Effects of ions and detergents in drug partition chromatography on liposomes

We have determined drug partitioning into phospholipid bilayers by immobilized-liposome chromatography (ILC). Electrostatic effects on the drug partitioning were observed on neutral bilayers at low ionic strength. The size of the counterions affected the partitioning. When liposomes were supplemented with ionic detergents the partitioning of charged drugs was strongly affected, allowing complete separation of drugs of different charges which showed similar retention on neutral bilayers. Partial separation was obtained on bilayers containing fatty acid. Detergent ions or fatty acid inserted into phospholipid bilayers affected the partitioning of drugs much more than did free ions or phospholipid head group charges.     

Effect of the electrostatic charge on the mechanism inducing liposome solubilization: a kinetic study by synchrotron radiation SAXS

The anionic surfactant sodium dodecyl sulfate (SDS) was used to induce the initial steps of the solubilization of liposomes. The structural transformations as well as the kinetics associated with this initial period were studied by means of time-resolved small-angle X-ray scattering (SAXS) using a synchrotron radiation source. Neutral and electrically charged (anionic and cationic) liposomes were used to investigate the effect of the electrostatic charges on the kinetics of these initial steps. The mechanism that induces the solubilization process consisted of adsorption of surfactant on the bilayers and desorption of mixed micelles from the liposomes surface to the aqueous medium. In all cases the time needed for desorption of the first mixed micelles was shorter than that for complete adsorption of the surfactant on the liposomes surface. The present work demonstrates that adsorption of the SDS molecules on negatively charged liposomes was slower and release of mixed micelles from the surface of these liposomes was faster than for neutral liposomes. In contrast, in the case of positively charged liposomes, the adsorption and release processes were, respectively, faster and slower than those for neutral vesicles.     

Alterations in the Detergent-Induced Membrane Permeability and Solubilization of Saturated Phosphatidylcholine/Cholesterol Liposomes: Effects of Poly(ethylene glycol)-Conjugated Lipid

We have investigated the effects of two bile salts, chenodeoxycholate (CDC) and ursodeoxycholate (UDC), and a widely used detergent, Triton X-100 (T(X-100)), on normal and poly(ethylene glycol)-modified liposomes (PEGylated liposomes). We tested various lipid compositions, including hydrogenated soybean phosphatidylcholine/cholesterol/PEG-conjugated lipid (HSPC/PEG-lipid). Alterations in permeability were determined by the rate of drug release from the liposomes and solubilization was assessed by measuring the particle size of liposomes. In addition, we attempted to observe interactions between the detergents and lipid bilayers by using surface plasmon resonance (SPR). CDC induced drug release from liposomes in a dose-dependent manner, and the PEGylated liposomes tended to be susceptible to CDC. While UDC did not strongly induce drug release from liposomes, UDC exhibited a similar tendency with CDC. In case of T(X-100), there were significant differences in the percentage of released drug between normal and PEGylated liposomes, and the percentage of T(X-100)-induced drug release further increased with an increased ratio of PEG-lipid. SPR analysis revealed that the lipid bilayer including PEG-lipid was selectively solubilized by T(X-100), correlating with the drug release data. These results suggest that the effect of detergents on the lipid bilayer of liposomes depends on both the kind of detergent and the lipid composition, including the presence or absence of PEG-lipid. Moreover, the effects of T(X-100) on the lipid bilayers of the PEGylated liposomes significantly differed from those on the lipid bilayers of the normal liposomes.     

Drug partition chromatography on immobilized porcine intestinal brush border membranes

We immobilized porcine intestinal brush border membrane vesicles (BBMVs) for chromatographic analyses of drug partitioning into the membranes determined as Ks, the drug retention per phospholipid amount. For positive and neutral drugs Ks decreased day by day, whereas Ks for negative drugs increased marginally. Similar results on vesicle-lipid liposomes indicated a gradual loss of negative charge from the columns. The Ks values for positive drugs were higher than those for negative drugs with the same octanol/water partitioning or the same Ks on egg yolk phospholipid bilayers. Electrostatic interactions seem to be important for the partitioning of charged drugs into brush border membranes.     

Studies on pectin coating of liposomes for drug delivery

The present study investigated the surface coating of charged liposomes by three different types of pectin (LM, HM and amidated pectin) by particle size determinations and zeta potential measurements. The pectins and the pectin coated liposomes were visualized by atomic force microscopy. The adsorption of pectin onto positive liposomes yielded a reproducible increase in particle size and a shift of the zeta potential from positive to negative side for all three pectin types, whereas the adsorption of pectin onto negative liposomes did not render any significant changes probably due to electrostatic repulsion. The positive liposomes coated with HM-pectin gave the largest pectin coated particles with the least negative zeta potential, while the opposite was observed for the LM-pectin coated positive liposomes. Furthermore, results from dynamic light scattering revealed narrow size distributions, indicating that the degree of aggregation was low for the pectin coated liposomes. As liposomes are able to encapsulate drugs and pectin has been found to be mucoadhesive, these pectin coated liposomes may be potential drug delivery systems.     

Influence of microenvironment and liposomal formulation on secondary structure and bilayer interaction of lysozyme

The conformation of peptide and protein drugs in various microenvironments and the interaction with drug carriers such as liposomes are of considerable interest. In this study the influence of microenvironments such as pH, salt concentration, and surface charge on the secondary structure of a model protein, lysozyme, either in solution or entrapped in liposomes with various molar ratios of phosphatidylcholine (PC):cholesterol (Chol) was investigated. It was found that entrapment efficiency was more pronounced in negatively charged liposomes than in non-charged liposomes, which was independent of Chol content and pH of hydration medium. The occurrence of aggregation, decrease in zeta potential, and alteration of ³¹P NMR chemical shift of negatively charged lysozyme liposomes compared to blank liposomes suggested that the electrostatic interaction plays a major role in protein–lipid binding. Addition of sodium chloride could impair the neutralizing ability of positively charged lysozyme on negatively charged membrane via chloride counterion binding. Neither lysozyme in various buffer solutions with sodium chloride nor that entrapped in liposomes showed any significant change in their secondary structures. However, significant decrease in α-helical content of lysozyme in non-charged liposomes at higher pH and salt concentrations was discovered.     

Molecular interactions between lipid bilayers and a water-soluble polymer

Molecular interactions between lipid bilayers (liposomes) and chondroitin sulfate C (CS), a water soluble polymer, have been investigated in terms of zeta-potential, particle size, microscopic-viscosity, microscopic-polarity of liposomes and permeability of calcein. Microscopic morphology is dramatically changed by the addition of CS to the positively charged liposomes (Pos.L), while it is not changed by the addition to uncharged liposomes (Unc.L) or negatively charged liposomes (Neg.L). The absolute value of the particle size of Pos.L increases with the addition of CS, while the zeta- potential of Pos.L decreases. Permeability of Pos.L decreases with an increase in the concentration of CS. Phase transition temperature of Pos.L is changed after the addition of CS. These values, however, are not changed for the other liposomes by the addition of CS. The results of gel filtration chromatography show that CS is absorbed on the Pos.L surface. Microscopic viscosity is also increased by the addition of CS to Pos.L due to the adsorption of CS.     

Substrate effects on interactions of lipid bilayer assemblies with bound nanoparticles

Understanding the interactions of nanoparticles with lipid membranes is crucial in establishing the mechanisms that govern assembly of membrane-based nanocomposites, nanotoxicology, and biomimetic inspired self-assembly. In this study, we explore binding of charged nanoparticles to lipid bilayers, both as liposomes and substrate supported assemblies. We find that the presence of a solid-support, regardless of curvature, eliminates the ability of zwitterionic fluid phase lipids to bind charged nanoparticles.     

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.     

PH effects on drug interactions with lipid bilayers by liposome electrokinetic chromatography

Liposome electrokinetic chromatography (LEKC) provides convenient and rapid methods for studying drug interactions with lipid bilayers using liposomes as a pseudostationary phase. LEKC was used to determine the effects of pH on the partitioning of basic drugs into liposomes composed of zwitterionic phosphatidylcholine (PC), anionic phosphatidylglycerol (PG), and cholesterol, which mimic the composition of natural cell membranes. An increase in pH results in a smaller degree of ionization of the basic drugs and consequently leads to a lower degree of interaction with the negatively charged membranes. From the LEKC retention data, the fractions of drugs distributed in the bulk aqueous and the liposome phase were determined at various pH values. Finally, lipid mediated shifts in the ionization constants of drugs were examined.     

Enhancing physical stability of positively charged catanionic vesicles in the presence of calcium chloride via cholesterol-induced fluidic bilayer characteristic

An ion pair amphiphile (IPA), hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS), and a double-chained cationic surfactant, dimethyldimyristylammonium bromide (DTDAB), could form positively charged catanionic vesicles with a potential application in gene delivery. To improve the gene delivery efficiency, the addition of CaCl₂ into cationic liposomal systems has been proposed in the literature. In this study, detrimental effect of calcium chloride on the physical stability of the positively charged HTMA-DS/DTDAB catanionic vesicles was demonstrated by the size and zeta potential analyses of the vesicles. It was noted that the reduced electrostatic interaction between the catanionic vesicles could not fully explain the lowered physical stability of the vesicles in the presence of CaCl₂. Apparently, the molecular packing/interaction in the vesicular bilayers played an important role in the vesicle physical stability. To modify the molecular packing/interaction in the vesicular bilayers, cholesterol was adopted as an additive to form catanionic vesicles with HTMA-DS/DTDAB. It was found that the physical stability of the catanionic vesicles was significantly improved with the presence of cholesterol in the vesicular bilayers even in the presence of 50 mM CaCl₂. An infrared analysis suggested that with the incorporation of cholesterol into HTMA-DS/DTDAB vesicular bilayers, the alkyl chain motion was enhanced, and the molecular packing became less ordered. The cholesterol-induced fluidic bilayer characteristic allowed the vesicular bilayers to be adjusted to a stable status, resulting in improved physical stability of the catanionic vesicles even in the presence of CaCl₂ with a high concentration.     

Detection of supported lipid bilayers using their electric charge

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.     

Effects of lactose permease on the phospholipid environment in which it is reconstituted: a fluorescence and atomic force microscopy study

The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

The action of indomethacin on neutral and positively charged monolayers

The penetration ability of indomethacin in neutral and positively charged monolayers has been studied. Neutral monolayers of cholesterol and dipalmitoyl phosphatidyl choline present a slight but significant increase of surface pressure. The presence of stearylamine in the films results in an increase of surface pressure due to an electrostatic effect between the carboxylic anion of indomethacin and the polar head group of the stearylamine. These values can afford a reference point to choose the best lipid composition of liposomes encapsulating indomethacin to avoid the drug causing leakage of liposomes.     

Development of non-phospholipid liposomes containing a high cholesterol concentration

We present a novel formulation of non-phospholipid liposomes formed from cholesterol and palmitic acid. Despite the fact that these two lipidic species do not form individually fluid bilayers, we show that once mixed together, fluid bilayers can be obtained and, moreover, these can be extruded using classical extrusion processes to form liposomes. The chemical analysis indicates that these liposomes contain 70 mol % cholesterol, a content that is considerably higher that the saturation limit generally reported for phospholipid bilayers. These cholesterol-rich liposomes, formed with molecules that have low toxicity in vivo, display an improved impermeability relative to that of traditional phospholipid liposomes. In addition, because of the presence of palmitic acid, the stability of the liposomes is pH-dependent, and it is possible to trigger the release of encapsulated materials by pH stimuli.     

Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes

The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide.     

Sequestration of amitriptyline by liposomes

We study the uptake of amitriptyline, which is a common cause of overdose-related fatalities, in aqueous solutions by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes and liposomes composed of a mixture of DMPC and 1,2-dioleoyl-sn-glycero-3-[phospho-rac(1-glycerol)] (DOPG) lipids. The effect of drug concentration, liposomal charge, pH, salt, and protein presence on the drug uptake is investigated using two different methodologies, a precipitation and a centrifugation method. Furthermore, the time scale of the drug uptake is studied through qualitative observations at high pH and through conductivity measurements at neutral pH and found to be <5 s. The results of the quantitative studies show that the fractional drug uptake decreases with increasing drug concentration, and for a given concentration it increases with the pH and decreases in the presence of salt. We find that a larger amount of drug is sequestered by negatively charged liposomes (those containing DOPG) than liposomes with no net charge (DMPC). We speculate that the mechanism of drug uptake is due to both electrostatic interactions as well as hydrophobic effects. The fractional uptake by DMPC:DOPG in a 70:30 ratio is as high as 95% in water and about 90% in physiological buffer. The fractional uptake is also measured in presence of 2% (w/w) bovine serum albumin (BSA), which is approximately the protein concentration in the intercellular fluid. In presence of protein the fractional uptakes by 70:30 DMPC:DOPG liposomes and 50:50 DMPC:DOPG liposomes are 82 and 90%, respectively, at 125 muM drug amitriptyline. In the absence of liposomes, 67% of the drug is taken up by the protein in a 2% (w/w) BSA, 125 muM amitriptyline solution. Thus, addition of 50:50 DMPC:DOPG liposomes reduces the free drug concentration by a factor of about 3.5, making them attractive candidates for drug detoxification.     

Arrays of lipid bilayers and liposomes on patterned polyelectrolyte templates

This paper presents novel methods to produce arrays of lipid bilayers and liposomes on patterned polyelectrolyte multilayers. We created the arrays by exposing patterns of poly(dimethyldiallylammonium chloride) (PDAC), polyethylene glycol (m-dPEG) acid, and poly(allylamine hydrochloride) (PAH) on polyelectrolyte multilayers (PEMs) to liposomes of various compositions. The resulting interfaces were characterized by total internal reflection fluorescence microscopy (TIRFM), fluorescence recovery after pattern photobleaching (FRAPP), quartz crystal microbalance (QCM), and fluorescence microscopy. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) (DOPA) were found to preferentially adsorb on PDAC and PAH surfaces. On the other hand, liposome adsorption on sulfonated poly(styrene) (SPS) surfaces was minimal, due to electrostatic repulsion between the negatively charged liposomes and the SPS-coated surface. Surfaces coated with m-dPEG acid were also found to resist liposome adsorption. We exploited these results to create arrays of lipid bilayers by exposing PDAC, PAH and m-dPEG patterned substrates to DOPA/DOPC vesicles of various compositions. The patterned substrates were created by stamping PDAC (or PAH) on SPS-topped multilayers, and m-dPEG acid on PDAC-topped multilayers, respectively. This technique can be used to produce functional biomimetic interfaces for potential applications in biosensors and biocatalysis, for creating arrays that could be used for high-throughput screening of compounds that interact with cell membranes, and for probing, and possibly controlling, interactions between living cells and synthetic membranes.     

Electrostatics of PEGylated micelles and liposomes containing charged and neutral lipopolymers

The electrostatics of large unilamellar vesicles (LUVs) of various lipid compositions were determined and correlated with steric stabilization. The compositional variables studied include (a) degree of saturation, comparing the unsaturated egg phosphatidylcholine (EPC) and the fully hydrogenated soy phosphatidylcholine (HSPC) as liposome-forming lipids; (b) the effect of 40 mol % cholesterol; (c) the effect of mole % of three methyl poly(ethylene glycol) (mPEG)-lipids (the negatively charged mPEG-distearoyl phosphoethanolamine (DSPE) and two uncharged lipopolymers, mPEG-distearoyl glycerol (DSG) and mPEG-oxycarbonyl-3-amino-1,2-propanediol distearoyl ester (DS)); and (d) the negatively charged phosphatidyl glycerol (PG). The lipid phases were as follows: liquid disordered (LD) for the EPC-containing LUV, solid ordered (SO) for the HSPC-containing LUV, and liquid ordered (LO) for either of those LUV with the addition of 40 mol % cholesterol. The LUV zeta potential and electrical surface potential (psi(0)) were determined. It was found that progressive addition of mPEG(2k)-DSPE to liposomes increases negative surface potential and reduces surface pH to a similar extent as the addition of PG. However, due to the "hidden charge effect", zeta potential was more negative for liposomes containing PG than for those containing mPEG(2k)-DSPE. Replacing mPEG-DSPE with mPEG(2k)-DS or mPEG-DSG had no effect on surface pH and surface potential, and zeta potential was approximately zero. Addition of low concentrations of cationic peptides (protamine sulfate and melittin) to PG- or mPEG-DSPE-containing liposomes neutralized the liposome negative surface potential to a similar extent. However, only in liposomes containing PG, did liposome aggregation occur. Replacing the negatively charged lipopolymer mPEG-DSPE with the neutral lipopolymers mPEG-DS or mPEG-DSG eliminates or reduces such interactions. The relevance of this effect to the liposome performance in vivo is discussed.