Amitriptyline overdose treatment by pegylated anionic liposomes

Liposomes composed of DOPG, 50:50 DMPC:DOPG, 95:5 and 85:15 DOPG:DPPE-mPEG-2000, and 55:15:30 DMPC:DPPE-mPEG-2000:CH were studied for their ability to sequester amitriptyline in human serum. The effects of lipid type and loading, liposome size, PEG inclusion, protein interaction and storage were considered. Liposome size had no effect on drug uptake, as 40, 100, and 284 nm liposomes bound similar amounts of drug in buffer. The optimal amount of PEG-modified lipid incorporated into liposomes was found to be 5%. 95:5 DOPG:DPPE-mPEG-2000 liposomes loaded at 1.44 mg lipid/mL were most effective at shielding protein interactions while still allowing amitriptyline to diffuse to the bilayer surface and bind. Absolute reductions of 99% in buffer and human serum samples were observed, while the free drug concentration reduction relative to binding in serum without liposomes was nearly 90% across a drug concentration range of 1-20 muM. With such reductions, serum drug concentrations could be rapidly reduced from toxic to therapeutic levels. Furthermore, storage tests revealed that such liposomes may be stored for at least one month without a change in drug binding ability. These findings strongly suggest that predominantly anionic liposomes incorporated with PEG are excellent candidates for amitriptyline overdose treatment.     

pH-driven assembly of various supported lipid platforms: a comparative study on silicon oxide and titanium oxide

Supported lipid platforms are versatile cell membrane mimics whose structural properties can be tailored to suit the application of interest. By identifying parameters that control the self-assembly of these platforms, there is potential to develop advanced biomimetic systems that overcome the surface specificity of lipid vesicle interactions under physiological conditions. In this work, we investigated the adsorption kinetics of vesicles onto silicon and titanium oxides as a function of pH. On each substrate, a planar bilayer and a layer of intact vesicles could be self-assembled in a pH-dependent manner, demonstrating the role of surface charge density in the self-assembly process. Under acidic pH conditions where both zwitterionic lipid vesicles and the oxide films possess near-neutral electric surface charges, vesicle rupture could occur, demonstrating that the process is driven by nonelectrostatic interactions. However, we observed that the initial rupturing process is insufficient for propagating bilayer formation. The role of electrostatic interactions for propagating bilayer formation differs for the two substrates; electrostatic attraction between vesicles and the substrate is necessary for complete bilayer formation on titanium oxide but is not necessary on silicon oxide. Conversely, in the high pH regime, repulsive electrostatic interactions can result in the irreversible adsorption of intact vesicles on silicon oxide and even a reversibly adsorbed vesicle layer on titanium oxide. Together, the results show that pH is an effective tool to modulate vesicle-substrate interactions in order to create various self-assembled lipid platforms on hydrophilic substrates.     

Competitive Reactions in Three-Component System Cationic Colloid–Anionic Liposome–Protein

The formation of complexes of anionic liposomes (50 nm) and polymer microspheres with grafted polycationic chains with a diameter of 240 nm (spherical polycationic brushes) in a physiological solution at a NaCl concentration of 0.15 mol/L is investigated. Liposomes are quantitatively adsorbed on the surface of brushes; every brush can bind up to 24 intact liposomes. The saturated brush–liposome complex is able to additionally bind negatively charged protein albumin; the excess of protein does not displace liposomes from the complex with brushes. The obtained results are important for understanding the mechanism of formation and functioning of electrostatic multiliposomal containers in biological media containing a high amount of protein.     

Scattering studies of hydrophobic monomers in liposomal bilayers: an expanding shell model of monomer distribution

Hydrophobic monomers partially phase separate from saturated lipids when loaded into lipid bilayers in amounts exceeding a 1:1 monomer/lipid molar ratio. This conclusion is based on the agreement between two independent methods of examining the structure of monomer-loaded bilayers. Complete phase separation of monomers from lipids would result in an increase in bilayer thickness and a slight increase in the diameter of liposomes. A homogeneous distribution of monomers within the bilayer would not change the bilayer thickness and would lead to an increase in the liposome diameter. The increase in bilayer thickness, measured by the combination of small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS), was approximately half of what was predicted for complete phase separation. The increase in liposome diameter, measured by dynamic light scattering (DLS), was intermediate between values predicted for a homogeneous distribution and complete phase separation. Combined SANS, SAXS, and DLS data suggest that at a 1.2 monomer/lipid ratio approximately half of the monomers are located in an interstitial layer sandwiched between lipid sheets. These results expand our understanding of using self-assembled bilayers as scaffolds for the directed covalent assembly of organic nanomaterials. In particular, the partial phase separation of monomers from lipids corroborates the successful creation of nanothin polymer materials with uniform imprinted nanopores. Pore-forming templates do not need to span the lipid bilayer to create a pore in the bilayer-templated films.     

Complexation of polycations to anionic liposomes: composition and structure of the interfacial complexes

Poly(N-ethyl-4-vinylpyridinium bromide) (a polycation with a degree of polymerization of 1100) was adsorbed onto liposomes composed of egg lecithin with a 0.05-0.20 molar fraction (nu) of anionic headgroups provided by cardiolipin (a doubly anionic lipid). According to electrophoretic mobility data, this led to total charge neutralization of the liposomes, whereupon the liposomes adopted a positive charge as additional polymer continued to adsorb. Although the liposomes aggregated at the charge-neutralization point, they disassembled into individual liposomes after becoming positively charged. The degree of polymer adsorption was shown to reach a limit. Thus, by measuring the free polymer content in a liposome suspension, it was possible to determine the polymer concentration at which the liposome surface became saturated with polymer. Beyond this point, an electrostatic/steric barrier at the surface suppressed further adsorption. Dynamic light scattering studies of liposomes with and without adsorbed polymer allowed calculation of the polymer film thickness which ranged from 22 to 35 nm as the molar fraction of cardiolipin (nu) increased from 0.05 to 0.20. The greater the content on the anionic lipid in the bilayer, the thicker the polymer film. The maximum number of polymer molecules adsorbed onto the liposomes was estimated: 1-2 molecules for nu = 0.05; 3 molecules for nu = 0.1; 4- molecules for nu = 0.15; and 6 molecules for nu = 0.2. The polymer appears to lie on the liposome surface, rather than embedding into the bilayer, because addition of NaCl easily dislodges the polymer from the liposome into the bulk water.     

Supramolecular assembly of functionalized lipids

This article describes recent developments in the studies of Supramolecular assemblies of three types of functionalized lipids, focusing on the interactions of their liposomes with biological cells: an artificial boundary lipid, 1,2-dimyristamido-1,2-deoxyphosphatidylcholine (D₁₄DPC) enhances transfer of membrane proteins from cells and tissues to the liposome; an artificial lipid bearing a poly(ethylene oxide) moiety (PEO-lipid) induces membrane fusion between cells and the liposome; and gangliosides and their artificial analogues (sialyl cholesterols), reconstituted in the liposome, directly stimulate T-lymphocytes.     

Probing mechanical properties of liposomes using acoustic sensors

Acoustic devices were employed to characterize variations in the mechanical properties (density and viscoelasticity) of liposomes composed of 1-oleoyl-2-palmitoyl- sn-glycero-3-phosphocholine (POPC) and cholesterol. Liposome properties were modified in three ways. In some experiments, the POPC/cholesterol ratio was varied prior to deposition on the device surface. Alternatively, the ratio was changed in situ via either insertion of cholesterol or removal of cholesterol with beta-cyclodextrin. This was done for liposomes adsorbed directly on the device surface and for liposomes attached via a biotin-terminated poly(ethylene glycol) linker. The acoustic measurements make use of two simultaneous time-resolved signals: one signal is related to the velocity of the acoustic wave, while the second is related to dissipation of acoustic energy. Together, they provide information not only about the mass (or density) of the probed medium but also about its viscoelastic properties. The cholesterol-induced increase in the surface density of the lipid bilayer was indeed observed in the acoustic data, but the resulting change in signal was larger than expected from the change in surface density. In addition, increasing the bilayer resistance to stretching was found to lead to a greater dissipation of the acoustic energy. The acoustic response is assessed in terms of the possible distortions of the liposomes and the known effects of cholesterol on the mechanical properties of the lipid bilayer that encloses the aqueous core of the liposome. To aid the interpretation of the acoustic response, it is discussed how the above changes in the lipid bilayer will affect the effective viscoelastic properties of the entire liposome/solvent film on the scale of the acoustic wavelength. It was found that the acoustic device is very sensitive to the mechanical properties of lipid vesicles; the response of the acoustic device is explained, and the basic underlying mechanisms of interaction are identified.     

Formation of an intact liposome layer adsorbed on oxidized gold confirmed by three complementary techniques: QCM‐D,AFM and confocal fluorescence microscopy

Supported layers of vesicles of dimyristoyl and dipalmitoyl phosphatidylcholine (DMPC and DPPC) containing cholesterol (CHOL) are adequate models for eukaryotic plasma membranes. Among the possible substrates to support these layers, gold offers the possibility of being used as an electrode for application in sensors. However, the formation of intact liposome layers on gold is not completely understood and several authors use more or less complex strategies to bind the liposomes. In this work we investigate the adsorption of unilamellar vesicles of DMPC, DMPC/CHOL and DMPC/DPPC/CHOL on the surface of oxidized gold using a quartz crystal microbalance with dissipation, atomic force microscopy and laser scanning confocal fluorescence microscopy. The results of all techniques indicate that for lipid concentrations ≥ 0.7 mg?mL^–1 a dense layer of intact liposomes irreversibly adsorbs on the gold surface. Copyright © 2011 John Wiley & Sons, Ltd.     

Biodegradable Electrostatic Complexes of Chitosan Cationic Microparticles and Anionic Liposomes

Using the electrostatic adsorption of anionic liposomes on the surface of cationic microparticles of ion-crosslinked chitosan, complexes in which each microparticle can bind up to 110 intact (undestroyed) liposomes are prepared. The saturated complex 350–400 nm in diameter does not dissociate to initial components in aqueous solutions with pH 7 and a NaCl concentration of 0.15 mol/L, but decomposes to 10-nm particles in the presence of proteolytic enzymes. The chitosan–liposome complex and its biodegradation products are characterized by a low cytotoxicity. The described technique may be used to obtain biodegradable multiliposomal containers for the encapsulation and delivery of drugs.     

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers

The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, circular dichroism, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes containing phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer negative charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin positive charges and the phospholipid negative charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed molecular analyses of melittin pore formation are incomplete without considering also these colloidal aspects.     

Effect of light-harvesting complex II on ion transport across model lipid membranes

The effect of the incorporation of the major light-harvesting complex of photosystem II (LHCII) to planar bilayer lipid membranes (BLMs) formed from soybean asolectin and unilamellar small liposomes formed from egg-yolk phosphatidylcholine on ion transport across the lipid bilayer has been studied. The specific conductivity of the BLM rises from 5.2 +/- 0.8 x 10(-9) up to 510 x 10(-9) O(-1) cm(-2) upon the incorporation of LHCII. The conductivity of the membrane with LHCII depends upon the ionic strength of the bathing solution and is higher by a factor of five when the KCl concentration increases from 0.02 to 0.22 M. Such a strong effect has not been observed in the same system without LHCII. The liposome model is also applied to analyse the effect of LHCII on the bilayer permeability to protons. Unilamellar liposomes with a diameter less than 50 nm have been prepared, containing (trapped inside) Neutral Red, a pigment sensitive to proton concentration. A gradient of protons on the membrane is generated by the acidification of the liposome suspension and spectral changes of Neutral Red are recorded in time, reflecting the penetration of protons into the internal space of liposomes. Two components of proton permeation across liposome membranes are observed: a fast one (proceeding within seconds) and a slow one (operating on the time scale of minutes). The rate of both components of proton transport across LHCII-containing membranes is higher than for liposomes alone. The enhancement effect of LHCII on the ion transport across the lipid membrane is discussed in terms of aggregation of the pigment-protein complexes. The possible physiological importance of such an effect in controlling ion permeability across the thylakoid membrane is discussed.     

Binding of Lipid Vesicles to Protein-Coated Solid Polymer Surfaces: A Model for Cell Adhesion to Artificial Biocompatible Materials

The adhesion of lipid vesicles (liposomes) having controlled chemical and physical structure to polymer supported human serum albumin (HSA) thin layers was investigated by a spectrofluorimetric technique. The vesicle lipid bilayer was labeled with a small amount of an apolar fluorescent probe (diphenylexathriene) and the vesicle suspension was set in contact with the protein film. After washing and drying, the adhering vesicles containing sample was dissolved in chloroform and the homogeneous solution was analyzed by standard spectrofluorimetric techniques. Different parameters of the lipid bilayer, suspending solution, and protein film were varied and their influence on the liposome binding was investigated. Concerning the lipid bilayer, we studied the effect of liposome surface charge by using different mixtures of neutral (dipalmitoyl-phosphatidylcholine) and charged (dipalmitoyl-phosphatidic acid) phospholipids and the fluid or gel nature of the lipid bilayer (switched on and off by temperature variation). Variations of the local environment involve Ca(2+) and H(+) changes in the millimolar range as well as different hydrodynamical flows (in the range 0.1-10 cm/s). Preliminary measurements using different protein layers were also performed. Results show: (a) negligible adhesion without the protein layer, (b) the presence of a maximum for the liposome adhesion vs ion concentration (depending on the liposome composition and kind of the adsorbed ions), (c) a much stronger adhesion for vesicles in the fluid phase (overcoming the entropy-driven desorption increase with temperature), and (d) a dramatic lowering of the adhesion capability under hydrodynamic flow. Points a-c have been interpreted on the basis of a simple mechanoelectrical model. Copyright 2000 Academic Press.     

A reduction-triggered delivery by a liposomal carrier possessing membrane-permeable ligands and a detachable coating

To control the cellular uptake of drugs and genes, we synthesized a liposomal carrier possessing membrane-permeable ligands and a detachable poly(ethylene glycol) (PEG) coating. For the detachable coating, a lipid having a thiolytic cleavable spacer (PEG-S-S-DOPE) was synthesized by the reaction of dioleoylphosphatidylethanolamine (DOPE) with a PEG chain via a disulfide linkage. The liposomes were prepared from a mixture of dipalmitoylphosphatidylcholine (DPPC), DOPE, PEG-S-S-DOPE, and cholesteryl hemisuccinate (CHEMS). The octamer (R8 peptide) of arginine was chosen as the membrane-permeable ligand and covalently immobilized onto the CHEMS portion of the liposome surface (PEG-S-S-R8-liposome). The disulfide bond of the PEG chain was cleaved to display the R8 peptides on the liposome surface by adding a reducing agent such as L-cysteine, and thereby internalization of the liposomes was significantly facilitated. When L-cysteine was added to the mixture of cells and the liposome that incorporated plasmids encoding the enhanced green fluorescence protein (pEGFP), the expression of EGFP was low but could be observed in almost 100% of the cells.     

Attenuated total reflectance infrared studies of liposome adsorption at the solid-liquid interface

Interfacial interactions between liposomes and the solid–liquid interface (i.e. a ZnSe internal reflection element, modified to mimic a biological surface) were studied by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflectance (ATR) mode. Both conventional liposomes, containing lecithin and cholesterol and Stealth^® liposomes containing poly(ethylene)glycol (PEG)5000- or PEG2000-lipids were investigated. IR bands due to the liposome components were observed to increase with time and enabled the liposome adsorption kinetics and thermodynamics to be quantified. The liposome solution conditions, surface properties and compositions have all been shown to influence liposome adsorption. Free energies of adsorption were determined to be in the range from −10.0 to −11.0 kJ mol⁻¹ and slightly reduced by PEG incorporation. The adsorption rate constant is decreased with increased solution pH and decreased ionic strength; this reflects the importance of electrostatics in controlling liposome adsorption. Increasing the level and molecular weight of PEG incorporation in the liposomes significantly reduced both the rate and extent of liposome adsorption; steric hindrance is considered to play a key role. Findings from this research will improve the understanding of liposome interaction during drug delivery, give insight into the actions of liposomes in the body and may form the basis for improved liposome formulations.     

Encapsulation of drugs by lyophilized empty dipalmitoylphosphatidylcholine liposomes: effect of calcium ion

The effects of divalent cation (Ca2+) on the characteristics of dipalmitoylphosphatidylcholine (DPPC) liposomes regenerated from lyophilized empty liposomes by rehydration and warming were investigated. The results showed that the volume (ml) of internal aqueous compartment per g lipid (captured volume; Vcap) has a maximum at a certain concentration range of calcium chloride and the maximal value is more than ten times the minimal value. This phenomenon can be explained by considering that binding of Ca2+ to phosphate groups in DPPC molecules induces an increase in the distance (r) between adjacent bilayer membranes in multilamellar liposomes through electrostatic force and causes an increase in Vcap. The dynamic properties of lyophilized liposomes in the rehydration process were examined using a multilamellar vesicle model. The results of simulation suggested that a repulsive force induced between the adjacent bilayer membranes causes rearrangement of the constituent lipid molecules in a liposome followed by an increase in the distance r, a decrease in the internal lamellar number, a smaller increase in liposome size and finally a significant increase in Vcap.     

Molecular interactions between phospholipids and mangostin in a lipid bilayer

Molecular interactions between phospholipids and mangostin in a lipid bilayer have been investigated in terms of the maximum additive concentration (MAC) of mangostin in liposomes, the surface potential, particle size, microscopic-viscosity and microscopic-polarity of liposomes, and the permeability of glucose. The mangostin used is a natural product extract: 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9-xanthenenone.

The MAC of mangostin was fairly dependent upon the nature of the liposomes (uncharged, negatively charged or positively charged). Solubilization of mangostin in the liposomal bilayer resulted in both an increase in the negative charge on the liposomal surface, strenghthening the state of the bilayer membrane, and a depression in the release of the glucose involved. Mangostin was found to temporarily stabilize the liposomal bilayer, although the bilayer membrane is still unstable in the long run.     

Multinuclear NMR studies of single lipid bilayers supported in cylindrical aluminum oxide nanopores

Lipid bilayers were deposited inside the 0.2 microm pores of anodic aluminum oxide (AAO) filters by extrusion of multilamellar liposomes and their properties studied by 2H, 31P, and 1H solid-state NMR. Only the first bilayer adhered strongly to the inner surface of the pores. Additional layers were washed out easily by a flow of water as demonstrated by 1H magic angle spinning NMR experiments with addition of Pr3+ ions to shift accessible lipid headgroup resonances. A 13 mm diameter Anopore filter of 60 microm thickness oriented approximately 2.5 x 10(-7) mol of lipid as a single bilayer, corresponding to a total membrane area of about 500 cm2. The 2H NMR spectra of chain deuterated POPC are consistent with adsorption of wavy, tubular bilayers to the inner pore surface. By NMR diffusion experiments, we determined the average length of those lipid tubules to be approximately 0.4 microm. There is evidence for a thick water layer between lipid tubules and the pore surface. The ends of tubules are well sealed against the pore such that Pr3+ ions cannot penetrate into the water underneath the bilayers. We successfully trapped poly(ethylene glycol) (PEG) with a molecular weight of 8000 in this water layer. From the quantity of trapped PEG, we calculated an average water layer thickness of 3 nm. Lipid order parameters and motional properties are unperturbed by the solid support, in agreement with existence of a water layer. Such unperturbed, solid supported membranes are ideal for incorporation of membrane-spanning proteins with large intra- and extracellular domains. The experiments suggest the promise of such porous filters as membrane support in biosensors.     

Impact of membrane fluidity on steric stabilization by lipopolymers

In this work, the impact of lipid lateral mobility on the steric interaction between membranes containing poly(ethylene glycol) (PEG) functionalized lipids was investigated using the surface force apparatus. The force-distance profiles show the presence of electrostatic and steric repulsion that arise from the presence of negatively charged PEG functionalized lipids. Fluid-phase bilayers have high lateral diffusion relative to gel-phase bilayers; however, a quantitative comparison of the interaction forces between membranes in these two different phase states demonstrates a reduced rate of diffusion in the fluid phase for the PEG-lipids under constrained geometries. Thus, the amount of polymer in the contact zone can be modulated and is reduced with fluid membranes; however, complete exclusion was not achieved. As a result, the steric repulsion afforded by PEG chains or binding affinity of ligated PEG chains can only be modestly tailored by the phase state of the liposome.     

Structure of small actin-containing liposomes probed by atomic force microscopy: effect of actin concentration & liposome size

Actin-containing liposomes were prepared via extrusion through 400 and 600 nm pore diameter membranes at different monomeric actin concentrations in low ionic strength buffer (G-buffer). After subjecting the liposome dispersions to high ionic strength polymerization buffer (F-buffer), topological changes in liposome structure were studied using atomic force microscopy (AFM). Paired dumbbell, horseshoelike, and disklike assemblies were observed for actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes. The topology of actin-containing liposomes was found to be highly dependent on both liposome size and actin concentration. At 1 mg/mL actin, the actin-containing liposomes transformed into a disklike shape, whereas, at 5 mg/mL actin, the actin-containing liposomes retained a spherical shape. On the basis of these observations, we hypothesize that actin could either polymerize on the surface of the inner leaflet of the liposome membrane or polymerize in the aqueous core of the liposome. We explain the associated shape changes induced in actin-containing liposomes on the basis of the hypothesized mechanism of actin polymerization inside the liposomes. At higher actin concentrations (5 mg/mL), we observed membrane-induced actin self-assembly in G-buffer, which implies that G-actin is able to interact directly with lipid bilayers at sufficiently high concentrations.     

The surface properties of phospholipid liposome systems and their characterisation

The field of liposome (vesicle) research has expanded considerably over the last 30 years. In physical chemical terms liposomes have many of the characteristics of colloidal particles and their stability is determined in part by the classical surface forces. It is now possible to engineer a wide range of liposomes varying in size, phospholipid composition and surface characteristics. The surfaces of liposomes can be modified by the choice of bilayer lipid as well as by the incorporation and covalent linkage of proteins (e.g. antibodies and sugar binding proteins [lectins]), glycoproteins and synthetic polymers. Much of the impetus for liposome design has come from their potential value as drug delivery systems. The development of technologies for the production of such a range of liposome systems has presented interesting problems in the characterisation of their properties. The review addresses the progress that has been made in characterising the surfaces of different types of liposomes with specific reference to their electrophoretic properties and their interpretation and the physical interactions between liposomal bilayers.