0.09?µg/ml), respectively, was observed. ICP-MS was also utilized for analyzing BCH in a brain distribution study. The detection limit of boron analysis by ICP-MS is at least three orders of magnitude lower than of that of ICP-AES (1?ng B/ml). The method was linear in the range of 500-1?ng B/ml and the linearity correlation coefficient was 1. In addition, an ultracentrifugation method was developed to separate liposomes from low-density lipoprotein (LDL). Factors such as density gradient and size of liposomes were adjusted to optimize separation and it was observed that in conjunction to time, speed and density gradient, size of the liposome also had impact on the separation using centrifugation method. These findings show the importance of ICP-AES as an analytical method for the analysis of element-based compounds encapsulated in phospholipid vesicles.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

The effect of gentamicin on the biophysical properties of phosphatidic acid liposomes is influenced by the O-C = O group of the lipid

We previously reported that gentamicin binds to liposomes composed of anionic phospholipids and depresses glycerol permeability and raises the activation energy for glycerol permeation in these liposomes. We postulated that these changes in the glycerol permeability and in the activation energy (Ea) for glycerol permeation were due to hydrogen bonding between O-C = O groups in the hydrogen belt and one or more amino groups of gentamicin. To test this hypothesis, we examined the effects of gentamicin on the membrane surface potential, the glycerol permeability coefficient (p), the Ea for glycerol permeation, and the aggregation of liposomes composed of 1:1 phosphatidylcholine (PC) and phosphatidic acid with the acyl chains of phosphatidic acid in either an ester (PA) or an ether (PA*) linkage. Gentamicin depressed the membrane surface electrostatic potential, measured by the partitioning of methylene blue between the bulk solution and the liposomal membrane, to an equivalent degree in PC-PA and PC-PA* liposomes, which indicates that substitution of the ether for the ester linkage did not interfere with the electrostatic interaction between the cationic drug and the negatively charged phosphate head group. Gentamicin caused a temperature-dependent decrease of p and raised Ea for glycerol permeation from 17.7 +/- 0.3 to 21.6 +/- 0.4 kcal/mol in PC-PA liposomes but had little or no effect on these parameters in PC-PA* liposomes. In contrast, gentamicin induced a significantly greater degree of aggregation of PC-PA* liposomes compared to that of PC-PA liposomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of loaded liposomes by size exclusion chromatography

This review focuses on the use of conventional (SEC) and high performance (HPSEC) size exclusion chromatography for the analysis of liposomes. The suitability of both techniques is examined regarding the field of liposome applications. The potentiality of conventional SEC is strongly improved by using a HPLC system associated to gel columns with a size selectivity range allowing liposome characterization in addition to particle fractionation. Practical aspects of size exclusion chromatography are described and a methodology based on HPSEC coupled to multidetection modes for on-line analysis of liposomes via label or substance encapsulation is presented. Examples of conventional SEC and HPSEC applications are described which concern polydispersity, size and encapsulation stability, bilayer permeabilization, liposome formation and reconstitution, incorporation of amphiphilic molecules. Size exclusion chromatography is a simple and powerful technique for investigation of encapsulation, insertion/interaction of substances from small solutes (ions, surfactants, drugs, etc.) up to large molecules (proteins, peptides and nucleic acids) in liposomes.     

Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.     

Selective partitioning of cholesterol and a model drug into liposomes of varying size

The resistance of a lipid bilayer with respect to a bending deformation generally depends on the presence of membrane additives such as sterols, cosurfactants, peptides, and drugs. As a consequence, the partitioning of membrane additives into liposomes becomes selective with respect to liposome size; i.e., membrane rigidification depletes the membrane additives in the smaller (more strongly curved) liposomes. We have measured this liposome size-selective partitioning for two membrane additives - cholesterol and the porphyrin-based photosensitizer temoporfin - using asymmetrical flow field-flow fractionation (AF4) of liposomes and radioactive labeling of the membrane additive and lipid. The method yields either the molar cholesterol-to-lipid or the temoporfin-to-lipid ratio as a function of liposome size, from which we calculate the corresponding change of the membrane bending stiffness. For small unilamellar fluid-phase liposomes composed of palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylglycerol (POPG), we find that cholesterol rigidifies the host membrane in a manner consistent with previously reported measurements. In contrast, temoporfin softens this membrane. Partitioning results for gel-phase liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) are also curvature-sensitive but cannot be interpreted on the basis of the bending stiffness alone.     

Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors

Although small, 100-nm liposomes are known to selectively accumulate in solid tumors, the individual contributions of liposome influx and egress rates are not well understood. The aim of this work was to determine influx and efflux kinetics for 100-nm, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol) liposomes by inducing aggregate formation of biotinylated liposomes upon administering avidin. Injecting 50 microg of neutravidin intravenously to mice that had previously been administered 100 mg/kg DPSC/Chol liposomes containing 0.5 mol% biotin-conjugated lipid resulted in >90% elimination of the liposomes from plasma within 1 h. This rapid removal by the reticuloendothelial system (RES) permitted the determination of the tumor efflux kinetics due to negligible tumor influx after neutravidin injection. The tumor efflux rate constant (k(-1)) was determined to be 0.041 h(-1) when neutravidin was injected 4 h after liposome injection. This allowed the determination of the tumor influx rate constant (k(1)), which under these conditions was 0.022 h(-1). Therefore, DSPC/Chol liposomal accumulation, in LS180 solid tumors, is dictated primarily by plasma liposome concentrations and liposome egress is comparable or slightly faster than influx into the tumors. This method is applicable for a wide range of lipid doses, and can be used to characterize influx and efflux parameters at different time points after accumulation. The application, therefore, has the potential to be used to fully characterize the impact of different liposome parameters such as lipid composition, steric stabilization, size and dose on tumor accumulation kinetics.     

Entrapment of ovalbumin into liposomes--factors affecting entrapment efficiency, liposome size, and zeta potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB + 0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential--the factors that are of great importance for the use of liposomes as drug carriers.     

Preparation of submicron liposomes exhibiting efficient entrapment of drugs by freeze-drying water-in-oil emulsions

A novel liposome preparation method is described as freeze-drying of water-in-oil emulsions containing sucrose in the aqueous phase (W) and phospholipids and poly(ethylene glycol)₁₅₀₀ (PEG) in the oil phase (O). The water-in-oil emulsions were prepared by sonication and then lyophilized to obtain dry products. Upon rehydration, the dry products formed liposomes with a size smaller than 200 nm and an encapsulation efficiency (EE) higher than 60% for model drugs. The presence of lyoprotectant and PEG was found to be a prerequisite for the formation of liposomes with desirable properties, such as a small particle size and high EE. The lyophilates were stable and could be rehydrated to form liposomes without any change in size or EE even after a storage period of 6 months. Also, the lipophilic drug-containing FWE liposomes were stable and could be stored for at least 6 months although the liposomes containing hydrophilic drugs showed significant leakage. Based on the vesicle size and EEs of the model drugs, as well as the scanning electron micrograph (SEM) and small angle X-ray scattering (SAXS) pattern of the lyophilates, a possible mechanism for the liposome formation is proposed.     

Properties of liposomes that contain chloroplast pigments: Photosensitivity and efficiency of energy conversion

Liposomes that contain chlorophyll and carotene are photosensitive. If a gradient of redox potential exists across the liposome membrane, illumination causes charge transport. The quantum efficiency of energy conversion in liposomes is about 0.075. It appears that chlorophyll aggregates are present in the liposomes and that these aggregates are involved in energy conversion.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

Physicochemical and safety evaluation of 5-aminolevulinic acid in novel liposomes as carrier for skin delivery

In this study we successfully entrapped 5-aminolevulinic acid (ALA) in liposome, although it exists as a zwitter ion. A molar ratio of 2:1:2.5 phosphatidyle-thanolamine (PE)/cholesterol/sodium stearate represented the best condition to achieve high entrapment efficiency (29.37 +/- 1.21%), and the average vehicle size was 133.6 +/- 2.8 nm. After 32 days of storage, the vehicle sizes of formulations with PE series were still approximately less than 200 nm. The safety of liposomes was tested and ensured both with regard to cellular cytotoxicity and erythrocyte hemolysis. Safety studies showed that liposome formulations did not affect cell viability except when both potassium stearate and sodium oleate were added. Moreover, PE and PE/cholesterol did not damage human erythrocytes in this study. The range of the hemolytic effect caused by liposomes was 5 to 37% and the effect was dependent on the amount of sodium stearate added to the formulation. According to the release rates and skin penetration of ALA liposomes in vitro, PE/cholesterol/sodium stearate liposomes might increase skin penetration, and it was shown that penetration across the stratum-corneum (sc) layer was the rate-limiting process. Images from confocal laser scanning microscopy (CLSM) confirmed the great potency of liposomes for delivering ALA into skin.     

Surface changes induced by osmotic stress and its influence on the glycerol permeability in lipid bilayers

The penetration rate of glycerol across lipid bilayers can be assayed dispersing liposomes filled with a 0.1 M glucose solution in an isotonic or a hypertonic solution of glycerol. The kinetic of glycerol permeation is found to be different in each of those cases. Liposomes dispersed above the phase transition temperature in hypertonic solutions show an increase in the surface polarization as measured by means of merocyanine 540. Under this condition, the permeation of glycerol shows a two-step kinetic which is indicative of a non-fickean diffusion process. In contrast, liposomes dispersed in isotonic solutions of the permeant show a fickean behavior. The changes in polarization of the membrane interface are ascribed to variations in the surface potential due to the osmotic collapse and the glycerol concentration in contact with the outer surface. The permeability of polar molecules can, in consequence, be considered as a function of the surface potential of the liposome which is congruent with previous data in literature reporting that water permeability increases as a function of the zeta potential of liposomes shrunken in hypertonic solutions.     

Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein

Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.