Stabilized liposomes with phospholipid polymers and their interactions with blood cells

To stabilize a phospholipid liposome, addition of various water-soluble polymers into a liposomal aqueous suspension was investigated. The water-soluble polymers were poly(ethylene oxide) (PEO), poly(N-vinyl pyrrolidone) (PVPy) and poly[2-methacryloyloxyethyl phosphorylcholine(MPC)], and poly[MPC-co-n-butyl methacrylate(BMA)]. The gel–liquid crystal transition temperature (T_c) of the diparmitoylphosphatidylcholine (DPPC) liposome was not changed by addition of these polymers significantly. However, membrane fluidity of DPPC liposome treated with water-soluble polymers, which was measured with fluorescence probe, depended on the chemical structure of the water-soluble polymers. In the case of PEO and PVPy, the temperature dependence of membrane fluidity was the same as that of the original DPPC liposome, on the other hand, poly(MPC) and poly(MPC-co-BMA) induced a rise in the temperature where an increase in the membrane fluidity was observed. The release of carboxy fluorescein from the DPPC liposome was suppressed by the addition of the MPC polymers. The liposomes in the MPC polymer solution were stable compared with those in water when plasma was added into the suspension. Interactions with stabilized liposome with blood cells such as platelets and erythrocytes were evaluated. Activation of platelets in contact with liposome covered with poly(MPC) or poly(MPC-co-BMA) was less than PEO-stabilized liposome. On the other hand, no hemolysis of erythrocytes was observed when every polymer-treated liposome was added in the suspension of erythrocytes. Based on these results, the MPC polymers could interact with the liposome surface, adsorb on the liposomes and stabilize them, and had no adverse effect to the blood cells even when they were in a physiological environment.     

Specific interaction between water-soluble phospholipid polymer and liposome

A water-soluble polymer having both a phospholipid polar group and an azobenzene group, poly(2-methacryloyloxyethyl phosphorylcholine-co-p-phenylazoacrylanilide) (poly(MPC-co-PAAn)) was synthesized and the interaction between the liposome of the phospholipid was investigated in comparison with other water-soluble azoaromatic polymers. When the poly(MPC-co-PAAn) was incubated with dipalmitoylphosphatidylcholine (DPPC) lipo-somes above these gel-liquid crystalline temperatures, an electronic spectrum of azobenzene moiety, which is a hydrophobic probe, showed that the a copolymer chain exists under nonpolar circumstances. Thus, poly(MPC-co-PAAn) interacts with DPPC liposomes. On the other hand, other copolymers do not interact with the liposomes. Moreover, heat of the gel-liquid crystalline transition of DPPC liposomes was increased by the presence of poly(MPC-co-PAAn). These findings clearly indicate that the poly(MPC-co-PAAn) has an affinity to the DPPC liposomes and hybridizes with liposomes based on the phospholipid polar group of the copolymer.     

Preparation and physicochemical properties of various soybean lecithin liposomes using supercritical reverse phase evaporation method

Three kinds of soybean lecithin liposomes composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidic acid (PA), were prepared by using the previously developed supercritical reverse phase evaporation method (Langmuir 17 (2001) 3898). The effect of phospholipid composition on the formation of liposomes and their physicochemical properties were examined by means of trapping efficiency measurements, transmission electron microscopy, dynamic light scattering and zeta potential measurements. The trapping efficiency of liposomes for d-(+)-Glucose made of Lecinol S-10EX which contains approximately 95% PC is higher than that of Lecinol S-10 and SLP white SP which contain approximately 31% PC. However there is not any difference between the trapping efficiency of liposomes for d-(+)-Glucose made of Lecinol S-10 which has saturated hydrocarbons tails and that of liposomes made of SLP white SP which has unsaturated hydrocarbon chains. The electron micrographs of liposomes made of Lecinol S-10 and SLP white SP show small spherical liposomes with diameter of 0.1–0.25 μm, while that of Lecinol S-10EX shows large unilamellar liposomes (LUV) with diameter of 0.2–1.2 μm. These results clearly show that phospholipid structure of PC allows an efficient preparation of LUV and a high trapping efficiency for water-soluble substances. Liposomes made of Lecinol S-10 and SLP white SP remained well-dispersed for at least 14 days, while liposome suspension made of Lecinol S-10EX separated in two phase at 14 days due to aggregation and fusion of liposomes. The dispersibility of liposomes made of Lecinol S-10EX is lower than that of Lecinol S-10 and SLP white SP due to the smaller zeta potential of Lecinol S-10EX.     

Liposome formation of selectively-polymerized diene-containing phospholipids and their postpolymerization

Small and large unilamellar liposomes composed of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) are prepared by sonication and extrusion, respectively. They are polymerized with water-insoluble radical initiator, azobis(isobutyronitrile) (AIBN) which can selectively polymerize diene groups in 1-acyl chains of the lipids. Polymerized liposomes are freeze-dried to obtain the polymerized liposome powder. There are two methods to redisperse lyophilized liposomes into water. The extrusion is an effective method to disperse them because the energy at extrusion is necessary only for redispersion, whereas the excess energy at sonication gives damage on liposome structure. There is no difference in stability between polymerized liposomes before and after redispersion with extrusion. DODPC polymers, obtained from free radical-initiated polymerization with AIBN, are linear and have polymerizable diene groups in 2-acyl chains. The liposome powder is therefore soluble in organic solvents. Reconstruction of polymerized liposomes is performed with lipid polymers having low or high molecular weight. The lipid polymers having high molecular weight provide stable large unilamellar liposomes by ethanol injection, but unstable small unilamellar liposomes are formed by sonication. The liposomes reconstructed from lipid polymers having low molecular weight are unstable regardless of their size. After reconstruction of liposomes selectively polymerized by AIBN, diene groups in 2-acyl chains are polymerized by water-soluble radical initiator or UV-irradiation to yield highly crosslinked structure. Their stability is improved remarkably by this postpolymerization.     

Nanoformulation and Evaluation of Oral Berberine-Loaded Liposomes

Berberine (BBR) is a poorly water-soluble quaternary isoquinoline alkaloid of plant origin with potential uses in the drug therapy of hypercholesterolemia. To tackle the limitations associated with the oral therapeutic use of BBR (such as a first-pass metabolism and poor absorption), BBR-loaded liposomes were fabricated by ethanol-injection and thin-film hydration methods. The size and size distribution, polydispersity index (PDI), solid-state properties, entrapment efficiency (EE) and in vitro drug release of liposomes were investigated. The BBR-loaded liposomes prepared by ethanol-injection and thin-film hydration methods presented an average liposome size ranging from 50 nm to 244 nm and from 111 nm to 449 nm, respectively. The PDI values for the liposomes were less than 0.3, suggesting a narrow size distribution. The EE of liposomes ranged from 56% to 92%. Poorly water-soluble BBR was found to accumulate in the bi-layered phospholipid membrane of the liposomes prepared by the thin-film hydration method. The BBR-loaded liposomes generated by both nanofabrication methods presented extended drug release behavior in vitro. In conclusion, both ethanol-injection and thin-film hydration nanofabrication methods are feasible for generating BBR-loaded oral liposomes with a uniform size, high EE and modified drug release behavior in vitro.     

On the effect of Ca2+ and La3+ on the colloidal stability of liposomes

This work deals with the effect of Ca2+ and La3+ on the colloidal stability of phosphatidylcholine (PC) liposomes in aqueous media. As physical techniques, nephelometry, photon correlation spectroscopy, electrophoretic mobility, and surface tension were used. The theoretical predictions of the colloidal stability of liposomes were followed using the Derjaguin-Landau-Verwey-Overbeek theory. Changes in the size of liposomes and high polydispersity values were observed as La3+ concentration increases, suggesting that this cation induces the aggregation of liposomes. However, changes in polydispersity were not observed with Ca2+, suggesting a coalescence mechanism or fusion of liposomes. The stability factor (W), calculated from the nephelometry measurements indicated that aggregation/fusion occurs at a critical concentration (c.c.) of 0.3 and 0.7 M for La3+ and Ca2+, respectively. To gain a better insight into the interaction mechanism between the liposomes and the studied ions, the interaction between PC monolayers and Ca2+ and La3+ was studied. Changes in the surface area per lipid molecule (A0) in the monolayer at the c.c. values were found for both ions, with a more pronounced effect in the case of Ca2+. This corresponds with a larger reduction of the steric repulsive interaction between the headgroups at the phospholipid membrane (pi(head)). The experimental result validates the hypothesis made on the liposome fusion in the presence of Ca2+ and liposome aggregation in the presence of La3+. These aggregation mechanisms have also been confirmed by transmission electron microscopy.     

Liposomes Assembled from a Dual Drug‐tailed Phospholipid for Cancer Therapy

We report a novel dual drug‐tailed phospholipid which can form liposomes as a combination of prodrug and drug carrier. An amphiphilic dual chlorambucil‐tailed phospholipid (DCTP) was synthesized by a straightforward esterification. With two chlorambucil molecules as hydrophobic tails and one glycerophosphatidylcholine molecule as a hydrophilic head, the DCTP, a phospholipid prodrug, undergoes assembly to form a liposome without any additives by the thin lipid film technique. The DCTP liposomes, as an effective carrier of chlorambucil, exhibited a very high loading capacity and excellent stability. The liposomes had higher cytotoxic effects to cancer cell lines than free DCTP and chlorambucil. The in vivo antitumor activity assessment indicated that the DCTP liposomes could inhibit the tumor growth effectively. This novel strategy of dual drug‐tailed phospholipid liposomes may be also applicable to other hydrophobic anticancer drugs which have great potential in cancer therapy.     

Polyelectrolyte-coated liposomes: stabilization of the interfacial complexes

Anionic liposomes, composed of egg lecithin (EL) or dipalmitoylphosphatidylcholine (DPPC) with 20 mol% of cardiolipin (CL(2-)), were mixed with cationic polymers, poly(4-vinylpyridine) fully quaternized with ethyl bromide (P2) or poly-l-lysine (PL). Polymer/liposome binding studies were carried out using electrophoretic mobility (EPM), fluorescence, and conductometry as the main analytical tools. Binding was also examined in the presence of added salt and polyacrylic acid (PAA). The following generalizations arose from the experiments: (a) Binding of P2 and PL to small EL/CL(2-) liposomes (60-80 nm in diameter) is electrostatic in nature and completely reversed by addition of salt or PAA. (b) Binding can be enhanced by hydrophobization of the polymer with cetyl groups. (c) Binding can also be enhanced by changing the phase state of the lipid bilayer from liquid to solid (i.e. going from EL to DPPC) or by increasing the size of the liposomes (i.e. going from 60-80 to 300 nm). By far the most promising systems, from the point of view of constructing polyelectrolyte multilayers on liposome cores without disruption of liposome integrity, involve small, liquid, anionic liposomes coated initially with polycations carrying pendant alkyl groups.     

Formulation and evaluation of celastrol-loaded liposomes

The main purpose of this study was to evaluate the intestinal absorption and the antineoplastic effect of the poorly water-soluble drug celastrol when liposomes were used as oral drug delivery system. Liposomes were prepared by the ethanol-injection method. An optimized liposome formulation composed of phospholipid, cholesterol and Tween-80 resulted in favorable encapsulation efficiency at 98.06 ± 0.94%. Homogeneous and stable particle size of 89.6 ± 7.3 nm and zeta potential of -(87.7 ± 5.8) mV were determined by laser particle size analyzer. Subsequently, the four-site perfusion rat intestinal model revealed that celastrol-loaded liposomes had improved effective permeability compared to the free drug in four intestinal segments (p < 0.05). Moreover, celastrol-loaded liposomes could also inhibit the tumor growth in C57BL/6 mice. These results suggest that liposomes could be a promising perioral carrier for celastrol.     

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.     

Polymerization of liposomes composed of diene-containing lipids by radical initiators. II. Polymerization of monodiene-type lipids as liposomes

1-Palmitoyl-2(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine (POPC), a polymerizable lipid that contains one diene group in only a 2-acyl chain, was polymerized as liposome in an aqueous medium. Polymerization was initiated by water-insoluble azobisisobutyronitrile (AIBN), or water-soluble azobis(2-amidinopropane) dihydrochloride (AAPD). AIBN was mixed with monomeric lipids, and the mixture was dispersed in an aqueous medium by sonication to prepare AIBN-containing monomeric lipid liposomes. On the other hand, AAPD was simply added to the liposome suspension. The POPC liposomes were easily polymerized by the addition of AAPD, a water-soluble radical initiator, but few were polymerized by AIBN. The results suggested that the diene group in the 2-acyl chain was in an aqueous phase and, therefore, easily polymerized by a water-soluble radical initiator. The polymerized POPC liposomes were revealed to be more stable than those of monomeric ones because the scattered-light intensity from the polymerized POPC liposome suspension changed a little by the addition of Triton X-100. For only the polymerized ones, the liposome structure was confirmed by TEM after addition of an excess amount of Triton X-100.     

Contrasting behavior of zwitterionic and cationic polymers bound to anionic liposomes

Zwitterionic polymers were prepared by quaternizing polyvinylpyridine (DP = 1100) with bromoacids (Br(CH2)nCOOH, where n = 1, 2, 3, and 5). The resulting polymers were then added to unilamellar liposomes composed of egg lecithin or dipalmitoylphosphatidylcholine admixed with 20 mol % of cardiolipin (a phospholipid with two negative charges). These systems were compared (along with polyethylvinylpyridinium chloride, a polycation) by light scattering, electrophoretic mobility, fluorescence, and high-sensitivity differential scanning calorimetry. The external zwitterionic polymers induce no flip-flop of cardiolipin from the inner leaflet to the outer leaflet as does the polycation. Aside from this similarity, the four zwitterionic polymers all behave differently from each other toward the anionic liposomes: (a) For n = 1, there is no detectable interaction between the polymer and the liposomes. (b) For n = 2, electrostatic attraction induces polymer-liposome association (reversed by the addition of NaCl) that maintains the original negative charge on the liposome. Aggregation of the liposomes accompanies polymer adsorption. (c) For n = 3, electrostatic binding also occurs along with aggregation. However, the binding is so strong that NaCl is unable to induce polymer/liposome dissociation. (d) For n = 5, there is polymer binding and NaCl-promoted dissociation but no substantial aggregation. These differences among the closely related polymers are discussed and analyzed in molecular terms.     

Evaluation of fusion between liposomes and erythrocytes for intracellular derivatization of amino acids in cells

Erythrocytes were fused with liposome for intracellular derivatization of amino acids in cells. The fusion efficiency was evaluated with capillary electrophoresis (CE) and laser-induced fluorescence (LIF) detection. Reagent fluorescein isothiocyanate (FITC) was enveloped in liposomes and introduced into erythrocytes by fusion between liposomes and erythrocytes. The amino acids in the fused cells were derivated by the introduced FITC and the derivated amino acids were extracted for detection by capillary electrophoresis equipped with laser-induced fluorescence detector. The fusion conditions were investigated. It was found that incubation of liposome and erythrocytes in the presence of 13% polyethylene glycol 6000 (PEG 6000) for 15min produced the highest fusion efficiency and kept the erythrocytes stability.     

STUDIES ON THE MECHANISM OF THE HEMATOPORPHYRIN-SENSITIZED PHOTOOXIDATION OF 1,3-DIPHENYLISOBENZOFURAN IN ETHANOL and UNILAMELLAR LIPOSOMES

The 5 microM hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran (DPBF) was studied in homogeneous ethanolic solutions and in aqueous dispersions of unilamellar liposomes of dipalmitoylphosphatidylcholine; both the porphyrin and DPBF are embedded in the phospholipid bilayer. The rate and quantum yield of DPBF photooxidation were found to increase upon increasing the substrate concentration and were higher in the liposome system, while they were unaffected by the fluidity of the phospholipid bilayer. Time-resolved spectroscopic measurements showed that the photooxidation of DPBF in ethanol solution proceeds by a type II O2(1 delta g)-involving mechanism. In the liposomal vesicles the high local concentration of hematoporphyrin (Hp) and DPBF in the phospholipid bilayer (ca 2000-fold higher than the stoichiometric concentration) enhances the probability of energy transfer from triplet Hp to DPBF with generation of triplet DPBF; hence O2 (1 delta g) formation can be promoted by both triplet Hp and triplet DPBF. A minor fraction of triplet DPBF quenchings appears to generate radical species which propagate DPBF damage by chain reaction.     

Adsorption of liposomes and emulsions studied with a quartz crystal microbalance

The adsorption from phospholipid liposome solutions (1.2%) and phospholipid stabilized oil-in-water emulsions (20% purified soybean oil) with the same phospholipid liposome concentration, has been followed by means of a quartz crystal microbalance allowing the simultaneous determination of changes in resonance frequency and energy dissipation. Both the fundamental resonance frequency and the third overtone were used for following the interfacial processes. The adsorption from the liposome solution resulted in formation of a phospholipid bilayer with an additional and incomplete outer layer of liposomes. The outer layer was removed by dilution leaving a bilayer of phospholipids on the surface. The adsorption process observed from the concentrated emulsion solution was considerably more complex. A slow spreading process that also resulted in some expulsion of material from the interface followed the rapid initial adsorption of emulsion droplets. After rinsing with water a phospholipid bilayer was retained on the surface.     

Possibility of Targeting Treatment for Ischemic Heart Disease With Liposome (Ⅰ)

This paper reports the basic research on the possibility of using targeting treatment for ischemic heart disease with liposome as drug carrier. Studies have been performed on isolated rat cardiomyocytes, or isolated perfused rat and rabbit hearts. Results show that cardiomyocytes may interact with liposome through fusion, endocytosis, adsorption and molecular exchange of phospholipid. Forms of cellular uptake of liposome depend chiefly on the physicochemical properties of liposomes. Anoxia changes the pattern of liposome uptake by cardiomyocytes and increases uptake of liposomes. Uptake of liposomes, especially of positively charged liposomes by ischemic myocardium is significantly increased. The quantity of increase of liposome uptake is in the following order: ischemia-reperfusion area>peripheral area of the infarct>non-ischemic area>infarcted area. The above results indicate that liposome as drug carrier might promote the delivery of drug into ischemic myocardium and cardiomyocytes.     

Effect of lipid composition on phospholipase A2-catalyzed membrane leakage in immobilized liposomes: sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.     

Thermodynamics of Chitinase Partitioning in Soy Lecithin Liposomes and Their Storage Stability

The goal of this study was to define the partitioning behavior of chitinase from Trichoderma spp. in soy lecithin liposomes, using a thermodynamic approach based on the partitioning variation with temperature. An effort has been made to define the liposomes, as well as free and immobilized enzyme stability during storage at 4 and 25 °C. The partition coefficients (K (o/w)) were greater than 1; therefore, the standard free energies of the enzyme transfer were negative, indicating an affinity of the enzymes for encapsulation in liposomes. The enthalpy calculation led to the conclusion that the process is exothermic. The presence of enzyme decreased the liposome storage stability from 70 days to an approximately 20 days at 25 °C and 30 days at 4 °C. Monitoring of the liposome's diameter demonstrated that their size and concentration decreased during storage. The liposome's diameters ranged from 1.06 to 3.30 μm. The higher percentage of liposome corresponded to a diameter range from 1.06 to 1.34 μm. This percentage increased during storage. There were no evidences for liposome fusion process. The stability of immobilized enzyme was increased in comparison with free chitinase.     

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.     

Anomalous solubilization behavior of dimyristoylphosphatidylcholine liposomes induced by sodium dodecyl sulfate micelles

The solubilization dynamics of dimyristoylphosphatidylcholine (DMPC) liposomes, as induced by sodium dodecyl sulfate (SDS), were investigated; this investigation was motivated by several types of atypical behavior that were observed in the solubilization in this system. The liposomes and surfactants were mixed in a microchip, and the solubilization reaction of each liposome was observed using a microscope. We found that solubilization occurred not only via a uniform dissolution of the liposome membrane, but also via a dissolution involving the rapid motion of the liposome, or via active emission of protrusions from the liposome surface. We statistically analyzed the distribution of these patterns and considered hypotheses accounting for the solubilization mechanism based on the results. When the SDS concentration was lower than the critical micelle concentration (CMC), the SDS monomers entered the liposome membrane, and mixed micelles were emitted. When the SDS concentration was higher than the CMC, the SDS micelles directly attacked the liposome membrane, and many SDS molecules were taken up; this caused instability, and atypical solubilization patterns were triggered. The size dependence of the solubilization patterns was also investigated. When the particle size was smaller, the SDS molecules were found to be homogeneously dispersed throughout the whole membrane, which dissolved uniformly. In contrast, when the particle size was larger, the density of SDS molecules increased locally, instability was induced, and atypical dissolution patterns were often observed.