Temperature-dependent osmotic permeability in glycoproteion containing liposomes

The osmotic water outflow of large multilamellar liposomes containing ₁-acid glycoprotein was measured at a temperature near the lipid's phase transition temperature. The liposomes were formed from a mixture of DPPC, cholesterol and glycoprotein in molar ratios 100:20:1, by continuous sucrose density gradient centrifugation. These liposomes captured 35% of the radiolabeled glycoprotein. The temperature-dependent experiments showed that near phase transition temperature the initial rate of water outflow increased drastically in comparison with glycoprotein free liposomes incubated in buffer containing glycoprotein. We suggested that eventual a channel mechanism may be involved due to spontaneous incorporation of glycoprotein into the bilayer.     

Effect of microwave radiation on the permeability of carbonic anhydrase loaded unilamellar liposomes

The influence of 2.45 GHz microwave exposure (6 mW/g) on the diffusion processes in enzyme-loaded unilamellar liposomes as bioreactors was studied. The enzyme carbonic anhydrase (CA) was entrapped into cationic unilamellar vesicles. Previous kinetic experiments showed a very low self-diffusion rate of the substrate p-nitrophenyl acetate (PNPA) across intact liposome bilayer. A twofold increase in the diffusion rate of PNPA through the lipid bilayer was observed after 120 min of microwave radiation compared to temperature control samples. The microwave effect was time dependent. The enzyme activity, as a function of increased diffusion of PNPA, rises over 120 min from 22.3% to 80%. The increase in stearylamine concentration reduces the enzyme activity from 80% to 65% at 120 min. No enzyme leakage was observed. © 1994 Wiley-Liss, Inc.     

Effects of 2.45-GHz microwave and 100-MHz radiofrequency radiation on liposome permeability at the phase transition temperature

Large unilamellar dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) liposomes loaded with an aqueous chemotherapeutic drug, cytosine arabinofuranoside (ARA-C), were exposed for 30 min to 60 W/kg continuous-wave (CW) 100-MHz or 2.45-GHz radiation in vitro at temperatures between 37 degrees C and 43 degrees C. Liposomes were exposed in HEPES buffer or in HEPES buffer supplemented with 44% by volume fetal calf serum (FCS). Characteristic phase transition responses were detected in the range of 39 degrees C to 40 degrees C with the presence of FCS, increasing maximum % release of 3H-ARA-C by 20% relative to HEPES suspension. Neither frequency of electromagnetic radiation had any detectable effect on liposome permeability or the location of the phase transition in the presence or absence of FCS.     

Amine spin probe permeability in sonicated liposomes

Summary Permeabilities for an homologous series of amine nitroxide spin probes were measured in liposomes of varying composition by an electron paramagnetic resonance (EPR) method. Results show that the rate-limiting step in permeation is not adsorption/desorption at the aqueous/membrane interface for two probes in phosphatidylcholine/phosphatidic acid liposomes and for one probe in phosphatidylcholine/cholesterol/phosphatidic acid liposomes. Accordingly, we interpret observed selectivity patterns for the entire series of probes in liposomes and red cells in terms of the properties of the bilayer interior.Results are inconsistent with simple applications of either free volume or hydrocarbon sheet models of nonelectrolyte permeation. In the former case, it was found that liposomes do not select against these probes on the basis of molecular volume. In the latter case, probe permeabilities are all much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. Together with the results of previous studies of spin-labeled solutes in membranes, as well as studies of lipid dynamics in membranes, these latter results suggest that the rate-limiting region in nonelectrolyte permeation is not in the center of the bilayer, but in the relatively ordered acyl chain segments near the glycerol backbone.     

Extemporaneous preparation of large unilamellar liposomes

Direct contact between lipids solubilized by octyl glucoside and Amberlite XAD-2 beads yielded large liposomes (240 nm diameter) with no residual detergent molecules, in less than 10 min. This extemporaneous preparation of liposomes was prepared with a detergent/bead ratio no higher than 0.12 (mumol/mg) and a phosphatidylcholine/phosphatidylserine/cholesterol molar ratio of 1:1:1. The liposomes were mainly unilamellar, as deduced from thin section and freeze-fracture electron micrographs and from measurement of calcein incorporation into the vesicles. The relatively large internal volume of these vesicles (8.9 l/mol lipid) accounts for the high percentage of entrapped material observed. The percentage increased with lipid concentration, but could not be increased above 20% corresponding to 20 mM total lipids.     

Diffusion of hydrogen peroxide across DPPC large unilamellar liposomes

The decomposition of hydrogen peroxide catalyzed by catalase entrapped in the pool of di-palmitoylphosphatidyl choline unilamellar liposomes has been studied. The rate of the process was evaluated by following the production of oxygen as a function of time. Under the experimental conditions employed the rate of oxygen production was controlled by the diffusion of hydrogen peroxide, allowing for the estimation of the diffusion coefficient of hydrogen peroxide across the liposome bilayer. The rate of diffusion across the bilayer increases with the temperature and the presence of fluidizers (n-nonanol), according with changes in the bilayer fluidity, as sensed by 1,6-diphenyl hexatriene (DPH) fluorescence anisotropy. A peculiar aspect of the data is the fast hydrogen peroxide diffusion observed at the bilayer phase transition temperature. This fast diffusion is associated to rafts fluctuations that take place in the partially melted bilayer. These fluctuations have no effect on the microviscosity sensed by DPH.     

Measurement of net proton-hydroxyl permeability of large unilamellar liposomes with the fluorescent pH probe, 9-aminoacridine

The fluorescent probe 9-aminoacridine was used to measure the rate of decay of experimentally established pH gradients across liposome membranes. From the rate of decay, separate permeability coefficients for protons (PH) and hydroxyls (POH) were calculated and summed to yield the net proton-hydroxyl permeability (Pnet). The net permeability of protons and hydroxyls was found to be approximately 10(-4) cm/s, six orders of magnitude greater than that measured for sodium and pyrophosphate ions under similar conditions. This suggests that protons and/or hydroxyls cross lipid bilayers by a different mechanism than do other monovalent cations and anions. In addition, the measurements provide a standard for net proton-hydroxyl permeability in pure phospholipid bilayers for comparison with biological membranes.     

Hyaluronan is a key component in cryoprotection and formulation of targeted unilamellar liposomes

Lyophilized unilamellar liposomes (ULV), the dosage form of choice for shelf-life, revert upon reconstitution to the larger multilamellar liposomes (MLV), which is detrimental to the many carrier-mediated therapies that require small particles. High doses of sugars such as trehalose, sucrose and others, included in the original formulations for cryoprotection, were shown to prevent the conversion to MLV. In this study we set out to test whether hyaluronan (HA), the surface-bound ligand in our previously developed targeted bioadhesive liposomes (BAL), can also act as a cryoprotectant. The studies included structural and physicochemical characterization of original and reconstituted hyaluronan-ULV (HA-ULV). For each HA-ULV, similar regular ULV (RL-ULV) served as controls. Four properties were tested: particle size, zeta potential, encapsulation efficiency and half-life of drug release (τ_1/2), for three drugs—chloramphenicol (CAM), vinblastine (VIN) and mitomycin C (MMC). Encapsulation efficiencies of the original systems were quite alike for similar RL-ULV and HA-ULV ranging from 25% to 70%. All systems acted as sustained-release drug depots, τ_1/2 ranging from 1.3 to 5.3 days. Drug species and lipid composition were the major determinants of encapsulation and release magnitudes. By all tests, as anticipated, lyophilization generated significant changes in the reconstituted RL-ULV: 17-fold increase in diameter; tripling of zeta potential; 25-60% drop in encapsulation efficiencies; 25-30% decrease in τ_1/2. In contrast, the reconstituted HA-ULV retained the same dimensions, zeta potentials, encapsulation efficiencies and τ_1/2 of the original systems. These data clearly show HA to be a cryoprotectant, adding another clinically relevant advantage to HA-BAL. We propose that, like the sugars, HA cryoprotects by providing substitute structure-stabilizing H-bonds.     

Entrapping of hydrophobized plague capsular antigen into large unilamellar liposomes

The entrapping of a plague capsular antigen into the liposomes, prepared by the "reverse phase" evaporating method, was investigated. Both the native antigen and the antigen modified by palmitoylchloride were used. The entrapping of the modified antigen into the liposomes was greater than that of the native one: 46.1 +/- 5.4% and 3.7 +/- 2.7%, respectively. The highest content of the antigen on the membrane surface was observed when using palmitoylized protein and liposomes with egg lecithin, cholesterol and dicethylphosphate in the molar ratio 7:2:1.     

Osmotic water permeability in glycoprotein containing liposomes

The kinetics of osmotic water permeability in proteoliposomes containing ₁-acid glycoprotein was investigated by means of stopped-flow spectrophotometry. A biphasic time-course of scattered light with time was registered. The rate constants calculated from fits to an exponential function in the first phase were proportional to the final medium osmolarity. The apparent second order rate constants K_app (Osm^-1 sec^-1) were determined at different glycoprotein concentrations in the original mixture for preparation of proteoliposomes. The value of K_app at lipid:glycoprotein weight ratio = 1 was plotted in Arrhenius coordinates. The calculated activation energy for water permeation through the lipid bilayer suggests that eventual channel mechanism may be involved due to the presence of glycoprotein molecule in the liposomes.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 °C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [³¹P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H_II) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation

The reverse phase evaporation procedure was used to prepare large unilamellar liposomes containing bacteriorhodopsin. Electron microscopy showed that proteoliposomes were unilamellar and fairly uniform in size provided the preparation was extruded through calibrated nucleopore membranes : the vesicles have diameters around 200 nm. The spectral properties of the bacteriorhodopsin in the large liposomes resembled those of bacteriorhodopsin in purple membrane. Furthermore, the chromoprotein in the reconstituted vesicles had an inside-out orientation and on illumination, translocated protons efficiently from the external medium into the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, a light-independent transmembrane potential of about 60 mV was measured from thiocyanate distribution. In the presence of valinomycin, this transmembrane electrical potential was abolished and then a light-dependent transmembrane pH gradient of about 2 pH units could be generated.     

Biophysical aspects of using liposomes as delivery vehicles

Liposomes are used as biocompatible carriers of drugs, peptides, proteins, plasmic DNA, antisense oligonucleotides or ribozymes, for pharmaceutical, cosmetic, and biochemical purposes. The enormous versatility in particle size and in the physical parameters of the lipids affords an attractive potential for constructing tailor-made vehicles for a wide range of applications. Some of the recent literature will be reviewed here and presented from a biophysical point of view, thus providing a background for the more specialized articles in this special issue on liposome technology. Different properties (size, colloidal behavior, phase transitions, and polymorphism) of diverse lipid formulations (liposomes, lipoplexes, cubic phases, emulsions, and solid lipid nanoparticles) for distinct applications (parenteral, transdermal, pulmonary, and oral administration) will be rationalized in terms of common structural, thermodynamic and kinetic parameters of the lipids. This general biophysical basis helps to understand pharmaceutically relevant aspects such as liposome stability during storage and towards serum, the biodistribution and specific targeting of cargo, and how to trigger drug release and membrane fusion. Methods for the preparation and characterization of liposomal formulations in vitro will be outlined, too.     

Proton permeability of liposomes from natural phospholipid mixtures

A method to determine the proton permeability coefficient of phospholipid membrane with the fluorescent probe pyranine is described. Very high proton permeability coefficients of liposomes from natural extracts are measured with great accuracy. The proton permeability appears to be linearly related to the fluidity of the bilayers. This relation as well as the comparison of the activation energies of proton permeability and fluidity support the hypothesis (Nichols J.W. and Deamer D.W. (1980) Proc. Natl. Acad. Sci. USA 77, 2038–2042) of a transfer process along a network of hydrogen bonded water molecules. It is suggested that the common lipid characteristics of biological membranes (net surface charge and unsaturation) favor a high proton permeability.     

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.     

载药脂质体的研究与应用进展

载药脂质体给药系统已成为国内外的研究热点。传统脂质体经修饰和改良后表现出良好的生物相容性,缓释性和靶向性。新型脂质体在经皮给药,肺部给药,脑部靶向治疗,基因治疗等方面的应用研究结果显示,集药物缓释、靶向于一体的具有良好生物安全性的脂质体给药系统具有很大发展潜力。本文综述了该领域中的最新研究进展。     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

In vivo fate of large unilamellar sphingomyelin-cholesterol liposomes after intraperitoneal and intravenous injection into rats

We investigated the fate of intraperitoneally and intravenously injected reverse phase evaporation vesicles of fairly uniform size (100–200 m) with respect to blood celarance, tissue distribution and integrity in vivo. The vesicles are composed of sphingomyelin and cholesterol in a molar ratio 3 : 2 and contain ¹²⁵I-labeled poly(vinyl pyrrolidone) in the aqueous compartment. It is shown that following an intrapersoneal injection the vesicles are transported intact, and not associated with cells, from the peritoneal activity to the blood and are subsequently taken up mainly by liver and spleen, where, particularly in liver, the phospholipid is partially metabolized. After an intraperitoneal injection the rate of vesicle-uptake by liver and spleen is reduced by a factor of 2–3 compared to the rate of vesicle-uptake by liver and spleen following an intravenous injection. The peritoneal cavity functions as a reservior of vesicles for some hours. The rates of blood clearance and uptake of the vesicles by liver and spleen appear to be slower than that found for vesicles of different lipid composition.