Interaction Of Liposomes Research Articles

Combined Effect of Surface Electrostatic Charge and Poly(ethyl glycol) on the Association of Liposomes with Colon Carcinoma Cells

Particulate drug formulations are considered to be a means that may improve the pharmacokinetics and biodistribution of active compounds. By using them, drug distribution is determined solely by the properties of the carrier. The surface properties of such supramolecular aggregates determine how they will interact with various biological structures. Among others, surface electrostatic charge and surface grafted polymers are considered to be among the major factors affecting its interaction with proteins and cells. In this article, we present experimental evidence that properly selected surface electrostatic charge and grafted polymers can alter the association of liposomes with colon cancer cells. The dependence of the adsorption of liposomes onto the cell surface on the quantity and length of surface grafted polymers for a certain surface charge density exhibits a distinct maximum. For example, when liposomes were formed with 20 mol% of DOTAP, PE-PEG350 increased liposome adsorption by up to 6 mol%. This adsorption maximum depends on both polymer length and charge type. Results presented in this article show that the interaction of liposomes with colon cancer cells can be tuned by a proper combination of liposome surface electrostatics and surface grafted polymers.

Interactions of drugs and an oligonucleotide with charged membranes analyzed by immobilized liposome chromatography

We studied the effect of charged lipids or detergent on the retention of drugs and an oligonucleotide by immobilized liposome chromatography to characterize solute-membrane interactions. This is a novel approach in analysis of oligonucleotide-liposome interactions. The charged lipids (phosphatidylserine or distearoyltrimethylammoniumpropane) or detergent (sodium dodecylsulfate) interacted electrostatically in a concentration-dependent matter with the solutes. The oligonucleotide ions presumably bound to the liposomes by multipoint interactions, which was saturable. Sodium dodecylsulfate seemed to affect the drug-membrane interactions more strongly than phosphatidylserine did, probably due to different positioning in the bilayer.

Engineering Lipid Vesicles of Enhanced Intratumoral Transport Capabilities: Correlating Liposome Characteristics with Penetration into Human Prostate Tumor Spheroids

Liposomes have been widely used delivery systems, particularly relevant to the development of cancer therapeutics. Numerous liposome-based drugs are in the clinic or in clinical trials today against multiple tumor types; however, systematic studies of liposome interactions with solid or metastatic tumor nodules are scarce. This study is describing the in vitro interaction between liposomes and avascular human prostate (LNCaP-LN3) tumor spheroids. The ability of fluorescently labelled liposomal delivery systems of varying physicochemical characteristics to penetrate within multicellular tumor spheroids has been investigated by confocal laser scanning microscopy. A variety of liposome characteristics and experimental parameters were investigated, including lipid bilayer composition, duration of liposome-spheroid interaction, mean liposome size, steric stabilization of liposomes. Electrostatic binding between cationic liposomes and spheroids was very efficient; however, it impeded any significant penetration of the vesicles within deeper layers of the tumor spheroid. Small unilamellar liposomes of neutral surface character did not bind as efficiently but exhibited enhanced penetrative transport capabilities closer to the tumor core. Polymer-coated (sterically stabilised) liposomes exhibited almost no interaction with the spheroid, indicating that their limited diffusion within avascular tissues may be a limiting step for their use against micrometastases. Multicellular tumor spheroids were used as models of solid tumor interstitium relevant to delivery systems able to extravasate from the microcapillaries or as models of prevascularized micrometastases. This study illustrates that interactions between liposomes and other drug delivery systems with multicellular tumor spheroids can offer critically important information with respect to optimizing solid or micrometastatic tumor delivery and targeting strategies.

Fibronectin conformational changes induced by adsorption to liposomes

One of the major drawbacks of drug delivery techniques that utilize liposomes as carriers is that they are often cleared from the body before they can deliver their therapeutic cargo. It is well known that serum proteins can adsorb to these drug delivery vehicles and influence their uptake by phagocytic cells. For this reason, protein adsorption to liposomes has been extensively quantified, and strategies have been developed to minimize protein adsorption to improve drug delivery. However, the conformation of proteins on surfaces can play an even greater role in controlling cell behavior than the quantity of adsorbed protein. We have therefore used fluorescence resonance energy transfer (FRET) to measure changes in the structure of fibronectin (Fn)—a key serum protein involved in phagocytosis—upon interaction with phosphatidylcholine (PC) liposomes. Our experiments reveal that fibronectin opens up from its inactive, compact conformation upon interaction with gel phase PC liposomes. We also used FRET to estimate a physiologically relevant dissociation constant, K D=1.1 nM, for the interaction. Conformational changes in serum proteins may result in the exposure of otherwise concealed recognition sites and therefore influence the interaction of liposomes with phagocytic cells.

The role of β 2-glycoprotein I in liposome–hepatocyte interaction

Adsorption of serum proteins to the liposomal surface plays a critical role in liposome clearance from the blood. The aim of this study was to investigate the role of liposome-adsorbed serum proteins in the interaction of liposomes with hepatocytes. We analyzed the serum proteins adsorbing to the surface of differently composed small unilamellar liposomes during incubation with human or rat serum, and found that one protein, with a molecular weight of around 55 kDa, adsorbed in a large amount to negatively charged liposomes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). The binding was dependent on the liposomal charge density. The ∼55-kDa protein was identified as β 2-glycoprotein I (β 2GPI) by Western blotting. Despite the high affinity of β 2GPI for strongly negatively charged liposomes, in vitro uptake and binding experiments with isolated rat hepatocytes, Kupffer cells or liver endothelial cells, and with HepG2 cells showed no enhancing effect of this protein on the association of negatively charged liposomes with any of these cells. On the contrary, an inhibitory effect was observed. We conclude that despite abundant adsorption to negatively charged liposomes, β 2GP1 inhibits, rather than enhances, liposome uptake by liver cells.

An investigation on the analytical potential of polymerized liposomes bound to lanthanide ions for protein analysis.

We present a promising approach to protein sensing based on Eu3+ ions incorporated into polymerized liposomes. The sensitization of Eu3+ is accomplished with 5-aminosalicylic acid, which provides energy transfer for a stable reference signal and a wide wavelength excitation range free from protein interference. The lipophilic character of polymerized liposomes provides the appropriate platform for protein interaction with the lanthanide ion. Quantitative analysis is based on the linear relationship between the luminescence signal of Eu3+ and protein concentration. Because no spectral shift of the lanthanide luminescence is observed upon protein interaction, qualitative analysis is based on the luminescence lifetime of polymerized liposomes. This parameter, which changes significantly upon protein-liposome interaction, follows a well-behaved single-exponential decay that might be useful for protein identification.

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.

Analyte–receptor binding on surface plasmon resonance biosensors: a fractal analysis of Cre– loxP interactions and the influence of Cl, O, and S on drug–liposome interactions

A fractal analysis of the association and dissociation (whereever applicable) of Cre– loxP interactions and drug–liposome interactions on a sensor chip surface is presented. In both of these cases a dual-fractal analysis is required to adequately describe the association kinetics. The dissociation kinetics for Cre– loxP interactions is adequately described by a single-fractal analysis. The dual-fractal analysis used to describe the association kinetics of Cre– loxP interactions is consistent with the original two-step mechanism presented using a surface plasmon resonance biosensor. Our analysis includes both diffusion and surface effects by introducing the fractal dimension which makes quantitative the degree of heterogeneity on the sensor chip surface. Affinities are provided. Only the association kinetics were analysed for drug–liposome interactions since the initial sections of the dissociation curves were too steep to obtain reasonable drug–liposome complex concentration values on the sensor chip with time. Attempts made to relate the association rate coefficients with the molecular weight of the drug were unsuccessful. On using desipramine and imipramine as “arbitrarily selected standards” or “references” (only C, H, and N atoms present), it was noticed from the data analysed that the inclusion of the O and S atoms in the drug leads to a decrease in the association rate coefficients, k a1 (or k 1) and k a2 (or k 2) (compared with the arbitrarily selected standards or references). Similarly, the addition of the Cl atom in the drug leads to an increase in the association rate coefficient (compared with the arbitrarily selected standards or references). More data needs to be analysed to determine whether this is true for other drugs also.

Interactions of complementary PEGylated liposomes and characterization of the resulting aggregates.

The interaction of complementary liposomes bearing both recognizable and protective ligands at their external surface has been investigated. Aggregation of hydrogenated phosphatidyl choline/cholesterol (2:1 molar ratio) based liposomes was mediated by the molecular recognition of the complementary phosphate and guanidinium groups incorporated in separate unilamellar liposomes. The phosphate group was incorporated in the bilayer employing dihexadecyl phosphate, while the guanidinium moiety was introduced in the membrane through the incorporation of various guanidinium lipids. For the latter, anchoring ability and primarily introduction of a spacer group between their lipophilic part and the guanidinium group was found to affect the ability for molecular recognition. Also, poly(ethylene glycol) (PEG) introduced in both types of liposomes at various concentrations and up to 15% with respect to cholesterol modifies the interaction effectiveness and morphology of the obtained aggregates. Interaction of these complementary liposomes leads to large precipitating aggregates or fused liposomes, as shown by phase contrast microscopy and dynamic light scattering. Specifically, fusion of liposomes takes place under a nonleaking process involving lipid mixing, as demonstrated by calcein entrapment and resonance energy transfer experiments. Calorimetric parameters also correlate with the processes of aggregation and fusion. The interactions of non-PEGylated liposomes involve exothermic processes of higher enthalpic content than those of the PEGylated counterparts.

Physicochemical Aspects of the Liposome−Wool Interaction in Wool Dyeing

Despite the promising application of liposomes in wool dyeing, little is known about the mechanism of liposome interactions with the wool fiber and dyestuffs. The kinetics of wool dyeing by two dyes, Acid Green 27 (hydrophobic) and Acid Green 25 (hydrophilic), were compared in three experimental protocols: (1) without liposomes, (2) in the presence of phosphatidylcholine (PC) liposomes, and (3) with wool previously treated with PC liposomes. Physicochemical interactions of liposomes with wool fibers were studied under experimental dyeing conditions with particular interest in the liposome affinity to the fiber surface and changes in the lipid composition of the wool fibers. The results obtained indicate that the presence of liposomes favors the retention of these two dyes in the dyeing bath, this effect being more pronounced in case of the hydrophobic dye. Furthermore, the liposome treatment is accompanied by substantial absorption of PC by wool fibers with simultaneous partial solubilization of their polar lipids (more evident at higher temperatures). This may result in structural modification of the cell membrane complex of wool fibers, which could account for a high level of the dye exhaustion observed at the end of the liposome dyeing process.

Peripheral and Integral Binding of Cytochrome c to Phospholipids Vesicles

The interactions of ferric cytochrome c (Cyt-c) with dioleoyl-phosphatidylglycerol (DOPG) at low ionic strength have been studied by viscosity and turbidity measurements as well as by resonance Raman, circular dichroism, and UV−vis-absorption spectroscopy to monitor the structural changes of the liposomes and the protein upon complex formation. The observed mutual structural changes in the liposomes and the protein are associated with three different modes of protein binding. At high lipid/protein (L/P) ratios, Cyt-c binds electrostatically to the anionic headgroups of the phospholipids which induces structural changes of the protein. Decreasing the L/P-ratio weakens the electrostatic interactions such that membrane anchoring of Cyt-c can effectively compete with peripheral binding. This mode of binding is accompanied by an increase of long-range liposome−liposome interactions. Upon lowering the L/P-ratio below the ratio for full protein coverage of the vesicles, further Cyt-c binding is achieved via interactions with the protein monolayer. This mode of binding initiates phase separation of the liposome aggregates from the aqueous buffer. Our results indicate that the crucial parameter controlling the interplay between the binding modes appears to be the membrane surface potential which in turn sensitively depends on the protein coverage.

Hydrogen bonding interactions of liposomes simulating cell-cell recognition. A review.

Amphiphiles bearing polar heads with the property to form hydrogen bond(s) exhibit unique organizational and aggregational behaviour. Thus appropriate amphiphilic molecules assemble and form liposomes, which further interact through hydrogen bonding with complementary molecules or liposomal counterparts affording larger and more elaborated aggregates. A number of examples are demonstrating the interaction mode of liposomes and of associated phenomena as related to the structural features of the supramolecular aggregates obtained. The recognition between cells incorporating recognizable amphiphiles in their membranes has shown similarities to the analogous interactions between liposomes. Thus molecular recognition between liposomes can be used in modeling recognitions occurring between cells. Designed experiments in this area can support the Lipid World Model proposed for the origin of life.

Interactions of Hyaluronan-Targeted Liposomes with Cultured Cells: Modeling of Binding and Endocytosis

Interactions of Hyaluronan-Targeted Liposomes with Cultured Cells: Modeling of Binding and Endocytosis

Distal Cationic Poly(Ethylene Glycol) Lipid Conjugates in Large Unilamellar Vesicles Prepared by Extrusion Enhance Liposomal Cellular Uptake

Cationic poly(ethylene glycol)‐lipid conjugates (CPLs), a class of lipid designed to enhance the interaction of liposomes with cells, possess the following architectural features: 1) a hydrophobic lipid anchor of distearoylphosphatidylethanolamine (DSPE); 2) a hydrophilic spacer of poly(ethylene glycol); and 3) a cationic head group prepared with 0, 1, 3, or 7 lysine residues located at the distal end of the PEG chain, giving rise to CPL possessing 1, 2, 4, or 8 positive charges, respectively (CPL1 to CPL8). Previously we have described the synthesis of CPL, have characterized the postinsertion of CPL into PEG‐containing LUVs and SPLP (stabilized plasmid‐lipid particles), have shown significant increases in the binding of CPL‐LUV to cells, and have observed dramatically enhanced transfection (up to a million‐fold) of cells with CPL‐SPLP in the presence of calcium [Chen et al. (2000) Bioconjugate Chem. 11, 433–437; Fenske et al. (2001) Biochim. Biophys. Acta 1512, 259–272; Palmer et al. (2003) Biochim. Biophys. Acta 1611, 204–216]. In the present study, we examine a variety of CPL properties (such as polarity and CMC) and characterize CPL‐vesicular systems formed by extrusion and examine their interaction with cells. While CPL polarity was observed to increase dramatically with increasing charge number, CMC values were all found to be low, in the range of other PEGylated lipids, and exhibited only a small increase, going from CPL1 (1.3 µM) to CPL8 (2 µM). The CPLs were almost quantitatively incorporated into large unilamellar vesicles (LUVs) prepared by the extrusion method and were evenly distributed across the lipid bilayer. Lower levels of incorporation were obtained when CPLs were incubated with preformed liposomes (DSPC/Chol, 55:45) at 60°C. The binding of CPL‐LUVs to BHK cells in vitro was found to be dependent on the distal charge density of the CPL rather than total surface charge. Liposomes possessing CPL4 or CPL8 were observed to bind efficiently to cell surfaces and enhance cellular uptake in BHK cells (as observed with both lipid and aqueous content markers), whereas those possessing CPL1 or CPL2 exhibited little or no binding. These results suggest new directions for the design of liposomal systems capable of in vivo delivery of both conventional and genetic (plasmid and antisense) drugs.

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability.

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.

Interaction of dendritic cells with antigen-containing liposomes: effect of bilayer composition

Vaccine efficacy might be improved by exploiting the potent antigen presenting properties of dendrite cells (DCs), since their ability to stimulate specific major histocompatibility complex-restricted immune responses has been well documented during the recent years. In that light, we investigated how the interaction of antigen-containing liposomes with DCs was affected by the bilayer composition. Monocyte-derived human DCs and murine bone marrow-derived DCs were analysed and compared upon in vitro incubation with liposomes by flow cytometry and confocal microscopy. Anionic liposomes with a bilayer composition of phosphatidylcholine, cholesterol and phosphatidylglycerol or phosphatidylserine interacted with a limited fraction of the total DC population in case of both DC types. Inclusion of mannosylated phosphatidylethanolamine (Man-PE) for targeting to the mannose receptor (MR) increased the interaction of negatively charged liposomes with both human and murine DCs. This increase could be blocked in human DCs by addition of the polysaccharide mannan indicating that uptake might be mediated by the mannose receptor. Cationic liposomes containing trimethyl ammonium propane interacted with a very high percentage of both DC types and could be detected in high amounts intracellularly. In conclusion, liposome bilayer composition has an important effect on interaction with DCs and might be critical for the vaccination outcome.

Binding parameters for the interaction between Candida rugosa lipase and DPPC liposomes

The binding interactions of Candida rugosa lipase (CRL) to dipalmitoylphosphatidylcholine (DPPC) liposomes in light of this lipase's aggregation behavior were characterized. The affinity of CRL aggregates for DPPC liposomes was higher than the affinity of CRL monomeric forms. The specific activity of CRL aggregates was higher than that of the monomeric form. It is possible that enzyme aggregates provide a hydrophobic environment for lipase catalysis, leading to an enhancement in lipase activity. This activation mechanism would be similar to lipase interfacial activation upon binding to an oil–water interface. Both the number of lipid molecules associated with a lipid–enzyme complex and the number of enzyme binding sites doubled when lipase concentration was increased from 2.27 to 10.33 μg/ml. Neither a change in the phase transition temperature nor a broadening of the phase transition was detected as a result of the CRL–DPPC liposome interactions. Fluorescence resonance energy transfer spectroscopy proved to be a rapid and efficient method for the determination of lipase–liposome binding parameters.

Permeability investigations of phospholipid liposomes by adding cholesterol

Abstract The sublytic changes caused by sodium dodecyl sulfate (SDS) in egg phosphatidylcholine (PC) liposomes with rising concentrations of cholesterol (CH) (PC:CH mole ratios from 9:1 to 7:3) were investigated. The effective molar ratios Re of membrane-bound surfactant to lipid and the bilayer/aqueous phase partition coefficients (K) were determined. A linear increase and decrease in Re and K, respectively, take place as the CH proportion increase. Given that the surfactant capacity to interact with liposomes is inversely related to the Re value, the rise of CH reduces linearly both the SDS activity against vesicles (increase in Re) and its affinity with these structures (decrease in K). These linear variations may be due to the linear increase of hydrophobicity in the interfacial region of bilayers and to their reduced translational fluidity by changes in the bilayer packing and ordering. The increase in Re always results in two opposite effects on K. At low Re, K firstly increases because only the outer vesicle leaflet is available for interaction with SDS. The following abrupt fall in K is due to the fact that the binding of additional SDS molecules to bilayer is hampered. The K peaks obtained are correlated in all cases with the saturation of the outer vesicle leaflet by SDS. Increasing Re values, lead to an increased rate of flip–flop of the surfactant molecules (or permeabilization of the bilayers to SDS), thus also making the inner monolayer available for interaction with added SDS. The free SDS concentrations are always lower than its critical micelle concentration indicating that the SDS–liposome interaction is mainly ruled by the action of surfactant monomers regardless of the CH concentration in bilayers.

Measurement of diffusion of fluorescent molecules in living cells

The possibilities of the method of fluorescence correlation spectroscopy for studying the molecular dynamics in living cells are demonstrated. The method provides point measurements of extremely low concentrations of fluorescent molecules and their diffusion coefficients with a high time resolution in a microscopic volume, which is especially important in pharmacological investigations. A biological model of the interaction of liposomes with a cellular membrane is considered. The diffusion coefficients of fluorescent molecules are measured directly in the living cell cytoplasm.

Application of capillaries with minimized electroosmotic flow to the electrokinetic study of acidic drug–β-oleoyl-γ-palmitoyl- l-α-phosphatidyl choline liposome interactions

Interaction of a model set of common drugs varying widely in their polarity as well as in their chemical structure (salicylic acid, acetylsalicylic acid, ketoprofen, phenytoin and propranolol) with β-oleoyl-γ-palmitoyl- l-α-phosphatidyl choline (POPC) liposomes was investigated by means of capillary electrophoresis. Two phosphate buffers differing in their pH (50 m M, pH 7.5 and 9.2) were used both for liposome reconstitution and as background electrolytes for capillary electrophoresis using capillaries with minimised electroosmotic flow (EOF). The liposomes showed practically no electrophoretic mobility and formed a stable plug in the capillary. At alkaline pH (9.2), the polyimide coated capillary exhibited residual endoosmotic flow (the EOF marker appeared before the detection window around 40 min as compared to 2.2 min in the untreated capillary; attempts to reveal endoosmotic flow at pH 7.5 were unsuccessful). The concentration of the mixture of the test compounds was 50 μg/ml (except for ketoprofen concentration of which was 5 μg/ml due to the lower solubility of the drug), i.e. large enough to exceed the binding capacity of the injected liposome plug at least at the neutral pH (7.5) which consequently resulted in two regions in the electropherogram, namely that which contained the unbound species and that corresponding to the liposome (lipid)-bound fraction. On the other hand in runs done at high pH of the background electrolyte (9.2) the whole amount injected interacted with the liposomes. Acidic drugs and phenytoin were run with negative polarity at the injection site. It was documented that both at pH 7.5 and 9.2 the investigated solutes interacted with POPC liposomes, though at pH 7.5 the equilibrium between the bound and unbound drugs was in favor of the unbound species. On the contrary, at pH 9.2 binding was considerably stronger and only the liposome bound fraction was seen upon electrophoresis. The well-known instability of phenytoin at room temperature resulted in the formation of an acidic hydrolytic product which was strongly bound to liposomes at the higher pH value. While no binding of phenytoin could be established at pH 7.5, at pH 9.2 this compound was degraded (hydrolyzed) and its degradation product was clearly bound to liposomes. It has to be emphasized that binding experiments must be done separately for acidic/neutral and basic drugs; binding of acidic/neutral drugs must be done at reversed polarity, while in order to reveal binding of basic drugs, positive polarity at the injection site must be used.