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.     

Effect of water-soluble polymers on fusion of egg yolk and soybean phospholipid liposomes

The efficiencies of polyelectrolytes, i.e., polycations and polyanions, and several kinds of water-soluble polymers as fusogens on soybean phospholipid liposome (SL) and egg yolk phospholipid liposome (EL) were investigated by the fluorescence quenching method. There were optimal concentrations for the induction of fusion in every system. Polycations induced fusion of liposomes at very low concentration in comparison with other polymers. Poly(carboxylic acid)s induced fusion at relatively high concentration. A strong acidic polyanion with high molecular weight also induced fusion of liposomes. The induction efficiency of poly(ethylene glycol) on fusion was higher than other nonionic polymers. The efficiency of fusion of EL was lower than that of SL in all systems because of the higher stability of EL membrane. It was found that electrostatic interactions, hydrogen bonding and/or hydrophobic interaction between these water-soluble polymers and liposomal membranes played an important role on aggregation and fusion of liposomes.     

Optimization and characterization of liposome formulation by mixture design

This study presents the application of the mixture design technique to develop an optimal liposome formulation by using the different lipids in type and percentage (DOPC, POPC and DPPC) in liposome composition. Ten lipid mixtures were generated by the simplex-centroid design technique and liposomes were prepared by the extrusion method. Liposomes were characterized with respect to size, phase transition temperature, ζ-potential, lamellarity, fluidity and efficiency in loading calcein. The results were then applied to estimate the coefficients of mixture design model and to find the optimal lipid composition with improved entrapment efficiency, size, transition temperature, fluidity and ζ-potential of liposomes. The response optimization of experiments was the liposome formulation with DOPC: 46%, POPC: 12% and DPPC: 42%. The optimal liposome formulation had an average diameter of 127.5 nm, a phase-transition temperature of 11.43 °C, a ζ-potential of -7.24 mV, fluidity (1/P)(TMA-DPH)((?)) value of 2.87 and an encapsulation efficiency of 20.24%. The experimental results of characterization of optimal liposome formulation were in good agreement with those predicted by the mixture design technique.     

Effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the permeability of liposomal bilayer membranes

The effects of poly(ethylene glycol) (PEG) chain length of PEG-lipid on the membrane characteristics of liposomes were investigated by differential scanning calorimetry (DSC), freeze-fracture electron microscopy (FFEM), fluorescence polarization measurement and permeability measurement using carboxyfluorescein (CF). PEG-liposomes were prepared from mixtures of dipalmitoyl phosphatidylcholine (DPPC) and distearoyl phosphatidylethanolamines with covalently attached PEG molecular weights of 1000, 2000, 3000 and 5000 (DSPE-PEG). DSC and FFEM results showed that the addition of DSPE-PEG to DPPC in the preparation of liposomes caused the lateral phase separation both in the gel and liquid-crystalline states. The fluidity in the hydrocarbon region of liposomal bilayer membranes was not significantly changed by the addition of DSPE-PEG, while that in the interfacial region was markedly increased. From these results, it was anticipated that the CF leakage from PEG-liposomes is accelerated compared with DPPC liposomes. However, CF leakage from liposomes containing DSPE-PEG with a 0.060 mol fraction was depressed compared with regular liposomes, and the leakage decreased with increasing PEG chain length. Furthermore, the CF leakage from liposomes containing DSPE-PEG with a 0.145 mol fraction was slightly increased compared with that of liposomes containing DSPE-PEG with a 0.060 mol fraction. It is suggested that the solute permeability from the PEG-liposomes was affected by not only properties of the liposomal bilayer membranes such as phase transition temperature, phase separation and membrane fluidity, but also the PEG chain of the liposomal surface.     

Electron spin resonance studies of dipalmitoylphosphatidylcholine liposomes containing soybean-derived sterylglucoside

The effects of soybean-derived sterylglucoside (SG) on the fluidity of liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) were investigated compared with those of soybean-derived sterol (SS) and cholesterol (Ch) using an electron spin resonance spectrometer. Three kinds of liposomes were prepared in the molar ratio of DPPC/X=7/4, where X is SS, Ch or SG. The fluidity close to the polar head groups increased with an increase of temperature in the DPPC membrane containing SS, Ch and SG in the range 35 to 45 degrees C. Those near the hydrophobic end changed with an increase in temperature in liposomes containing SS, Ch and SG, which had a fluidizing effect on the DPPC membrane below the transition temperature (Tm, 41.9 degrees C) and a condensing effect over the Tm. The fluidizing effects of these compounds around 37 degrees C near the polar head group and the hydrophobic end increased in the following order: Ch < SG < or = SS and SS < Ch < SG, respectively. SG increased the fluidity of liposomal membrane dramatically above the Tm (35.4 degrees C). These results suggest that the high fluidity close to the hydrophobic end of the liposomal membranes around 37 degrees C, the decrease of Tm, and the sigmoidal nature of fluidity vs. temperature are important factors in the effectiveness of liposomes containing SG as a carrier of drugs.     

Calorimetric and spin-label ESR studies of PEG:2000-DPPE containing DPPC/lyso-PPC mixtures

The thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) multibilayers containing up to 10 mol% of lyso-palmitoylphosphatidylcholine (lyso-PPC) with and without low content of poly(ethylene glycol:2000)-grafted dipalmitoylphosphatidylethanolamine (PEG:2000-DPPE) has been studied by high sensitivity differential scanning calorimetry (DSC) and electron spin resonance (ESR) using the spin probe di-tert-butyl-nitroxide (DTBN). The three lipids, dispersed in buffer at appropriate concentrations, form thermosensitive liposomes used as site-specific drug-delivery systems. Without polymer–lipids, the DPPC main transition temperature is downshifted of 1.2–1.3 °C at the highest lyso-PPC content. The molar enthalpy and the cooperative unit of the DPPC main transition first decrease rapidly, then more slowly and finally slightly increase with lyso-PPC content. Moreover, in the mixed dispersions, the membrane fluidity increases at any temperature. The addition up to 5 mol% of PEG:2000-DPPE to DPPC/10 mol% lyso-PPC mixtures does not affect neither the thermotropic phase behavior nor the transition cooperativity and the fluidity of the dispersions.     

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.     

Electron spin resonance studies of phosphatidylcholine interacted with cholesterol and with a hopanoid in liposomal membrane.

The effects of bacteriohopane-32-ol (Monol) on liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (egg PC) were compared with those of cholesterol (Chol) in the change of fluidity using a spin label. The fluidity change close to the polar head groups caused by temperature increase in the DPPC membrane containing Monol was different from that of Chol. Chol had a condensing effect on DPPC membrane, whereas Monol had only a slight effect except when used at 20 mol%. Near the hydrophobic end, Chol incorporation into DPPC led to fluidization below transition temperature (Tm) and condensation above Tm. Monol incorporation into DPPC had only a fluidizing effect below Tm. On the other hand, in egg PC membrane Chol had the condensing effect at any temperature, whereas Monol had only slight effect. These results suggest that Monol may have a role in supporting constant membrane fluidity under drastic conditions.     

Effects of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers on structure and stability of liposomal dioleoylphosphatidylethanolamine

The effects caused by poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO; Pluronic) copolymers on the structure and stability of dioleoylphosphatidylethanolamine (DOPE) liposomes were studied by means of turbidity, leakage, and cryo-transmission electron microscopy investigations. The results show that by inclusion of Pluronics in the DOPE dispersion it is possible to stabilize the lamellar Lalpha phase and to produce liposomes that are stable and nonleaky at low pH (pH 5). The stabilizing capacity was observed to depend critically on the molecular composition of the Pluronics. Block copolymers with comparably long PPO and PEO segment lengths, such as F127 and F108, most effectively protected DOPE liposomes prepared at high pH from aggregation and subsequent structural rearrangements induced by acidification. A sufficiently long PPO block was found to be the most decisive parameter in order to obtain adequate coverage of the liposome surface at low Pluronic concentrations. Upon increasing the copolymer concentration, however, Pluronics with comparably short PPO and PEO segment lengths, such as F87 and P85, could also be used to stabilize the DOPE liposomes. Essentially the same trends were observed when the Pluronics were added to preformed DOPE liposomes instead of being included in the preparation mixture. In this case the least effective copolymers failed, however, to completely prevent the DOPE liposomes from releasing encapsulated hydrophilic markers.     

Stable polymeric nanoballoons: lyophilization and rehydration of cross-linked liposomes

The cross-linking of supramolecular assemblies of hydrated lipids is an effective method to stabilize these assemblies to disruption by surfactants or aqueous alcohol. The heterobifunctional lipids, Acryl/DenPC(16,18) and Sorb/DenPC(18,21), are examples of a new class of polymerizable lipid designed for the creation of cross-linked lipid structures. The robust nature of cross-linked liposomes was demonstrated by lyophilization of the liposomes followed by their essentially complete redispersion in water. The resulting liposomes were compared to the original sample by quasi-elastic light scattering and transmission electron microscopy. There was no major change in the size or structure of the cross-linked liposomes after rehydration of the freeze-dried powder of liposomes. Moreover, the rehydrated cross-linked liposomes continued to be resistant to surfactant solubilization. Neutral cross-linked liposomes were predominantly redispersed after freeze-drying with the aid of bath sonication. The small amount of residual liposome aggregation observed with neutral liposomes could be prevented by incorporating a surface charge into the liposome or attaching hydrophilic polymers, for example, poly(ethylene glycol), onto the liposome.     

Interaction of Dipalmitoyl Phosphatidylcholine with n‐Hexadecanol in Monolayer and Liposome

The monolayer collapse behavior of n‐hexadecanol/dipalmitoyl phosphatidylcholine (DPPC) was investigated in this study at the air/water interface at 37 °C. Surface pressure variations with time for the mixed monolayers of DPPC with 20 mol% and 50 mol% n‐hexadecanol at corresponding collapse points were recorded by a Langmuir trough system. In addition, the interaction of n‐hexadecanol with a pure DPPC monolayer was identified by fluorescence microscopy (FM). The results demonstrated distinct differences between these systems; according to our observation, the higher the ratio of n‐hexadecanol to DPPC, the more nucleation domains can be induced. The FM images demonstrated that pronounced domain formation was associated with a longer relaxation time of the collapsed DPPC and DPPC/n‐hexadecanol monolayers, and the presence of n‐hexadecanol appeared to enhance the relaxation processes. The liposome was prepared by the thin‐film hydration method. The average diameter of DPPC and DPPC/n‐hexadecanol liposomes was investigated by dynamic light scattering. It is shown that the diameter of DPPC liposome with n‐hexadecanol is smaller than pure DPPC liposome at the initial state. After 24 hours, DPPC/n‐hexadecanol liposome became larger than pure DPPC liposome and lasted for the next four days. The effects of a greater ratio of n‐hexadecanol did not play an important role in DPPC liposome formation based on our dynamic light scattering analysis. Our result demonstrated that n‐hexadecanol might affect the DPPC liposome stability. The increased ratio of n‐hexadecanol in DPPC liposomes could only a play a minor role in DPPC liposome fusion.     

Influence of some physical properties of 5-fluorouracil on encapsulation efficiency in liposomes

The aim of this study is to encapsulate two drugs: 5-fluorouracil (5-FU) with the hydrophobic properties and 1-β-D-arabinofuranosylcytosine (Ara-C) with the amphiphilic properties into liposomes prepared by the modified reverse-phase evaporation method (mREV) from L-α-phosphatidylcholine dipalmitoyl (DPPC). We studied the thermotropic phase behavior of liposome entrapped 5-FU and Ara-C. It is known that the stability of liposomes depends not only on the method of chemical gradient loading, the use of membrane stabilizer such as sterols, but also on the phase transition temperature (T _c) of phospholipids, which undergoes an alteration after encapsulation of drugs to liposomes. The competition of these two drugs entrapped in liposomes was analyzed by the use of two spectroscopies: ¹H NMR and UV on the basis of the analysis of the signals of each drug in the liposome—drug system. The percent of encapsulation in DPPC/Ara-C/5-FU liposome obtained by the use of UV spectroscopy amounted 93.84 and 96.05% for 5-FU and Ara-C, respectively. Phase transition temperature T _c of liposomes containing Ara-C did not significantly change while for the liposomes containing 5-FU it increased in comparison with T _c of the reference liposomes formed from DPPC.     

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.     

Fluorescence studies on radiation oxidative damage to membranes with implications to cellular radiosensitivity

Radiation oxidative damage to plasma membrane and its consequences to cellular radiosensitivity have received increasing attention in the past few years. This review gives a brief account of radiation oxidative damage in model and cellular membranes with particular emphasis on results from our laboratory. Fluorescence and ESR spin probes have been employed to investigate the structural and functional alterations in membranes after y-irradiation. Changes in the lipid bilayer in irradiated unilamellar liposomes prepared from egg yolk lecithin (EYL) were measured by using diphenylhexatriene (DPH) as a probe. The observed increase in DPH polarization and decrease in fluorescence intensity after γ-irradiation of liposomes imply radiation-induced decrease in bilayer fluidity. Inclusion of cholesterol in liposome was found to protect lipids against radiation damage, possibly by modulation of bilayer organization e.g. lipid packing. Measurements on dipalmitoyl phosphatidylcholine (DPPC) liposomes loaded with 6-carboxyfluorescein (CF) showed radiation dose-dependent release of the probe indicating radiation-induced increased permeability. Changes in plasma membrane permeability of thymocytes were monitored by fluorescein diacetate (FDA) and induced intracellular reactive oxygen species (ROS) were determined by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA). Results suggest a correlation between ROS generation and membrane permeability changes induced by radiation within therapeutic doses (0-10 Gy). It is concluded that increase in membrane permeability was the result of ROS-mediated oxidative reactions, which might trigger processes leading to apoptotic cell death after radiation exposure.     

Thermotropic behaviour of dipalmitoylphosphatidylcholine liposomes containing retinoids

Differential scanning calorimetry has been used to investigate the thermotropic behaviour of DPPC liposomes in the presence of different amounts of retinoids in a study of the sites concerned in the mutual lipid-retinoid interaction. The perturbing effect of retinal and retinol on DPPC liposome gel-liquid crystal phase transition has been related to the difference in the polar end group of retinoid. The hydrophilic polar group prevalence over the apolar tail has been evidentiated. Membrane fluidity increases by increasing the retinoid amount. These liposomes displayed a phase separation at high retinal or retinol concentrations.     

Magnolol Encapsulated by Liposome in Inhibiting Smooth Muscle Cell Proliferation

Magnolol, a pure compound extracted from Magnolia officinalis, encapsulated by liposome was investigated for inhibiting vascular smooth muscle cell (VSMC) proliferation leading to restenosis by Percutaneous Transluminal Coronary Angioplasty (PTCA). 1,2‐Diacyl‐Sn‐glycero‐3‐phosphocholine (EPC) and 1,2‐dipalmitoyl‐Sn‐glycero‐3‐phosphocholine (DPPC) liposomes were utilized to encapsulate the magnolol in this study. The inhibitory efficiency of the liposome encapsulated magnolol on cell viability was higher than the pure magnolol. EPC liposome was found to have higher efficiency in inhibiting VSMCs than DPPC. The diameters of EPC and DPPC liposome which encapsulated magnolol became larger than pure EPC and DPPC liposomes. The photos from transmission electron microscopy (TEM) were demonstrated that the EPC and DPPC liposomes could be interfered by magnolol to form a homogeneous liposome. Addition of cholesterol to EPC and DPPC liposome could reduce the liposome diameter.     

Stabilization of small unilamellar DMPC-liposomes by uncharged polymers

 Polymer-free and polymer-bearing small unilamellar (SUV) liposomes from dimyristoyl-phosphatidylcholine (DMPC) were prepared under standardized conditions. Polymer-bearing liposomes were formed by incorporating an uncharged polymer [hydrolyzed poly(vinyl alcohol) (PVA), poly(vinyl alcohol-co-vinylacetal) (PVA-Al), poly(vinyl alcohol-co-vinyl propional) (PVA-Prol) poly(vinyl alcohol-co-vinyl butiral) (PVA-Bul) copolymer or poly(vinyl pyrrolidone) (PVP)] into the membrane bilayer of vesicles. The kinetic (long-term) stability of the liposome dispersions stored in distilled water, in physiological NaCl solution and at various pH values, respectively, were studied. The physical stability of vesicles was tested by measuring the size and the zeta potential of liposomes by means of a Malvern Zetasizer 4 apparatus. It was shown that most of these polymers are effective steric stabilizers for the DMPC-liposomes. Among the polymers, the PVA-Bul and PVA-Prol copolymers and the PVP of high molecular mass exhibited the most efficient stabilizing effect at each pH studied, indicating that the formation of a relatively thick polymer layer around the lipid bilayers ensures an enhanced and prolonged physical stability of liposomes. Also, the butiral or propional side chain in the PVA-based copolymers presumably promotes the anchoring of macromolecules to the vesicles. Using these macromolecules, the colloidal interactions between vesicles can be modified and so the physical stability of liposomes and the kinetic stability of liposome dispersions can also be controlled. Received: 20 May 1997 Accepted: 03 September 1997     

Characterisation of electroosmotic flow in capillary electrochromatography columns

Immobilized liposome chromatography (ILC) has been proven to be a useful method for the study or rapid screening of drug-membrane interactions. To obtain an adequate liposomal membrane phase for ILC, unilamellar liposomes were immobilized in gel beads by avidin-biotin binding. The retardation of 15 basic drugs on the liposome column could be converted to membrane partitioning coefficients, K(LM). The effects of small or large unilamellar liposomes and multilamellar liposomes on the drug-membrane partitioning were compared. The K(LM) values for both small and large liposomes were similar, but higher than those for the multilamellar liposomes. The basic drugs showed stronger partitioning into negatively charged liposomes than into either neutral liposomes or positively charged liposomes. The membrane fluidity of the immobilized liposomes was modulated by incorporating cholesterol into the liposomal membranes, by changing the acyl chain length and degree of unsaturation of the phospholipids, and by changing the temperature for ILC runs. Our data show that K(LM) obtained using ILC correlated well with those reported by batch studies using free liposomes. It is concluded that negatively charged or cholesterol-containing large unilamellar liposomes are suitable models for the ILC analysis of drug-membrane interactions.     

Effects of silver nanoparticles on the fluidity of bilayer in phospholipid liposome

This paper describes the formation and characterization of liposome entrapping the silver nanoparticles in bilayer. Silver nanoparticles were entrapped in the bilayer of dipalmitoylphosphatidylcholine (DPPC) liposome, named as silver-loaded liposome. Specifically, above the gel to liquid-crystalline phase transition temperature of this lipid (i.e., 41 degrees C), it was observed that membrane fluidities of silver-loaded liposomes were increased, and fluorescence anisotropy values were reduced from 0.114 to 0.097. This might be due to the structural modifications and interactions between DPPC molecules and silver nanoparticles within the bilayer. It was also confirmed that silver nanoparticles were entrapped in hydrophobic region of lipid bilayer with transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) measurements.     

Liposome modified with Mn-porphyrin complex can simultaneously induce antioxidative enzyme-like activity of both superoxide dismutase and peroxidase

An antioxidative liposome catalysis that mimics both superoxide dismutase (SOD) and peroxidase (POD) activities has been developed by using the liposomes modified with lipophilic Mn-(5,10,15,20-tetrakis[1-hexadecylpyridium-4-yl]-21H,23H-porphyrin) (Mn-HPyP). The SOD- and POD-like activities of the Mn-HPyP-modified liposome were first investigated by varying the type of phospholipid, such as 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). Higher SOD-like activity was obtained in the case of DLPC and DMPC liposomes, in which the ligands were well-dispersed on the membrane in the liquid crystalline phase. The POD-like activity was maximal in the case of DMPC liposome, in which the Mn-HPyP complex was appropriately clustered on the membrane in the gel phase. On the basis of the above results, the co-induction of the SOD and POD activities to eliminate the superoxide and also hydrogen peroxide as a one-pot reaction was finally performed by using the Mn-HPyP-modified DMPC liposome, resulting in an increase in the efficiency of the elimination of both superoxide and hydrogen peroxide.